Science.gov

Sample records for adh enzyme activity

  1. Ethanol-Induced ADH Activity in Zebrafish: Differential Concentration-Dependent Effects on High- Versus Low-Affinity ADH Enzymes.

    PubMed

    Tran, Steven; Nowicki, Magda; Facciol, Amanda; Chatterjee, Diptendu; Gerlai, Robert

    2016-04-01

    Zebrafish express enzymes that metabolize ethanol in a manner comparable to that of mammals, including humans. We previously demonstrated that acute ethanol exposure increases alcohol dehydrogenase (ADH) activity in an inverted U-shaped dose-dependent manner. It was hypothesized that the biphasic dose-response was due to the increased activity of a high-affinity ADH isoform following exposure to low concentrations of ethanol and increased activity of a low-affinity ADH isoform following exposure to higher concentrations of ethanol. To test this hypothesis, we exposed zebrafish to different concentrations of ethanol (0%, 0.25%, 0.5%, and 1.0% v/v) for 30 min and measured the total ADH activity in the zebrafish liver. However, we also repeated this enzyme activity assay using a low concentration of the substrate (ethanol) to determine the activity of high-affinity ADH isoforms. We found that total ADH activity in response to ethanol induces an inverted U-shaped dose-response similar to our previous study. Using a lower substrate level in our enzyme assay targeting high-affinity isozymes, we found a similar dose-response. However, the difference in activity between the high and low substrate assays (high substrate activity - low substrate activity), which provide an index of activity for low-affinity ADH isoforms, revealed no significant effect of ethanol exposure. Our results suggest that the inverted U-shaped dose-response for total ADH activity in response to ethanol is driven primarily by high-affinity isoforms of ADH.

  2. Increased Variation in Adh Enzyme Activity in Drosophila Mutation-Accumulation Experiment Is Not Due to Transposable Elements at the Adh Structural Gene

    PubMed Central

    Aquadro, C. F.; Tachida, H.; Langley, C. H.; Harada, K.; Mukai, T.

    1990-01-01

    We present here a molecular analysis of the region surrounding the structural gene encoding alcohol dehydrogenase (Adh) in 47 lines of Drosophila melanogaster that have each accumulated mutations for 300 generations. While these lines show a significant increase in variation of alcohol dehydrogenase enzyme activity compared to control lines, we found no restriction map variation in a 13-kb region including the complete Adh structural gene and roughly 5 kb of both 5' and 3' sequences. Thus, the rapid accumulation of ADH activity variation after 28,200 allele generations does not appear to have been due to the mobilization of transposable elements into or out of the Adh structural gene region. PMID:1963870

  3. Cloning of the Arabidopsis and Rice Formaldehyde Dehydrogenase Genes: Implications for the Origin of Plant Adh Enzymes

    PubMed Central

    Dolferus, R.; Osterman, J. C.; Peacock, W. J.; Dennis, E. S.

    1997-01-01

    This article reports the cloning of the genes encoding the Arabidopsis and rice class III ADH enzymes, members of the alcohol dehydrogenase or medium chain reductase/dehydrogenase superfamily of proteins with glutathione-dependent formaldehyde dehydrogenase activity (GSH-FDH). Both genes contain eight introns in exactly the same positions, and these positions are conserved in plant ethanol-active Adh genes (class P). These data provide further evidence that plant class P genes have evolved from class III genes by gene duplication and acquisition of new substrate specificities. The position of introns and similarities in the nucleic acid and amino acid sequences of the different classes of ADH enzymes in plants and humans suggest that plant and animal class III enzymes diverged before they duplicated to give rise to plant and animal ethanol-active ADH enzymes. Plant class P ADH enzymes have gained substrate specificities and evolved promoters with different expression properties, in keeping with their metabolic function as part of the alcohol fermentation pathway. PMID:9215914

  4. [Effects of H2-blockers on alcohol dehydrogenase (ADH) activity].

    PubMed

    Jelski, Wojciech; Orywal, Karolina; Szmitkowski, Maciej

    2008-12-01

    First-pass metabolism (FPM) of alcohol is demonstrated by lower blood alcohol concentrations after oral than intravenous administration of the same dose. FPM occurs predominantly in the stomach and has been attributed to class IV of alcohol dehydrogenase (ADH) isoenzyme localizated in the gastric mucosa. A number of factors that influence on gastric ADH activity and thereby modulate FPM have been identified. These include age, sex, ethnicity, concentrations and amounts of alcohol consumed and drugs. Several H2-receptor antagonists, including cimetidine and ranitidine, inhibit gastric ADH activity and reduce FPM, resulting in higher blood alcohol concentrations after H2-blockers administration.

  5. The metabolic enzyme AdhE controls the virulence of Escherichia coli O157:H7

    PubMed Central

    Beckham, Katherine S H; Connolly, James P R; Ritchie, Jennifer M; Wang, Dai; Gawthorne, Jayde A; Tahoun, Amin; Gally, David L; Burgess, Karl; Burchmore, Richard J; Smith, Brian O; Beatson, Scott A; Byron, Olwyn; Wolfe, Alan J; Douce, Gillian R; Roe, Andrew J

    2014-01-01

    Classical studies have focused on the role that individual regulators play in controlling virulence gene expression. An emerging theme, however, is that bacterial metabolism also plays a key role in this process. Our previous work identified a series of proteins that were implicated in the regulation of virulence. One of these proteins was AdhE, a bi-functional acetaldehyde-CoA dehydrogenase and alcohol dehydrogenase. Deletion of its gene (adhE) resulted in elevated levels of extracellular acetate and a stark pleiotropic phenotype: strong suppression of the Type Three Secretion System (T3SS) and overexpression of non-functional flagella. Correspondingly, the adhE mutant bound poorly to host cells and was unable to swim. Furthermore, the mutant was significantly less virulent than its parent when tested in vivo, which supports the hypothesis that attachment and motility are central to the colonization process. The molecular basis by which AdhE affects virulence gene regulation was found to be multifactorial, involving acetate-stimulated transcription of flagella expression and post-transcriptional regulation of the T3SS through Hfq. Our study reveals fascinating insights into the links between bacterial physiology, the expression of virulence genes, and the underlying molecular mechanism mechanisms by which these processes are regulated. PMID:24846743

  6. Temperature and water loss affect ADH activity and gene expression in grape berry during postharvest dehydration.

    PubMed

    Cirilli, Marco; Bellincontro, Andrea; De Santis, Diana; Botondi, Rinaldo; Colao, Maria Chiara; Muleo, Rosario; Mencarelli, Fabio

    2012-05-01

    Clusters of Aleatico wine grape were picked at 18°Brix and placed at 10, 20, or 30°C, 45% relative humidity (RH) and 1.5m/s of air flow to dehydrate the berries up to 40% of loss of initial fresh weight. Sampling was done at 0%, 10%, 20%, 30%, and 40% weight loss (wl). ADH (alcohol dehydrogenase) gene expression, enzyme activity, and related metabolites were analysed. At 10°C, acetaldehyde increased rapidly and then declined, while ethanol continued to rise. At 20°C, acetaldehyde and ethanol increased significantly with the same pattern and declined at 40%wl. At 30°C, acetaldehyde did not increase but ethanol increased rapidly already at 10%wl. At the latter temperature, a significant increase in acetic acid and ethyl acetate occurred, while at 10°C their values were low. At 30°C, the ADH activity (ethanol to acetaldehyde direction), increased rapidly but acetaldehyde did not rise because of its oxidation to acetic acid, which increased together with ethyl acetate. At 10°C, the ADH activity increased at 20%wl and continued to rise even at 40%wl, meaning that ethanol oxidation was delayed. At 20°C, the behaviour was intermediate to the other temperatures. The relative expression of the VvAdh2 gene was the highest at 10°C already at 10%wl in a synchrony with the ADH activity, indicating a rapid response likely due to low temperature. The expression subsequently declined. At 20 and 30°C, the expression was lower and increased slightly during dehydration in combination with the ADH activity. This imbalance between gene expression and ADH activity at 10°C, as well as the unexpected expression of the carotenoid cleavage dioxygenase 1 (CCD1) gene, opens the discussion on the stress sensitivity and transcription event during postharvest dehydration, and the importance of carefully monitoring temperature during dehydration.

  7. A 5-hydroxymethyl furfural reducing enzyme encoded by the Saccharomyces cerevisiae ADH6 gene conveys HMF tolerance.

    PubMed

    Petersson, Anneli; Almeida, João R M; Modig, Tobias; Karhumaa, Kaisa; Hahn-Hägerdal, Bärbel; Gorwa-Grauslund, Marie F; Lidén, Gunnar

    2006-04-30

    The fermentation of lignocellulose hydrolysates by Saccharomyces cerevisiae for fuel ethanol production is inhibited by 5-hydroxymethyl furfural (HMF), a furan derivative which is formed during the hydrolysis of lignocellulosic materials. The inhibition can be avoided if the yeast strain used in the fermentation has the ability to reduce HMF to 5-hydroxymethylfurfuryl alcohol. To enable the identification of enzyme(s) responsible for HMF conversion in S. cerevisiae, microarray analyses of two strains with different abilities to convert HMF were performed. Based on the expression data, a subset of 15 reductase genes was chosen to be further examined using an overexpression strain collection. Three candidate genes were cloned from two different strains, TMB3000 and the laboratory strain CEN.PK 113-5D, and overexpressed using a strong promoter in the strain CEN.PK 113-5D. Strains overexpressing ADH6 had increased HMF conversion activity in cell-free crude extracts with both NADPH and NADH as co-factors. In vitro activities were recorded of 8 mU/mg with NADH as co-factor and as high as 1200 mU/mg for the NADPH-coupled reduction. Yeast strains overexpressing ADH6 also had a substantially higher in vivo conversion rate of HMF in both aerobic and anaerobic cultures, showing that the overexpression indeed conveyed the desired increased reduction capacity.

  8. Expression pattern, ethanol-metabolizing activities, and cellular localization of alcohol and aldehyde dehydrogenases in human large bowel: association of the functional polymorphisms of ADH and ALDH genes with hemorrhoids and colorectal cancer.

    PubMed

    Chiang, Chien-Ping; Jao, Shu-Wen; Lee, Shiao-Pieng; Chen, Pei-Chi; Chung, Chia-Chi; Lee, Shou-Lun; Nieh, Shin; Yin, Shih-Jiun

    2012-02-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are principal enzymes responsible for metabolism of ethanol. Functional polymorphisms of ADH1B, ADH1C, and ALDH2 genes occur among racial populations. The goal of this study was to systematically determine the functional expressions and cellular localization of ADHs and ALDHs in human rectal mucosa, the lesions of adenocarcinoma and hemorrhoid, and the genetic association of allelic variations of ADH and ALDH with large bowel disorders. Twenty-one surgical specimens of rectal adenocarcinoma and the adjacent normal mucosa, including 16 paired tissues of rectal tumor, normal mucosae of rectum and sigmoid colon from the same individuals, and 18 surgical mixed hemorrhoid specimens and leukocyte DNA samples from 103 colorectal cancer patients, 67 hemorrhoid patients, and 545 control subjects recruited in previous study, were investigated. The isozyme/allozyme expression patterns of ADH and ALDH were identified by isoelectric focusing and the activities were assayed spectrophotometrically. The protein contents of ADH/ALDH isozymes were determined by immunoblotting using the corresponding purified class-specific antibodies; the cellular activity and protein localizations were detected by immunohistochemistry and histochemistry, respectively. Genotypes of ADH1B, ADH1C, and ALDH2 were determined by polymerase chain reaction-restriction fragment length polymorphisms. At 33mM ethanol, pH 7.5, the activity of ADH1C*1/1 phenotypes exhibited 87% higher than that of the ADH1C*1/*2 phenotypes in normal rectal mucosa. The activity of ALDH2-active phenotypes of rectal mucosa was 33% greater than ALDH2-inactive phenotypes at 200μM acetaldehyde. The protein contents in normal rectal mucosa were in the following order: ADH1>ALDH2>ADH3≈ALDH1A1, whereas those of ADH2, ADH4, and ALDH3A1 were fairly low. Both activity and content of ADH1 were significantly decreased in rectal tumors, whereas the ALDH activity remained

  9. Locus Adh of Drosophila melanogaster under selection for delayed senescence

    SciTech Connect

    Khaustova, N.D.

    1995-05-01

    Dynamics of the Adh activity and frequencies of alleles Adh{sup F} and Adh{sup S} were analyzed under selection for delayed senescence. The experiments were performed on Drosophila melanogaster. Lines Adh{sup S}cn and Adh{sup F}vg and experimental populations cn` and vg`, selected for an increased duration of reproductive period (late oviposition) were used. Analysis of fertility, longevity, viability and resistance to starvation showed that selection for late oviposition resulted in delayed senescence of flies of the experimental populations. Genetic structure of population vg` changed considerably with regard to the Adh locus. This was confirmed by parameters of activity, thermostability, and electrophoretic mobility of the enzyme isolated from flies after 30 generations of selection. Analysis of frequencies of the Adh alleles showed that in both selected populations, which initially had different genetic composition, accumulated allele Adh{sup S}, which encodes the isozyme that is less active but more resistant to inactivation. Genetic mechanism of delayed senescence in Drosophila is assumed to involve selection at vitally important enzyme loci, including Adh. 18 refs., 2 tabs., 4 figs.

  10. Opposing--activating or inhibitory--effects of cimetidine and daidzein on human ADH1C activity depending on substrates and solvents.

    PubMed

    Kollock, Ronny; Glatt, Hansruedi

    2012-01-01

    Toxification of benzylic alcohols (e.g. hydroxymethylpyrenes) by sulfotransferases is efficiently competed by alcohol dehydrogenases (ADHs). We are interested in drugs and food constituents affecting this detoxification. Daidzein and cimetidine were reported to inhibit ADH1C-mediated ethanol oxidation. Surprisingly, we found that both modulators enhance the oxidation of 4-hydroxymethylpyrene by ADH1C. This activation was seen with either delivering solvents used, dimethylsulfoxide or acetonitrile. Addition of dimethylsulfoxide, but not acetonitrile, converted daidzein and cimetidine from inhibitors to activators of the ADH1C-mediated oxidation of the other substrate studied, ethanol (added in water). Other human ADH forms (ADH2, 3, 4) were inhibited by both agents independently of the substrate and the corresponding solvent used. Kinetic constants for the various reactions are presented. ADH1C was unique in its complex substrate-dependent interaction with daidzein/cimetidine and solvents.

  11. Genetic Variation in the Expression of ADH in DROSOPHILA MELANOGASTER

    PubMed Central

    Maroni, G.; Laurie-Ahlberg, C. C.; Adams, D. A.; Wilton, A. N.

    1982-01-01

    Several chromosomes derived from natural populations have been identified that affect the expression of alcohol dehydrogenase (ADH). Second chromosomes, which also carry the structural gene Adh, show a great deal of polymorphism of genetic elements that determine how much enzyme protein accumulates. The level of enzyme was measured in third instar larvae, 6-to-8-day-old males and in larval fat bodies and alimentary canals. In general, activities in the different organs and stages are highly correlated with one another. One line was found, however, in which the ADH level in the fat body is more than twice the level one would expect on the basis of the activity in alimentary canal. We have also found evidence of third-chromosome elements that affect the level of ADH. PMID:6816669

  12. Ethanol-induced alcohol dehydrogenase E (AdhE) potentiates pneumolysin in Streptococcus pneumoniae.

    PubMed

    Luong, Truc Thanh; Kim, Eun-Hye; Bak, Jong Phil; Nguyen, Cuong Thach; Choi, Sangdun; Briles, David E; Pyo, Suhkneung; Rhee, Dong-Kwon

    2015-01-01

    Alcohol impairs the host immune system, rendering the host more vulnerable to infection. Therefore, alcoholics are at increased risk of acquiring serious bacterial infections caused by Streptococcus pneumoniae, including pneumonia. Nevertheless, how alcohol affects pneumococcal virulence remains unclear. Here, we showed that the S. pneumoniae type 2 D39 strain is ethanol tolerant and that alcohol upregulates alcohol dehydrogenase E (AdhE) and potentiates pneumolysin (Ply). Hemolytic activity, colonization, and virulence of S. pneumoniae, as well as host cell myeloperoxidase activity, proinflammatory cytokine secretion, and inflammation, were significantly attenuated in adhE mutant bacteria (ΔadhE strain) compared to D39 wild-type bacteria. Therefore, AdhE might act as a pneumococcal virulence factor. Moreover, in the presence of ethanol, S. pneumoniae AdhE produced acetaldehyde and NADH, which subsequently led Rex (redox-sensing transcriptional repressor) to dissociate from the adhE promoter. An increase in AdhE level under the ethanol condition conferred an increase in Ply and H2O2 levels. Consistently, S. pneumoniae D39 caused higher cytotoxicity to RAW 264.7 cells than the ΔadhE strain under the ethanol stress condition, and ethanol-fed mice (alcoholic mice) were more susceptible to infection with the D39 wild-type bacteria than with the ΔadhE strain. Taken together, these data indicate that AdhE increases Ply under the ethanol stress condition, thus potentiating pneumococcal virulence.

  13. The diagnostic value of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) measurement in the sera of gastric cancer patients.

    PubMed

    Jelski, Wojciech; Orywal, Karolina; Laniewska, Magdalena; Szmitkowski, Maciej

    2010-12-01

    Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are present in gastric cancer cells (GC). Moreover, the activity of total ADH and class IV isoenzymes is significantly higher in cancer tissue than in healthy mucosa. The activity of these enzymes in cancer cells is probably reflected in the sera and could thus be helpful for diagnostics of gastric cancer. The aim of this study was to investigate a potential role of ADH and ALDH as tumor markers for gastric cancer. We defined diagnostic sensitivity, specificity, predictive value for positive and negative results, and receiver-operating characteristics (ROC) curve for tested enzymes. Serum samples were taken from 168 patients with gastric cancer before treatment and from 168 control subjects. Total ADH activity and class III and IV isoenzymes were measured by photometric but ALDH activity and ADH I and II by the fluorometric method, with class-specific fluorogenic substrates. There was significant increase in the activity of ADH IV isoenzyme and ADH total in the sera of gastric cancer patients compared to the control. The diagnostic sensitivity for ADH IV was 73%, specificity 79%, positive and negative predictive values were 81 and 72% respectively. Area under ROC curve for ADH IV was 0.67. The results suggest a potential role for ADH IV as marker of gastric cancer.

  14. Influence of fermentation conditions on specific activity of the enzymes alcohol and aldehyde dehydrogenase from yeasts.

    PubMed

    Mauricio, J C; Ortega, J M

    1993-01-01

    The effects of anaerobic, semi-aerobic and short aeration fermentation conditions and the addition of ergosterol and oleic acid to musts on the specific activity of alcohol and aldehyde dehydrogenase (ADH and ALDH) from two yeast species, Saccharomyces cerevisiae and Torulaspora delbrueckii, were studied. ADH I biosynthesis only occurred during the first few hours of fermentation. ADH II from S. cerevisiae and ALDH-NADP+ from the two yeast species behaved as constitutive enzymes under all fermentation conditions. ADH II from T. delbrueckii was only synthesized in small amounts, and its activity was always lower than in S. cerevisiae, where it was responsible for the termination of alcoholic fermentation during the steady growth phase.

  15. Inhibitory effects of the dietary flavonoid quercetin on the enzyme activity of zinc(II)-dependent yeast alcohol dehydrogenase: Spectroscopic and molecular docking studies.

    PubMed

    Bhuiya, Sutanwi; Haque, Lucy; Pradhan, Ankur Bikash; Das, Suman

    2017-02-01

    A multispectroscopic exploration was employed to investigate the interaction between the metallo-enzyme alcohol dehydrogenase (ADH) from yeast with bioflavonoid quercetin (QTN). Here, we have characterized the complex formation between QTN and Zn(2+) in aqueous solution and then examined the effect of such complex formation on the enzymatic activity of a zinc(II)-dependent enzyme alcohol dehydrogenase from yeast. We have observed an inhibition of enzymatic activity of ADH in presence of QTN. Enzyme inhibition kinetic experiments revealed QTN as a non-competitive inhibitor of yeast ADH. Perturbation of Circular dichroic (CD) spectrum of ADH in presence of QTN is observed due to the structural changes of ADH on complexation with the above flavonoid. Our results indicate a conformational change of ADH due to removal of Zn(2+) present in the enzyme by QTN. This was further established by molecular modeling study which shows that the flavonoid binds to the Zn(2+) ion which maintains the tertiary structure of the metallo-enzyme. So, QTN abstracts only half of the Zn(2+) ions present in the enzyme i.e. one Zn(2+) ion per monomer. From the present study, the structural alteration and loss of enzymatic activity of ADH are attributed to the complex formation between QTN and Zn(2+).

  16. Opossum alcohol dehydrogenases: Sequences, structures, phylogeny and evolution: evidence for the tandem location of ADH genes on opossum chromosome 5.

    PubMed

    Holmes, Roger S

    2009-03-16

    BLAT (BLAST-Like Alignment Tool) analyses and interrogations of the recently published opossum genome were undertaken using previously reported rat ADH amino acid sequences. Evidence is presented for six opossum ADH genes localized on chromosome 5 and organized in a comparable ADH gene cluster to that reported for human and rat ADH genes. The predicted amino acid sequences and secondary structures for the opossum ADH subunits and the intron-exon boundaries for opossum ADH genes showed a high degree of similarity with other mammalian ADHs, and four opossum ADH classes were identified, namely ADH1, ADH3, ADH6 and ADH4 (for which three genes were observed: ADH4A, ADH4B and ADH4C). Previous biochemical analyses of opossum ADHs have reported the tissue distribution and properties for these enzymes: ADH1, the major liver enzyme; ADH3, widely distributed in opossum tissues with similar kinetic properties to mammalian class 3 ADHs; and ADH4, for which several forms were localized in extrahepatic tissues, especially in the digestive system and in the eye. These ADHs are likely to perform similar functions to those reported for other mammalian ADHs in the metabolism of ingested and endogenous alcohols and aldehydes. Phylogenetic analyses examined opossum, human, rat, chicken and cod ADHs, and supported the proposed designation of opossum ADHs as class I (ADH1), class III (ADH3), class IV (ADH4A, ADH4B and ADH4C) and class VI (ADH6). Percentage substitution rates were examined for ADHs during vertebrate evolution which indicated that ADH3 is evolving at a much slower rate to that of the other ADH classes.

  17. Cloning and functional analysis of adhS gene encoding quinoprotein alcohol dehydrogenase subunit III from Acetobacter pasteurianus SKU1108.

    PubMed

    Masud, Uraiwan; Matsushita, Kazunobu; Theeragool, Gunjana

    2010-03-31

    The adhS gene which encodes the smallest subunit, subunit III, of quinoprotein alcohol dehydrogenase (PQQ-ADH) from Acetobacter pasteurianus SKU1108 has been cloned and characterized. The role of this subunit on the function of PQQ-ADH was investigated by construction of adhS gene disruptant and mutants. The adhS gene disruptant completely lost its PQQ-ADH activity and acetate-producing ability but retained acetic acid toleration. In contrast, this disruptant grew well, even better than the wild type, in the ethanol containing medium even though its PQQ-ADH activity and ethanol oxidizing ability was completely lost, while NAD(+)-dependent ADH (NAD(+)-ADH) was induced. Heme staining and immunoblot analysis of both membrane and soluble fractions with anti-ADH subunit III suggested that ethanol did not affect the adhS gene expression but induced PQQ-ADH activity. Over-expressed adhS did not enhance acetic acid production in both the wild type and the adhS disruptant. In addition, deletion analysis of upstream region of adhS gene suggested that its tentative promoter(s) might be located at around 118-268 bp upstream from an initiation codon. Random mutagenesis of adhS gene revealed that complete loss of PQQ-ADH activity and ethanol oxidizing ability were observed in the mutants' lack of the 140 and 73 amino acid residues at the C-terminal, whereas the lack of 22 amino acid residues at the C-terminal affected neither the PQQ-ADH activity nor ethanol oxidizing ability. In addition, some amino acid substitutions such as Leu18Gln, Ala26Val, Val36Ile, Val54Ile, Gly55Asp, Val70Ala and Val107Ala did not show any affect on PQQ-ADH activity and ethanol oxidizing ability. Interestingly, alteration of Thr104Lys led to a complete loss of ethanol oxidizing ability. However, point mutation at the possible promoter region also exhibited low PQQ-ADH activity and ethanol oxidizing ability. This result suggests that 104Thr might be involved in molecular coupling with subunit I in order

  18. Replication Study of ESCC Susceptibility Genetic Polymorphisms Locating in the ADH1B-ADH1C-ADH7 Cluster Identified by GWAS

    PubMed Central

    Xu, Xiaoling; Pan, Wenting; Ge, Yunxia; Zhou, Changchun; Liu, Chao; Gao, Jia; Yang, Ming; Mao, Weimin

    2014-01-01

    China was one of the countries with highest esophageal squamous cell carcinoma (ESCC) incidence and mortality worldwide. Alcohol drinking has been identified as a major environmental risk-factor related to ESCC. The alcohol dehydrogenase (ADH) family are major enzymes involved in the alcohol-metabolizing pathways, including alcohol dehydrogenase 1B (ADH1B) and ADH1C. Interestingly, ADH1B and ADH1C genes locate tandemly with ADH7 in a genomic segment as a gene cluster, and are all polymorphic. Several ESCC susceptibility single nucleotide polymorphisms (SNPs) of the ADH1B-ADH1C-ADH7 cluster have been identified previously through a genome-wide association study (GWAS). In the study, we examined the association between five ADH1B-ADH1C-ADH7 cluster SNPs (rs1042026, rs17033, rs1614972, rs1789903 and rs17028973) and risk of developing ESCC. Genotypes were determined in two independent case-control sets from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. Our data demonstrated that these ADH1B-ADH1C-ADH7 cluster SNPs confer susceptibility to ESCC in these two case-control sets, which were consistent to results of the previous GWAS. PMID:24722735

  19. Replication study of ESCC susceptibility genetic polymorphisms locating in the ADH1B-ADH1C-ADH7 cluster identified by GWAS.

    PubMed

    Wang, Jiwen; Wei, Jinyu; Xu, Xiaoling; Pan, Wenting; Ge, Yunxia; Zhou, Changchun; Liu, Chao; Gao, Jia; Yang, Ming; Mao, Weimin

    2014-01-01

    China was one of the countries with highest esophageal squamous cell carcinoma (ESCC) incidence and mortality worldwide. Alcohol drinking has been identified as a major environmental risk-factor related to ESCC. The alcohol dehydrogenase (ADH) family are major enzymes involved in the alcohol-metabolizing pathways, including alcohol dehydrogenase 1B (ADH1B) and ADH1C. Interestingly, ADH1B and ADH1C genes locate tandemly with ADH7 in a genomic segment as a gene cluster, and are all polymorphic. Several ESCC susceptibility single nucleotide polymorphisms (SNPs) of the ADH1B-ADH1C-ADH7 cluster have been identified previously through a genome-wide association study (GWAS). In the study, we examined the association between five ADH1B-ADH1C-ADH7 cluster SNPs (rs1042026, rs17033, rs1614972, rs1789903 and rs17028973) and risk of developing ESCC. Genotypes were determined in two independent case-control sets from two regions of China. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. Our data demonstrated that these ADH1B-ADH1C-ADH7 cluster SNPs confer susceptibility to ESCC in these two case-control sets, which were consistent to results of the previous GWAS.

  20. The diagnostic value of alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) measurement in the sera of patients with brain tumor

    PubMed Central

    Laniewska-Dunaj, Magdalena; Orywal, Karolina; Kochanowicz, Jan; Rutkowski, Robert; Szmitkowski, Maciej

    2017-01-01

    Introduction Alcohol dehydrogenase (ADH) isoenzymes and aldehyde dehydrogenase (ALDH) exist in the brain. Alcohol dehydrogenase and ALDH are also present in brain tumor cells. Moreover, the activity of class I isoenzymes was significantly higher in cancer than healthy brain cells. The activity of these enzymes in tumor tissue is reflected in the serum and could thus be helpful for diagnostics of brain neoplasms. The aim of this study was to investigate the potential role of ADH and ALDH as markers for brain tumors. Material and methods Serum samples were taken for routine biochemical investigation from 115 patients suffering from brain tumors (65 glioblastomas, 50 meningiomas). For the measurement of the activity of class I and II ADH isoenzymes and ALDH activity, fluorometric methods were used. The total ADH activity and activity of class III and IV isoenzymes were measured by the photometric method. Results There was a significant increase in the activity of ADH I isoenzyme and ADH total in the sera of brain tumor patients compared to the controls. The diagnostic sensitivity for ADH I was 78%, specificity 85%, and positive and negative predictive values were 86% and 76% respectively. The sensitivity and specificity of ADH I increased with the stage of the carcinoma. Area under receiver-operating characteristic curve for ADH I was 0.71. Conclusions The results suggest a potential role for ADH I as a marker for brain tumor. PMID:28261287

  1. A New View of Alcohol Metabolism and Alcoholism—Role of the High-Km Class III Alcohol Dehydrogenase (ADH3)

    PubMed Central

    Haseba, Takeshi; Ohno, Youkichi

    2010-01-01

    The conventional view is that alcohol metabolism is carried out by ADH1 (Class I) in the liver. However, it has been suggested that another pathway plays an important role in alcohol metabolism, especially when the level of blood ethanol is high or when drinking is chronic. Over the past three decades, vigorous attempts to identify the enzyme responsible for the non-ADH1 pathway have focused on the microsomal ethanol oxidizing system (MEOS) and catalase, but have failed to clarify their roles in systemic alcohol metabolism. Recently, using ADH3-null mutant mice, we demonstrated that ADH3 (Class III), which has a high Km and is a ubiquitous enzyme of ancient origin, contributes to systemic alcohol metabolism in a dose-dependent manner, thereby diminishing acute alcohol intoxication. Although the activity of ADH3 toward ethanol is usually low in vitro due to its very high Km, the catalytic efficiency (kcat/Km) is markedly enhanced when the solution hydrophobicity of the reaction medium increases. Activation of ADH3 by increasing hydrophobicity should also occur in liver cells; a cytoplasmic solution of mouse liver cells was shown to be much more hydrophobic than a buffer solution when using Nile red as a hydrophobicity probe. When various doses of ethanol are administered to mice, liver ADH3 activity is dynamically regulated through induction or kinetic activation, while ADH1 activity is markedly lower at high doses (3–5 g/kg). These data suggest that ADH3 plays a dynamic role in alcohol metabolism, either collaborating with ADH1 or compensating for the reduced role of ADH1. A complex two-ADH model that ascribes total liver ADH activity to both ADH1 and ADH3 explains the dose-dependent changes in the pharmacokinetic parameters (β, CLT, AUC) of blood ethanol very well, suggesting that alcohol metabolism in mice is primarily governed by these two ADHs. In patients with alcoholic liver disease, liver ADH3 activity increases, while ADH1 activity decreases, as alcohol

  2. Adh enhances Actinobacillus pleuropneumoniae pathogenicity by binding to OR5M11 and activating p38 which induces apoptosis of PAMs and IL-8 release.

    PubMed

    Wang, Lei; Qin, Wanhai; Zhang, Jing; Bao, Chuntong; Zhang, Hu; Che, Yanyi; Sun, Changjiang; Gu, Jingmin; Feng, Xin; Du, Chongtao; Han, Wenyu; Richard, Paul Langford; Lei, Liancheng

    2016-04-05

    Members of the Trimeric Autotransporter Adhesin (TAA) family play a crucial role in the adhesion of Gram-negative pathogens to host cells, but the immunopathogenesis of TAAs remains unknown. Our previous studies demonstrated that Adh from Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is required for full bacterial pathogenicity. Alveolar macrophages are the first line of defense against respiratory infections. This study compared the interactions between porcine alveolar macrophages (PAMs) and wild-type A. pleuropneumoniae (5b WT) or an Adh-deletion strain (5b ΔAdh) via gene microarray, immunoprecipitation and other technologies. We found that Adh was shown to interact with the PAMs membrane protein OR5M11, an olfactory receptor, resulting in the high-level secretion of IL-8 by activation of p38 MAPK signaling pathway. Subsequently, PAMs apoptosis via the activation of the Fax and Bax signaling pathways was observed, followed by activation of caspases 8, 9, and 3. The immunological pathogenic roles of Adh were also confirmed in both murine and piglets infectious models in vivo. These results identify a novel immunological strategy for TAAs to boost the pathogenicity of A. pleuropneumoniae. Together, these datas reveal the high versatility of the Adh protein as a virulence factor and provide novel insight into the immunological pathogenic role of TAAs.

  3. 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

  4. Activities of xenobiotic metabolizing enzymes in rat placenta and liver in vitro.

    PubMed

    Fabian, Eric; Wang, Xinyi; Engel, Franziska; Li, Hequn; Landsiedel, Robert; van Ravenzwaay, Bennard

    2016-06-01

    In order to assess whether the placental metabolism of xenobiotic compounds should be taken into consideration for physiologically-based toxicokinetic (PBTK) modelling, the activities of seven phase I and phase II enzymes have been quantified in the 18-day placenta of untreated Wistar rats. To determine their relative contribution, these activities were compared to those of untreated adult male rat liver, using commonly accepted assays. The enzymes comprised cytochrome P450 (CYP), flavin-containing monooxygenase (FMO), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), esterase, UDP-glucuronosyltransferase (UGT), and glutathione S-transferase (GST). In contrast to liver, no activities were measurable for 7-ethylresorufin-O-dealkylase (CYP1A), 7-pentylresorufin-O-dealkylase (CYP2B), 7-benzylresorufin-O-dealkylase (CYP2B, 2C and 3 A), UGT1, UGT2 and GST in placenta, indicating that the placental activity of these enzymes was well below their hepatic activity. Low activities in placenta were determined for FMO (4%), and esterase (8%), whereas the activity of placental ADH and ALDH accounted for 35% and 40% of the hepatic activities, respectively. In support of the negligible placental CYP activity, testosterone and six model azole fungicides, which were readily metabolized by rat hepatic microsomes, failed to exhibit any metabolic turnover with rat placental microsomes. Hence, with the possible exception of ADH and ALDH, the activities of xenobiotic-metabolizing enzymes in rat placenta are too low to warrant consideration in PBTK modelling.

  5. Ethanol formation in adh0 mutants reveals the existence of a novel acetaldehyde-reducing activity in Saccharomyces cerevisiae.

    PubMed Central

    Drewke, C; Thielen, J; Ciriacy, M

    1990-01-01

    A strain of Saccharomyces cerevisiae has been constructed which is deficient in the four alcohol dehydrogenase (ADH) isozymes known at present. This strain (adh0), being irreversibly mutated in the genes ADH1, ADH3, and ADH4 and carrying a point mutation in the gene ADH2 coding for the glucose-repressible isozyme ADHII, still produces up to one third of the theoretical maximum yield of ethanol in a homofermentative conversion of glucose to ethanol. Analysis of the glucose metabolism of adh0 cells shows that the lack of all known ADH isozymes results in the formation of glycerol as a major fermentation product, accompanied by a significant production of acetaldehyde and acetate. Treatment of glucose-growing adh0 cells with the respiratory-chain inhibitor antimycin A leads to an immediate cessation of ethanol production, demonstrating that ethanol production in adh0 cells is dependent on mitochondrial electron transport. Reduction of acetaldehyde to ethanol in isolated mitochondria could also be demonstrated. This reduction is apparently linked to the oxidation of acetaldehyde to acetate. Preliminary data suggest that this novel type of ethanol formation in S. cerevisiae is associated with the inner mitochondrial membrane. Images PMID:2193925

  6. ADH (Antidiuretic Hormone) Test

    MedlinePlus

    ... stored in the posterior pituitary gland at the base of the brain. ADH helps regulate water balance ... of ADH (vasopressin). Several blood and urine osmolality measurements are performed at timed intervals before and after ...

  7. Alcohol dehydrogenase gene ADH3 activates glucose alcoholic fermentation in genetically engineered Dekkera bruxellensis yeast.

    PubMed

    Schifferdecker, Anna Judith; Siurkus, Juozas; Andersen, Mikael Rørdam; Joerck-Ramberg, Dorte; Ling, Zhihao; Zhou, Nerve; Blevins, James E; Sibirny, Andriy A; Piškur, Jure; Ishchuk, Olena P

    2016-04-01

    Dekkera bruxellensis is a non-conventional Crabtree-positive yeast with a good ethanol production capability. Compared to Saccharomyces cerevisiae, its tolerance to acidic pH and its utilization of alternative carbon sources make it a promising organism for producing biofuel. In this study, we developed an auxotrophic transformation system and an expression vector, which enabled the manipulation of D. bruxellensis, thereby improving its fermentative performance. Its gene ADH3, coding for alcohol dehydrogenase, was cloned and overexpressed under the control of the strong and constitutive promoter TEF1. Our recombinant D. bruxellensis strain displayed 1.4 and 1.7 times faster specific glucose consumption rate during aerobic and anaerobic glucose fermentations, respectively; it yielded 1.2 times and 1.5 times more ethanol than did the parental strain under aerobic and anaerobic conditions, respectively. The overexpression of ADH3 in D. bruxellensis also reduced the inhibition of fermentation by anaerobiosis, the "Custer effect". Thus, the fermentative capacity of D. bruxellensis could be further improved by metabolic engineering.

  8. Adh1 and Adh1/4 knockout mice as possible rodent models for presymptomatic Parkinson's disease.

    PubMed

    Anvret, Anna; Ran, Caroline; Westerlund, Marie; Gellhaar, Sandra; Lindqvist, Eva; Pernold, Karin; Lundströmer, Karin; Duester, Gregg; Felder, Michael R; Galter, Dagmar; Belin, Andrea Carmine

    2012-02-01

    Alcohol dehydrogenases (ADH) catalyze the reversible metabolism of many types of alcohols and aldehydes to prevent the possible toxic accumulation of these compounds. ADHs are of interest in Parkinson's disease (PD) since these compounds can be harmful to dopamine (DA) neurons. Genetic variants in ADH1C and ADH4 have been found to associate with PD and lack of Adh4 gene activity in a mouse model has recently been reported to induce changes in the DA system. Adh1 knockout (Adh1-/-) and Adh1/4 double knockout (Adh1/4-/-) mice were investigated for possible changes in DA system related activity, biochemical parameters and olfactory function compared to wild-type (WT) mice. Locomotor activity was tested at ∼7 (adult) and >15 months of age to mimic the late onset of PD. Adh1-/- and Adh1/4-/- mice displayed a significantly higher spontaneous locomotor activity than WT littermates. Both apomorphine and d-amphetamine increased total distance activity in Adh1-/- mice at both age intervals and in Adh1/4-/- mice at 7 months of age compared to WT mice. No significant changes were found regarding olfactory function, however biochemical data showed decreased 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratios in the olfactory bulb and decreased homovanillic acid (HVA)/DA ratios in the olfactory bulb, frontal cortex and striatum of Adh1/4-/- mice compared to WT mice. Our results suggest that lack of Adh1 alone or Adh1 and Adh4 together lead to changes in DA system related behavior, and that these knockout mice might be possible rodent models to study presymptomatic PD.

  9. Transient overexpression of adh8a increases allyl alcohol toxicity in zebrafish embryos.

    PubMed

    Klüver, Nils; Ortmann, Julia; Paschke, Heidrun; Renner, Patrick; Ritter, Axel P; Scholz, Stefan

    2014-01-01

    Fish embryos are widely used as an alternative model to study toxicity in vertebrates. Due to their complexity, embryos are believed to more resemble an adult organism than in vitro cellular models. However, concerns have been raised with respect to the embryo's metabolic capacity. We recently identified allyl alcohol, an industrial chemical, to be several orders of magnitude less toxic to zebrafish embryo than to adult zebrafish (embryo LC50 = 478 mg/L vs. fish LC50 = 0.28 mg/L). Reports on mammals have indicated that allyl alcohol requires activation by alcohol dehydrogenases (Adh) to form the highly reactive and toxic metabolite acrolein, which shows similar toxicity in zebrafish embryos and adults. To identify if a limited metabolic capacity of embryos indeed can explain the low allyl alcohol sensitivity of zebrafish embryos, we compared the mRNA expression levels of Adh isoenzymes (adh5, adh8a, adh8b and adhfe1) during embryo development to that in adult fish. The greatest difference between embryo and adult fish was found for adh8a and adh8b expression. Therefore, we hypothesized that these genes might be required for allyl alcohol activation. Microinjection of adh8a, but not adh8b mRNA led to a significant increase of allyl alcohol toxicity in embryos similar to levels reported for adults (LC50 = 0.42 mg/L in adh8a mRNA-injected embryos). Furthermore, GC/MS analysis of adh8a-injected embryos indicated a significant decline of internal allyl alcohol concentrations from 0.23-58 ng/embryo to levels below the limit of detection (< 4.6 µg/L). Injection of neither adh8b nor gfp mRNA had an impact on internal allyl alcohol levels supporting that the increased allyl alcohol toxicity was mediated by an increase in its metabolization. These results underline the necessity to critically consider metabolic activation in the zebrafish embryo. As demonstrated here, mRNA injection is one useful approach to study the role of candidate enzymes involved in

  10. ADH5 — EDRN Public Portal

    Cancer.gov

    ADH5 is a member of the alcohol dehydrogenase family. This protein forms a homodimer. ADH5 is ineffective in oxidizing ethanol, but exhibits high activity for oxidation of long-chain primary alcohols and for oxidation of S-hydroxymethyl-glutathione, a spontaneous adduct between formaldehyde and glutathione. There are several non-transcribed pseuodogenes related to this gene.

  11. Characterization of alcohol dehydrogenase (ADH12) from Haloarcula marismortui, an extreme halophile from the Dead Sea.

    PubMed

    Timpson, Leanne M; Alsafadi, Diya; Mac Donnchadha, Cillín; Liddell, Susan; Sharkey, Michael A; Paradisi, Francesca

    2012-01-01

    Haloarchaeal alcohol dehydrogenases are of increasing interest as biocatalysts in the field of white biotechnology. In this study, the gene adh12 from the extreme halophile Haloarcula marismortui (HmADH12), encoding a 384 residue protein, was cloned into two vectors: pRV1 and pTA963. The resulting constructs were used to transform host strains Haloferax volcanii (DS70) and (H1209), respectively. Overexpressed His-tagged recombinant HmADH12 was purified by immobilized metal-affinity chromatography (IMAC). The His-tagged protein was visualized by SDS-PAGE, with a subunit molecular mass of 41.6 kDa, and its identity was confirmed by mass spectrometry. Purified HmADH12 catalyzed the interconversion between alcohols and aldehydes and ketones, being optimally active in the presence of 2 M KCl. It was thermoactive, with maximum activity registered at 60°C. The NADP(H) dependent enzyme was haloalkaliphilic for the oxidative reaction with optimum activity at pH 10.0. It favored a slightly acidic pH of 6.0 for catalysis of the reductive reaction. HmADH12 was significantly more tolerant than mesophilic ADHs to selected organic solvents, making it a much more suitable biocatalyst for industrial application.

  12. POZ domain transcription factor, FBI-1, represses transcription of ADH5/FDH by interacting with the zinc finger and interfering with DNA binding activity of Sp1.

    PubMed

    Lee, Dong-Kee; Suh, Dongchul; Edenberg, Howard J; Hur, Man-Wook

    2002-07-26

    The POZ domain is a protein-protein interaction motif that is found in many transcription factors, which are important for development, oncogenesis, apoptosis, and transcription repression. We cloned the POZ domain transcription factor, FBI-1, that recognizes the cis-element (bp -38 to -22) located just upstream of the core Sp1 binding sites (bp -22 to +22) of the ADH5/FDH minimal promoter (bp -38 to +61) in vitro and in vivo, as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The ADH5/FDH minimal promoter is potently repressed by the FBI-1. Glutathione S-transferase fusion protein pull-down showed that the POZ domains of FBI-1, Plzf, and Bcl-6 directly interact with the zinc finger DNA binding domain of Sp1. DNase I footprinting assays showed that the interaction prevents binding of Sp1 to the GC boxes of the ADH5/FDH promoter. Gal4-POZ domain fusions targeted proximal to the GC boxes repress transcription of the Gal4 upstream activator sequence-Sp1-adenovirus major late promoter. Our data suggest that POZ domain represses transcription by interacting with Sp1 zinc fingers and by interfering with the DNA binding activity of Sp1.

  13. Ectopic ADH secretion

    MedlinePlus

    ... ADH. Often, there are no symptoms from a low sodium level. When symptoms do occur, they may include ... Lab tests that can confirm and help diagnose low sodium include: Comprehensive metabolic panel (includes blood sodium) Osmolality ...

  14. Crystal structure of the vertebrate NADP(H)-dependent alcohol dehydrogenase (ADH8).

    PubMed

    Rosell, Albert; Valencia, Eva; Parés, Xavier; Fita, Ignacio; Farrés, Jaume; Ochoa, Wendy F

    2003-06-27

    The amphibian enzyme ADH8, previously named class IV-like, is the only known vertebrate alcohol dehydrogenase (ADH) with specificity towards NADP(H). The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP(+) have been now determined and refined to resolutions of 2.2A and 1.8A, respectively. The coenzyme and substrate specificity of ADH8, that has 50-65% sequence identity with vertebrate NAD(H)-dependent ADHs, suggest a role in aldehyde reduction probably as a retinal reductase. The large volume of the substrate-binding pocket can explain both the high catalytic efficiency of ADH8 with retinoids and the high K(m) value for ethanol. Preference of NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225 and the recruitment of conserved Lys228, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds to ADH8 in an extended conformation that superimposes well with the NAD(H) molecules found in NAD(H)-dependent ADH complexes. No additional reshaping of the dinucleotide-binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. The structural features support the classification of ADH8 as an independent ADH class.

  15. Molecular Characterization and Transcriptional Analysis of adhE2, the Gene Encoding the NADH-Dependent Aldehyde/Alcohol Dehydrogenase Responsible for Butanol Production in Alcohologenic Cultures of Clostridium acetobutylicum ATCC 824

    PubMed Central

    Fontaine, Lisa; Meynial-Salles, Isabelle; Girbal, Laurence; Yang, Xinghong; Croux, Christian; Soucaille, Philippe

    2002-01-01

    The adhE2 gene of Clostridium acetobutylicum ATCC 824, coding for an aldehyde/alcohol dehydrogenase (AADH), was characterized from molecular and biochemical points of view. The 2,577-bp adhE2 codes for a 94.4-kDa protein. adhE2 is expressed, as a monocistronic operon, in alcohologenic cultures and not in solventogenic cultures. Primer extension analysis identified two transcriptional start sites 160 and 215 bp upstream of the adhE2 start codon. The expression of adhE2 from a plasmid in the DG1 mutant of C. acetobutylicum, a mutant cured of the pSOL1 megaplasmid, restored butanol production and provided elevated activities of NADH-dependent butyraldehyde and butanol dehydrogenases. The recombinant AdhE2 protein expressed in E. coli as a Strep-tag fusion protein and purified to homogeneity also demonstrated NADH-dependent butyraldehyde and butanol dehydrogenase activities. This is the second AADH identified in C. acetobutylicum ATCC 824, and to our knowledge this is the first example of a bacterium with two AADHs. It is noteworthy that the two corresponding genes, adhE and adhE2, are carried by the pSOL1 megaplasmid of C. acetobutylicum ATCC 824. PMID:11790753

  16. Effects of endogenous antidiuretic hormone (ADH) on macrophage phagocytosis

    SciTech Connect

    Fernandez-Repollet, E.; Opava-Stitzer, S.; Tiffany, S.; Schwartz, A.

    1983-07-01

    Although several studies have indicated that antidiuretic hormone (ADH) enhances the phagocytic function of the reticuloendothelial system (RES) in shock syndromes, it remains unknown what influence ADH exerts upon the individual phagocytic components of this system. The present investigation was designed to evaluate the effects of endogenous ADH on the phagocytic activity of peritoneal macrophage cells. As a phagocytic stimuli, fluorescent methacrylate microbeads were injected intraperitoneally into Brattleboro (ADH deficient) and normal Long Evans rats in the presence and absence of exogenous ADH. Peritoneal cells were harvested 19-22 hr after the administration of the microbeads and the percent phagocytosis was determined in macrophage cells using a fluorescence-activated cell sorter (FACS II). Our results indicate that the percentage of peritoneal macrophages ingesting the fluorescent methacrylate microbeads was significantly reduced in the absence of ADH (Brattleboro rats: 5.4 +/- 0.6% versus Long Evans rats: 16.8 +/- 2.3%; p less than 0.001). In addition, our data demonstrate that exogenous administration of ADH significantly enhanced macrophage phagocytosis in Brattleboro (14.7 +/- 2.2%) and normal Long Evans (49.6 +/- 4.5%) rats. These data suggest, for the first time, that endogenous ADH might play a modulatory role in the phagocytic activity of a specific component of the RES, namely, the macrophage cell.

  17. Genetic polymorphisms of ADH1B, ADH1C and ALDH2 in Turkish alcoholics: lack of association with alcoholism and alcoholic cirrhosis.

    PubMed

    Vatansever, Sezgin; Tekin, Fatih; Salman, Esin; Altintoprak, Ender; Coskunol, Hakan; Akarca, Ulus Salih

    2015-05-17

    No data exists regarding the alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) gene polymorphisms in Turkish alcoholic cirrhotics. We studied the polymorphisms of ADH1B, ADH1C and ALDH2 genes in alcoholic cirrhotics and compared the results with non-cirrhotic alcoholics and healthy volunteers. Overall, 237 subjects were included for the study: 156 alcoholic patients (78 cirrhotics, 78 non-cirrhotic alcoholics) and 81 healthy volunteers. Three different single-nucleotide-polymorphism genotyping methods were used. ADH1C genotyping was performed using a polymerase chain reaction-restriction fragment length polymorphism method. The identified ADH1C genotypes were named according to the presence or absence of the enzyme restriction sites. ADH1B (Arg47Hys) genotyping was performed using the allele specific primer extension method, and ALDH2 (Glu487Lys) genotyping was performed by a multiplex polymerase chain reaction using two allele-specific primer pairs. For ADH1B, the frequency of allele *1 in the cirrhotics, non-cirrhotic alcoholics and healthy volunteers was 97.4%, 94.9% and 99.4%, respectively. For ADH1C, the frequency of allele *1 in the cirrhotics, non-cirrhotic alcoholics and healthy volunteers was 47%, 36.3% and 45%, respectively. There was no statistical difference between the groups for ADH1B and ADH1C (p>0.05). All alcoholic and non-alcoholic subjects (100%) had the allele *1 for ALDH2. The obtained results for ADH1B, ADH1C, and ALDH gene polymorphisms in the present study are similar to the results of Caucasian studies. ADH1B and ADH1C genetic variations are not related to the development of alcoholism or susceptibility to alcoholic cirrhosis. ALDH2 gene has no genetic variation in the Turkish population.

  18. The influence of Adh function on ethanol preference and tolerance in adult Drosophila melanogaster.

    PubMed

    Ogueta, Maite; Cibik, Osman; Eltrop, Rouven; Schneider, Andrea; Scholz, Henrike

    2010-11-01

    Preference determines behavioral choices such as choosing among food sources and mates. One preference-affecting chemical is ethanol, which guides insects to fermenting fruits or leaves. Here, we show that adult Drosophila melanogaster prefer food containing up to 5% ethanol over food without ethanol and avoid food with high levels (23%) of ethanol. Although female and male flies behaved differently at ethanol-containing food sources, there was no sexual dimorphism in the preference for food containing modest ethanol levels. We also investigated whether Drosophila preference, sensitivity and tolerance to ethanol was related to the activity of alcohol dehydrogenase (Adh), the primary ethanol-metabolizing enzyme in D. melanogaster. Impaired Adh function reduced ethanol preference in both D. melanogaster and a related species, D. sechellia. Adh-impaired flies also displayed reduced aversion to high ethanol concentrations, increased sensitivity to the effects of ethanol on postural control, and negative tolerance/sensitization (i.e., a reduction of the increased resistance to ethanol's effects that normally occurs upon repeated exposure). These data strongly indicate a linkage between ethanol-induced behavior and ethanol metabolism in adult fruit flies: Adh deficiency resulted in reduced preference to low ethanol concentrations and reduced aversion to high ones, despite recovery from ethanol being strongly impaired.

  19. Modeling Parkinson's disease genetics: altered function of the dopamine system in Adh4 knockout mice.

    PubMed

    Belin, Andrea Carmine; Westerlund, Marie; Anvret, Anna; Lindqvist, Eva; Pernold, Karin; Ogren, Sven Ove; Duester, Gregg; Galter, Dagmar

    2011-03-01

    Class IV alcohol dehydrogenase (ADH4) efficiently reduces aldehydes produced during lipid peroxidation, and may thus serve to protect from toxic effects of aldehydes e.g. on neurons. We hypothesized that ADH4 dysfunction may increase risk for Parkinson's disease (PD) and previously reported association of an ADH4 allele with PD. We found that a promoter polymorphism in this allele induced a 25-30% reduction of transcriptional activity. Based on these findings, we have now investigated whether Adh4 homo- (Adh4-/-) or heterozygous (Adh4+/-) knockout mice display any dopamine system-related changes in behavior, biochemical parameters or olfaction compared to wild-type mice. The spontaneous locomotor activity was found to be similar in the three groups, whereas administration of d-amphetamine or apomorphine induced a significant increase in horizontal activity in the Adh4-/- mice compared to wild-type mice. We measured levels of monoamines and their metabolites in striatum, frontal cortex and substantia nigra and found increased levels of dopamine and DOPAC in substantia nigra of Adh4-/- mice. Investigation of olfactory function revealed a reduced sense of smell in Adh4-/- mice accompanied by alterations in dopamine metabolite levels in the olfactory bulb. Taken together, our results suggest that lack of Adh4 gene activity induces changes in the function of the dopamine system, findings which are compatible with a role of loss-of-function mutations in ADH4 as possible risk factors for PD.

  20. 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.

  1. Activation of thiamin diphosphate in enzymes.

    PubMed

    Hübner, G; Tittmann, K; Killenberg-Jabs, M; Schäffner, J; Spinka, M; Neef, H; Kern, D; Kern, G; Schneider, G; Wikner, C; Ghisla, S

    1998-06-29

    Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.

  2. 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)…

  3. Genetic variants in or near ADH1B and ADH1C affect susceptibility to alcohol dependence in a British and Irish population.

    PubMed

    Way, Michael; McQuillin, Andrew; Saini, Jit; Ruparelia, Kush; Lydall, Gregory J; Guerrini, Irene; Ball, David; Smith, Iain; Quadri, Giorgia; Thomson, Allan D; Kasiakogia-Worlley, Katherine; Cherian, Raquin; Gunwardena, Priyanthi; Rao, Harish; Kottalgi, Girija; Patel, Shamir; Hillman, Audrey; Douglas, Ewen; Qureshi, Sherhzad Y; Reynolds, Gerry; Jauhar, Sameer; O'Kane, Aideen; Dedman, Alex; Sharp, Sally; Kandaswamy, Radhika; Dar, Karim; Curtis, David; Morgan, Marsha Y; Gurling, Hugh M D

    2015-05-01

    Certain single nucleotide polymorphisms (SNPs) in genes encoding alcohol dehydrogenase (ADH) enzymes confer a significant protective effect against alcohol dependence syndrome (ADS) in East Asian populations. Recently, attention has focused on the role of these SNPs in determining ADS risk in European populations. To further elucidate these associations, SNPs of interest in ADH1B, ADH1C and the ADH1B/1C intergenic region were genotyped in a British and Irish population (ADS cases n = 1076: controls n = 1027) to assess their relative contribution to ADS risk. A highly significant, protective association was observed between the minor allele of rs1229984 in ADH1B and ADS risk [allelic P = 8.4 × 10(-6) , odds ratio (OR) = 0.26, 95 percent confidence interval, 0.14, 0.49]. Significant associations were also observed between ADS risk and the ADH1B/1C intergenic variant, rs1789891 [allelic P = 7.2 × 10(-5) , OR = 1.4 (1.2, 1.6)] and three non-synonymous SNPs rs698, rs1693482 and rs283413 in ADH1C. However, these associations were not completely independent; thus, while the ADH1B rs1229984 minor allele association was independent of those of the intergenic variant rs1789891 and the three ADH1C variants, the three ADH1C variants were not individually independent. In conclusion, the rare ADH1B rs1229984 mutation provides significant protection against ADS in this British and Irish population; other variants in the ADH gene cluster also alter ADS risk, although the strong linkage disequilibrium between SNPs at this location precluded clear identification of the variant(s) driving the associations.

  4. Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) gene product as a broad specificity NADPH-dependent alcohol dehydrogenase: relevance in aldehyde reduction.

    PubMed Central

    Larroy, Carol; Fernández, M Rosario; González, Eva; Parés, Xavier; Biosca, Josep A

    2002-01-01

    YMR318C represents an open reading frame from Saccharomyces cerevisiae with unknown function. It possesses a conserved sequence motif, the zinc-containing alcohol dehydrogenase (ADH) signature, specific to the medium-chain zinc-containing ADHs. In the present study, the YMR318C gene product has been purified to homogeneity from overexpressing yeast cells, and found to be a homodimeric ADH, composed of 40 kDa subunits and with a pI of 5.0-5.4. The enzyme was strictly specific for NADPH and was active with a wide variety of substrates, including aliphatic (linear and branched-chain) and aromatic primary alcohols and aldehydes. Aldehydes were processed with a 50-fold higher catalytic efficiency than that for the corresponding alcohols. The highest k(cat)/K(m) values were found with pentanal>veratraldehyde > hexanal > 3-methylbutanal >cinnamaldehyde. Taking into consideration the substrate specificity and sequence characteristics of the YMR318C gene product, we have proposed this gene to be called ADH6. The disruption of ADH6 was not lethal for the yeast under laboratory conditions. Although S. cerevisiae is considered a non lignin-degrading organism, the catalytic activity of ADHVI can direct veratraldehyde and anisaldehyde, arising from the oxidation of lignocellulose by fungal lignin peroxidases, to the lignin biodegradation pathway. ADHVI is the only S. cerevisiae enzyme able to significantly reduce veratraldehyde in vivo, and its overexpression allowed yeast to grow under toxic concentrations of this aldehyde. The enzyme may also be involved in the synthesis of fusel alcohols. To our knowledge this is the first NADPH-dependent medium-chain ADH to be characterized in S. cerevisiae. PMID:11742541

  5. 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)

  6. Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase.

    PubMed

    Burdette, D; Zeikus, J G

    1994-08-15

    The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling.

  7. Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase.

    PubMed Central

    Burdette, D; Zeikus, J G

    1994-01-01

    The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling. Images Figure 1 PMID:8068002

  8. ADH and ALDH polymorphisms among Alaska Natives entering treatment for alcoholism.

    PubMed

    Segal, B

    1999-01-01

    The alcohol dehydrogenase (ADHs) and aldehyde dehydrogenases (ALDHs) involved in alcohol metabolism are polymorphic. Different alleles encode subunits of the enzymes that are related to differences in alcohol metabolism with different ethnic groups. This study examined the allele frequencies at the ADH1, ADH2, ADH3 and ALDH2 loci in Alaska Natives entering treatment for alcoholism to determine if allele frequencies at these loci differ among five distinct Alaska Native groups: Yupik and Inupiat Eskimos, Athabascan, Tlingit and Aleut. It was found that all persons were homozygous for the ADH1*1, ADH2*1 and ALDH2*1 alleles. Variations, however, were found for the allele distribution of the ADH3 genotype. Comparison with a general population sample found no differences in allele distributions for ADHs and ALDH2*1, but differences were found when comparisons were made with four Asian Groups. The study's findings suggest that the Alaska Natives are not protected from the risk of alcoholism in the same way that Asians who possess the ALDH2*2 genotype are considered to have a negative risk factor. Nor, does there appear to be any generalized differences between Alaska Native alcoholics and members of the general population with respect to the ALDH and ADH polymorphisms studied herein.

  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. Ethylene-responsive transcription factors interact with promoters of ADH and PDC involved in persimmon (Diospyros kaki) fruit de-astringency.

    PubMed

    Min, Ting; Yin, Xue-ren; Shi, Yan-na; Luo, Zheng-rong; Yao, Yun-cong; Grierson, Donald; Ferguson, Ian B; Chen, Kun-song

    2012-11-01

    The persimmon fruit is a particularly good model for studying fruit response to hypoxia, in particular, the hypoxia-response ERF (HRE) genes. An anaerobic environment reduces fruit astringency by converting soluble condensed tannins (SCTs) into an insoluble form. Although the physiology of de-astringency has been widely studied, its molecular control is poorly understood. Both CO(2) and ethylene treatments efficiently removed the astringency from 'Mopan' persimmon fruit, as indicated by a decrease in SCTs. Acetaldehyde, the putative agent for causing de-astringency, accumulated during these treatments, as did activities of the key enzymes of acetaldehyde synthesis, alcohol dehydrogenase (ADH), and pyruvate decarboxylase (PDC). Eight DkADH and DkPDC genes were isolated, and three candidates for a role in de-astringency, DkADH1, DkPDC1, and DkPDC2, were characterized by transcriptional analysis in different tissues. The significance of these specific isoforms was confirmed by principal component analysis. Transient expression in leaf tissue showed that DkPDC2 decreased SCTs. Interactions of six hypoxia-responsive ERF genes and target promoters were tested in transient assays. The results indicated that two hypoxia-responsive ERF genes, DkERF9 and DkERF10, were involved in separately regulating the DkPDC2 and DkADH1 promoters. It is proposed that a DkERF-DkADH/DkPDC cascade is involved in regulating persimmon de-astringency.

  11. Genes Encoding Enzymes Involved in Ethanol Metabolism

    PubMed Central

    Hurley, Thomas D.; Edenberg, Howard J.

    2012-01-01

    The effects of beverage alcohol (ethanol) on the body are determined largely by the rate at which it and its main breakdown product, acetaldehyde, are metabolized after consumption. The main metabolic pathway for ethanol involves the enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Seven different ADHs and three different ALDHs that metabolize ethanol have been identified. The genes encoding these enzymes exist in different variants (i.e., alleles), many of which differ by a single DNA building block (i.e., single nucleotide polymorphisms [SNPs]). Some of these SNPs result in enzymes with altered kinetic properties. For example, certain ADH1B and ADH1C variants that are commonly found in East Asian populations lead to more rapid ethanol breakdown and acetaldehyde accumulation in the body. Because acetaldehyde has harmful effects on the body, people carrying these alleles are less likely to drink and have a lower risk of alcohol dependence. Likewise, an ALDH2 variant with reduced activity results in acetaldehyde buildup and also has a protective effect against alcoholism. In addition to affecting drinking behaviors and risk for alcoholism, ADH and ALDH alleles impact the risk for esophageal cancer. PMID:23134050

  12. 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.

  13. How thiamine diphosphate is activated in enzymes.

    PubMed

    Kern, D; Kern, G; Neef, H; Tittmann, K; Killenberg-Jabs, M; Wikner, C; Schneider, G; Hübner, G

    1997-01-03

    The controversial question of how thiamine diphosphate, the biologically active form of vitamin B1, is activated in different enzymes has been addressed. Activation of the coenzyme was studied by measuring thermodynamics and kinetics of deprotonation at the carbon in the 2-position (C2) of thiamine diphosphate in the enzymes pyruvate decarboxylase and transketolase by use of nuclear magnetic resonance spectroscopy, proton/deuterium exchange, coenzyme analogs, and site-specific mutant enzymes. Interaction of a glutamate with the nitrogen in the 1'-position in the pyrimidine ring activated the 4'-amino group to act as an efficient proton acceptor for the C2 proton. The protein component accelerated the deprotonation of the C2 atom by several orders of magnitude, beyond the rate of the overall enzyme reaction. Therefore, the earlier proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.

  14. Normal Modes Expose Active Sites in Enzymes

    PubMed Central

    Glantz-Gashai, Yitav; Samson, Abraham O.

    2016-01-01

    Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427

  15. 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.

  16. 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.

  17. Effects of glucose, ethanol and acetic acid on regulation of ADH2 gene from Lachancea fermentati

    PubMed Central

    Yaacob, Norhayati; Salleh, Abu Bakar; Abdul Rahman, Nor Aini

    2016-01-01

    Background. Not all yeast alcohol dehydrogenase 2 (ADH2) are repressed by glucose, as reported in Saccharomyces cerevisiae. Pichia stipitis ADH2 is regulated by oxygen instead of glucose, whereas Kluyveromyces marxianus ADH2 is regulated by neither glucose nor ethanol. For this reason, ADH2 regulation of yeasts may be species dependent, leading to a different type of expression and fermentation efficiency. Lachancea fermentati is a highly efficient ethanol producer, fast-growing cells and adapted to fermentation-related stresses such as ethanol and organic acid, but the metabolic information regarding the regulation of glucose and ethanol production is still lacking. Methods. Our investigation started with the stimulation of ADH2 activity from S. cerevisiae and L. fermentati by glucose and ethanol induction in a glucose-repressed medium. The study also embarked on the retrospective analysis of ADH2 genomic and protein level through direct sequencing and sites identification. Based on the sequence generated, we demonstrated ADH2 gene expression highlighting the conserved NAD(P)-binding domain in the context of glucose fermentation and ethanol production. Results. An increase of ADH2 activity was observed in starved L. fermentati (LfeADH2) and S. cerevisiae (SceADH2) in response to 2% (w/v) glucose induction. These suggest that in the presence of glucose, ADH2 activity was activated instead of being repressed. An induction of 0.5% (v/v) ethanol also increased LfeADH2 activity, promoting ethanol resistance, whereas accumulating acetic acid at a later stage of fermentation stimulated ADH2 activity and enhanced glucose consumption rates. The lack in upper stream activating sequence (UAS) and TATA elements hindered the possibility of Adr1 binding to LfeADH2. Transcription factors such as SP1 and RAP1 observed in LfeADH2 sequence have been implicated in the regulation of many genes including ADH2. In glucose fermentation, L. fermentati exhibited a bell-shaped ADH2

  18. Metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, in mouse liver by alcohol dehydrogenase Adh1 and aldehyde reductase AKR1A4

    SciTech Connect

    Short, Duncan M.; Lyon, Robert; Watson, David G.; Barski, Oleg A.; McGarvie, Gail; Ellis, Elizabeth M. . E-mail: Elizabeth.ellis@strath.ac.uk

    2006-01-15

    The reductive metabolism of trans, trans-muconaldehyde, a cytotoxic metabolite of benzene, was studied in mouse liver. Using an HPLC-based stopped assay, the primary reduced metabolite was identified as 6-hydroxy-trans, trans-2,4-hexadienal (OH/CHO) and the secondary metabolite as 1,6-dihydroxy-trans, trans-2,4-hexadiene (OH/OH). The main enzymes responsible for the highest levels of reductase activity towards trans, trans-muconaldehyde were purified from mouse liver soluble fraction first by Q-sepharose chromatography followed by either blue or red dye affinity chromatography. In mouse liver, trans, trans-muconaldehyde is predominantly reduced by an NADH-dependent enzyme, which was identified as alcohol dehydrogenase (Adh1). Kinetic constants obtained for trans, trans-muconaldehyde with the native Adh1 enzyme showed a V {sub max} of 2141 {+-} 500 nmol/min/mg and a K {sub m} of 11 {+-} 4 {mu}M. This enzyme was inhibited by pyrazole with a K {sub I} of 3.1 {+-} 0.57 {mu}M. Other fractions were found to contain muconaldehyde reductase activity independent of Adh1, and one enzyme was identified as the NADPH-dependent aldehyde reductase AKR1A4. This showed a V {sub max} of 115 nmol/min/mg and a K {sub m} of 15 {+-} 2 {mu}M and was not inhibited by pyrazole.

  19. Metabolite Profiling of adh1 Mutant Response to Cold Stress in Arabidopsis

    PubMed Central

    Song, Yuan; Liu, Lijun; Wei, Yunzhu; Li, Gaopeng; Yue, Xiule; An, Lizhe

    2017-01-01

    As a result of global warming, vegetation suffers from repeated freeze-thaw cycles caused by more frequent short-term low temperatures induced by hail, snow, or night frost. Therefore, short-term freezing stress of plants should be investigated particularly in light of the current climatic conditions. Alcohol dehydrogenase (ADH) plays a central role in the metabolism of alcohols and aldehydes and it is a key enzyme in anaerobic fermentation. ADH1 responds to plant growth and environmental stress; however, the function of ADH1 in the response to short-term freezing stress remains unknown. Using real-time quantitative fluorescence PCR, the expression level of ADH1 was analyzed at low temperature (4°C). The lethal temperature was calculated based on the electrolyte leakage tests for both ADH1 deletion mutants (adh1) and wild type (WT) plants. To further investigate the relationship between ADH1 and cold tolerance in plants, low-Mr polar metabolite analyses of Arabidopsis adh1 and WT were performed at cold temperatures using gas chromatography-mass spectrometry. This investigation focused on freezing treatments (cold acclimation group: −6°C for 2 h with prior 4°C for 7 d, cold shock group: −6°C for 2 h without cold acclimation) and recovery (23°C for 24 h) with respect to seedling growth at optimum temperature. The experimental results revealed a significant increase in ADH1 expression during low temperature treatment (4°C) and at a higher lethal temperature in adh1 compared to that in the WT. Retention time indices and specific mass fragments were used to monitor 263 variables and annotate 78 identified metabolites. From these analyses, differences in the degree of metabolite accumulation between adh1 and WT were detected, including soluble sugars (e.g., sucrose) and amino acids (e.g., asparagine). In addition, the correlation-based network analysis highlighted some metabolites, e.g., melibiose, fumaric acid, succinic acid, glycolic acid, and xylose, which

  20. Metabolite Profiling of adh1 Mutant Response to Cold Stress in Arabidopsis.

    PubMed

    Song, Yuan; Liu, Lijun; Wei, Yunzhu; Li, Gaopeng; Yue, Xiule; An, Lizhe

    2016-01-01

    As a result of global warming, vegetation suffers from repeated freeze-thaw cycles caused by more frequent short-term low temperatures induced by hail, snow, or night frost. Therefore, short-term freezing stress of plants should be investigated particularly in light of the current climatic conditions. Alcohol dehydrogenase (ADH) plays a central role in the metabolism of alcohols and aldehydes and it is a key enzyme in anaerobic fermentation. ADH1 responds to plant growth and environmental stress; however, the function of ADH1 in the response to short-term freezing stress remains unknown. Using real-time quantitative fluorescence PCR, the expression level of ADH1 was analyzed at low temperature (4°C). The lethal temperature was calculated based on the electrolyte leakage tests for both ADH1 deletion mutants (adh1) and wild type (WT) plants. To further investigate the relationship between ADH1 and cold tolerance in plants, low-Mr polar metabolite analyses of Arabidopsis adh1 and WT were performed at cold temperatures using gas chromatography-mass spectrometry. This investigation focused on freezing treatments (cold acclimation group: -6°C for 2 h with prior 4°C for 7 d, cold shock group: -6°C for 2 h without cold acclimation) and recovery (23°C for 24 h) with respect to seedling growth at optimum temperature. The experimental results revealed a significant increase in ADH1 expression during low temperature treatment (4°C) and at a higher lethal temperature in adh1 compared to that in the WT. Retention time indices and specific mass fragments were used to monitor 263 variables and annotate 78 identified metabolites. From these analyses, differences in the degree of metabolite accumulation between adh1 and WT were detected, including soluble sugars (e.g., sucrose) and amino acids (e.g., asparagine). In addition, the correlation-based network analysis highlighted some metabolites, e.g., melibiose, fumaric acid, succinic acid, glycolic acid, and xylose, which

  1. 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.

  2. Effect of Laser Irradiation on Enzyme Activity

    NASA Astrophysics Data System (ADS)

    Murakami, Satoshi; Kashii, Masafumi; Kitano, Hiroshi; Adachi, Hiroaki; Takano, Kazufumi; Matsumura, Hiroyoshi; Inoue, Tsuyoshi; Mori, Yusuke; Doi, Masaaki; Sugamoto, Kazuomi; Yoshikawa, Hideki; Sasaki, Takatomo

    2005-11-01

    We previously developed a protein crystallization technique using a femtosecond laser and protein crystal processing and detaching techniques using a pulsed UV laser. In this study, we examine the effect of laser irradiation on protein integrity. After several kinds of laser were irradiated on part of a solution of glycerol-6-phosphate dehydrogenase from Leuconostoc mesenteroides, we measured the enzyme activity. Femtosecond and deep-UV laser irradiations have little influence on the whole enzyme activity, whereas the enzyme lost its activity upon high-power near-infrared laser irradiation at a wavelength of 1547 nm. These results suggest that suitable laser irradiation has no remarkable destructive influence on protein crystallization or crystal processing.

  3. [Effects of waterlogging on the growth and energy-metabolic enzyme activities of different tree species].

    PubMed

    Wang, Gui-Bin; Cao, Fu-Liang; Zhang, Xiao-Yan; Zhang, Wang-Xiang

    2010-03-01

    Aimed to understand the waterlogging tolerance and adaptation mechanisms of different tree species, a simulated field experiment was conducted to study the growth and energy-metabolic enzyme activities of one-year-old seedlings of Taxodium distichum, Carya illinoensis, and Sapium sebiferum. Three treatments were installed, i. e., CK, waterlogging, and flooding, with the treatment duration being 60 days. Under waterlogging and flooding, the relative growth of test tree species was in the order of T. distichum > C. illinoensis > S. sebiferum, indicating that T. distichum had the strongest tolerance against waterlogging and flooding, while S. sebiferum had the weakest one. Also under waterlogging and flooding, the root/crown ratio of the three tree species increased significantly, suggesting that more photosynthates were allocated in roots, and the lactate dehydrogenase (LDH) and alcohol dehydrogenase (ADH) activities of the tree species also had a significant increase. Among the test tree species, T. distichum had the lowest increment of LDH and ADH activities under waterlogging and flooding, but the increment could maintain at a higher level in the treatment duration, while for C. illinoensis and S. sebiferum, the increment was larger during the initial and medium period, but declined rapidly during the later period of treatment. The malate dehydrogenase (MDH), phosphohexose (HPI), and glucose-6-phosphate dehydrogenase (G6PDH) -6-phosphogluconate dehydrogenase (6PGDH) activities of the tree species under waterlogging and flooding had a significant decrease, and the decrement was the largest for T. distichum, being 35.6% for MDH, 21.0% for HPI, and 22.7% for G6PDH - 6PGDH under flooding. It was suggested that under waterlogging and flooding, the tree species with strong waterlogging tolerance had a higher ability to maintain energy-metabolic balance, and thus, its growth could be maintained at a certain level.

  4. Halophilic enzyme activation induced by salts

    PubMed Central

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

    2011-01-01

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

  5. Effect of hypovolemia, infusion, and oral rehydration on plasma electrolytes, ADH, renin activity, and +G/z/ tolerance

    NASA Technical Reports Server (NTRS)

    Greenleaf, J. E.; Brock, P. J.; Haines, R. F.; Rositano, S. A.; Montgomery, L. D.; Keil, L. C.

    1977-01-01

    Effects on plasma volume, electrolyte shifts, and +G(z) tolerance induced by: (1) blood withdrawal; (2) blood infusion; and (3) oral fluid intake, were determined at 0.5 G/min in centrifugation tests of six ambulatory male patients, aged 21 to 27 yrs. Hypovolemia induced by withdrawal of 400 ml blood, blood infusion followed by repeated centrifugation, effects of consuming an isotonic drink (0.9% NaCl) to achieve oral rehydration, and donning of red adaptation goggles were studied for effects on acceleration tolerance, pre-acceleration and post-acceleration plasma renin activity (PRA) and plasma vasopressin levels. No significant changes in post-acceleration PRA compared to pre-acceleration PRA were found, and administration of oral rehydration is found as effective as blood replacement in counteracting hypovolemic effects.

  6. 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)

  7. [Effects of catalase activators and inhibitors on ethanol pharmacokinetic characteristics and ethanol and aldehyde-metabolizing enzyme activities in the rat liver and brain].

    PubMed

    Bardina, L R; Pron'ko, P S; Satanovskaia, V I; Alieva, E V

    2010-01-01

    The effects of catalase regulators (aminotriazole, lead acetate, taurine, di-2-ethylhexylphthalate) on the preference for ethanol, its pharmacokinetics, and activities of rat liver and brain ethanol and acetaldehyde-metabolizing enzymes were studied. Lead acetate (100 mg/kg, i.p., 7 days), aminotriazole (1 g/kg, i.p., 7 days), and taurine (650 mg/kg, i.g., 14 days) decreased ethanol consumption under conditions of free choice (10% ethanol water), whereas di-2-ethylhexylphthalate (300 mg/kg, i.g., 7 days) did not exert any effect on this parameter. Taurine, lead acetate and di-2-ethylhexylphthalate significantly activated liver ADH, MEOS and catalase peroxidase activity. Aminotriazole also activated ADH and MEOS, but inhibited liver catalase. The activities of liver and brain A1DH as well as catalase were insignificantly changed by this treatment. The 7-day administration of lead acetate, di-2-ethylhexylphthalate and aminotriazole administrations significantly influenced the ethanol (2 g/kg., i.p.) pharmacokinetic parameters: the area under the pharmacokinetic curve and the elimination half-life time were significantly reduced, whereas the elimination constant and clearance were increased. This unequivocally indicates accelerated ethanol elimination. The 14-day ingestion of taurine insignificantly changed the parameters of ethanol pharmacokinetics in rats.

  8. Alcohol Dehydrogenase and Pyruvate Decarboxylase Activity in Leaves and Roots of Eastern Cottonwood (Populus deltoides Bartr.) and Soybean (Glycine max L.) 1

    PubMed Central

    Kimmerer, Thomas W.

    1987-01-01

    Pyruvate decarboxylase (PDC, EC 4.1.1.1) and alcohol dehydrogenase (ADH, EC 1.1.1.1) are responsible for the anaerobic production of acetaldehyde and ethanol in higher plants. In developing soybean embryos, ADH activity increased upon imbibition and then declined exponentially with development, and was undetectable in leaves by 30 days after imbibition. PDC was not detectable in soybean leaves. In contrast, ADH activity remained high in developing cottonwood seedlings, with no decline in activity during development. ADH activity in the first fully expanded leaf of cottonwood was 230 micromoles NADH oxidized per minute per gram dry weight, and increased with leaf age. Maximal PDC activity of cottonwood leaves was 10 micromoles NADH oxidized per minute per gram dry weight. ADH activity in cottonwood roots was induced by anaerobic stress, increasing from 58 to 205 micromoles NADH oxidized per minute per gram dry weight in intact plants in 48 hours, and from 38 to 246 micromoles NADH oxidized per minute per gram dry weight in detached roots in 48 hours. Leaf ADH activity increased by 10 to 20% on exposure to anaerobic conditions. Crude leaf enzyme extracts with high ADH activity reduced little or no NADH when other aldehydes, such as trans-2-hexenal, were provided as substrate. ADH and PDC are constitutive enzyme in cottonwood leaves, but their metabolic role is not known. PMID:16665586

  9. Molecular cloning of alcohol dehydrogenase genes of the yeast Pichia stipitis and identification of the fermentative ADH.

    PubMed

    Passoth, V; Schäfer, B; Liebel, B; Weierstall, T; Klinner, U

    1998-10-01

    Two Pichia stipitis ADH genes (PsADH1 and PsADH2) were isolated by complementation of a Saccharomyces cerevisiae Adh(-)-mutant. The genes enabled the transformants to grow in the presence of antimycin A on glucose, to use ethanol as sole carbon source and made them sensitive to allylalcohol. The sequences of the genes showed similarities of 70-77% to sequences of ADH genes of Candida albicans, Kluyveromyces lactis, K. marxianus, and S. cerevisiae and about 60% homology to those of Schizosaccharomyces pombe and Aspergillus flavus. Southern hybridization experiments suggested that P. stipitis has only these two ADH genes. Both genes are located on the largest chromosome of P. stipitis. PsADH2 encodes for the ADH activity that is responsible for ethanol formation at oxygen limitation. The gene is regulated at the transcriptional level. Moreover, also in cells grown on ethanol, only PsADH2 transcript was found. PsADH1 transcript was detected under aerobic conditions on fermentable carbon sources.

  10. A distinct type of alcohol dehydrogenase, adh4+, complements ethanol fermentation in an adh1-deficient strain of Schizosaccharomyces pombe.

    PubMed

    Sakurai, Masao; Tohda, Hideki; Kumagai, Hiromichi; Giga-Hama, Yuko

    2004-03-01

    In the fission yeast Schizosaccharomyces pombe, only one alcohol dehydrogenase gene, adh1(+), has been identified. To elucidate the influence of adh1(+) on ethanol fermentation, we constructed the adh1 null strain (delta adh1). The delta adh1 cells still produced ethanol and grew fermentatively as the wild-type cells. Both DNA microarray and RT-PCR analysis demonstrated that this ethanol production is caused by the enhanced expression of a Saccharomyces cerevisiae ADH4-like gene product (SPAC5H10.06C named adh4(+)). Since the strain lacking both adh1 and adh4 genes (delta adh1 delta adh4) showed non-fermentative retarded growth, only these two ADHs produce ethanol for fermentative growth. This is the first observation that a S. cerevisiae ADH4-like alcohol dehydrogenase functions in yeast ethanol fermentation.

  11. [Effects of Hg on soil enzyme activity].

    PubMed

    Yang, Chun-Lu; Sun, Tie-Heng; He, Wen-Xiang; Chen, Su

    2007-03-01

    With simulation test, this paper studied the effects of Hg on the activities of urease, invertase and neutral phosphotase in four soils. The results showed that Hg inhibited soil urease and invertase activities markedly, but its inhibitory effect differed with test soils. There was a significant logarithmic correlation between the concentration of HgCl2 and the activities of these two enzymes (P < 0.05). In test soils, the ED50 of urease activity was 87.99, 5.47, 24.05 and 19.88 mg x kg(-1), and that of invertase activity was 76.68, 727.49, 236.52 and 316.59 mg x kg(-1), respectively. Urease was more sensitive than invertase to Hg contamination, while organic matter had a protective effect on soil enzymes. Soil neutral phosphatase was not sensitive to Hg contamination, except that it was significantly activated by Hg in the meadow brown soil applied with plenty of organic fertilizer.

  12. Iodothyronine deiodinase enzyme activities in bone.

    PubMed

    Williams, Allan J; Robson, Helen; Kester, Monique H A; van Leeuwen, Johannes P T M; Shalet, Stephen M; Visser, Theo J; Williams, Graham R

    2008-07-01

    Euthyroid status is essential for normal skeletal development and maintenance of the adult skeleton, but the mechanisms which control supply of thyroid hormone to bone cells are poorly understood. Thyroid hormones enter target cells via monocarboxylate transporter-8 (MCT8), which provides a functional link between thyroid hormone uptake and metabolism in the regulation of T3-action but has not been investigated in bone. Most circulating active thyroid hormone (T3) is derived from outer ring deiodination of thyroxine (T4) mediated by the type 1 deiodinase enzyme (D1). The D2 isozyme regulates intra-cellular T3 supply and determines saturation of the nuclear T3-receptor (TR), whereas a third enzyme (D3) inactivates T4 and T3 to prevent hormone availability and reduce TR-saturation. The aim of this study was to determine whether MCT8 is expressed in the skeleton and whether chondrocytes, osteoblasts and osteoclasts express functional deiodinases. Gene expression was analyzed by RT-PCR and D1, D2 and D3 function by sensitive and highly specific determination of enzyme activities. MCT8 mRNA was expressed in chondrocytes, osteoblasts and osteoclasts at all stages of cell differentiation. D1 activity was undetectable in all cell types, D2 activity was only present in mature osteoblasts whereas D3 activity was evident throughout chondrocyte, osteoblast and osteoclast differentiation in primary cell cultures. These data suggest that T3 availability especially during skeletal development may be limited by D3-mediated catabolism rather than by MCT8 mediated cellular uptake or D2-dependent T3 production.

  13. A genetic analysis of Adh1 regulation

    SciTech Connect

    Freeling, M.

    1992-01-01

    The overall goal of our research proposal is to understand the meaning of the various cis-acting sites responsible for AdH1 expression in the entire maize plant. Progress is reported in the following areas: Studies on the TATA box and analysis of revertants of the Adh1-3F1124 allele; screening for more different mutants that affect Adh1 expression differentially; studies on cis-acting sequences required for root-specific Adh1 expression; refinement of the use of the particle gun; and functional analysis of a non- glycolytic anaerobic protein.

  14. Recombinant industrial brewing yeast strains with ADH2 interruption using self-cloning GSH1+CUP1 cassette.

    PubMed

    Wang, Zhao-Yue; Wang, Jin-Jing; Liu, Xi-Feng; He, Xiu-Ping; Zhang, Bo-Run

    2009-06-01

    A self-cloning module for gene knock-out and knock-in in industrial brewing yeast strain was constructed that contains copper resistance and gamma-glutamylcysteine synthetase gene cassette, flanked by alcohol dehydrogenase II gene (ADH2) of Saccharomyces cerevisiae. The module was used to obtain recombined strains RY1 and RY2 by targeting the ADH2 locus of host Y1. RY1 and RY2 were genetically stable. PCR and enzyme activity analysis of RY1 and RY2 cells showed that one copy of ADH2 was deleted by GSH1+CUP1 insertion, and an additional copy of wild type was still present. The fermentation ability of the recombinants was not changed after genetic modification, and a high level of glutathione (GSH) was secreted, resulting from GSH1 overexpression, which codes for gamma-glutamylcysteine synthetase. A pilot-scale brewing test for RY1 and RY2 indicated that acetaldehyde content in fermenting liquor decreased by 21-22%, GSH content increased by 20-22% compared with the host, the antioxidizability of the recombinants was improved, and the sensorial evaluation was also better than that of the host. No heterologous DNA was harbored in the recombinants; therefore, they could be applied in the beer industry in terms of their biosafety.

  15. 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.

  16. 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

  17. Angiotensin Converting Enzyme Activity in Alopecia Areata

    PubMed Central

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

    2014-01-01

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

  18. Characterization of the temperate bacteriophage phi adh and plasmid transduction in Lactobacillus acidophilus ADH.

    PubMed

    Raya, R R; Kleeman, E G; Luchansky, J B; Klaenhammer, T R

    1989-09-01

    Lactobacillus acidophilus ADH is lysogenic and harbors an inducible prophage, phi adh. Bacteriophage were detected in cell lysates induced by treatment with mitomycin C or UV light. Electron microscopy of lysates revealed phage particles with a hexagonal head (62 nm) and a long, noncontractile, flexible tail (398 nm) ending in at last five short fibers. Phage phi adh was classified within Bradley's B1 phage group and the Siphoviridae family. The phi adh genome is a linear double-stranded DNA molecule of 41.7 kilobase pairs with cohesive ends: a physical map of the phi adh genome was constructed. A prophage-cured derivative of strain ADH, designated NCK102, was isolated from cells that survived UV exposure. NCK102 did not exhibit mitomycin C-induced lysis, but broth cultures lysed upon addition of phage. Phage phi adh produced clear plaques on NCK102 in media containing 10 mM CaCl2 at pH values between 5.2 and 5.5. A relysogenized derivative (NCK103) of NCK102 was isolated that exhibited mitomycin C-induced lysis and superinfection immunity to phage phi adh. Hybridization experiments showed that the phi adh genome was present in the ADH and NCK103 chromosomes, but absent in NCK102. These results demonstrated classic lytic and lysogenic cycles of replication for the temperate phage phi adh induced from L. acidophilus ADH. Phage phi adh also mediates transduction of plasmid DNA. Transductants of strain ADH containing pC194, pGK12, pGB354, and pVA797 were detected at frequencies in the range of 3.6 x 10(-8) to 8.3 x 10(-10) per PFU. Rearrangements or deletions were not detected in these plasmids as a consequence of transduction. This is the first description of plasmid transduction in the genus Lactobacillus.

  19. [Impacts of root-zone hypoxia stress on muskmelon growth, its root respiratory metabolism, and antioxidative enzyme activities].

    PubMed

    Liu, Yi-Ling; Li, Tian-Lai; Sun, Zhou-Ping; Chen, Ya-Dong

    2010-06-01

    By using aeroponics culture system, this paper studied the impacts of root-zone hypoxia (10% O2 and 5% O2) stress on the plant growth, root respiratory metabolism, and antioxidative enzyme activities of muskmelon at its fruit development stage. Root-zone hypoxia stress inhibited the plant growth of muskmelon, resulting in the decrease of plant height, root length, and fresh and dry biomass. Comparing with the control (21% O2), hypoxia stress reduced the root respiration rate and malate dehydrogenase (MDH) activity significantly, and the impact of 5% O2 stress was more serious than that of 10% O2 stress. Under hypoxic conditions, the lactate dehydrogenase (LDH), alcohol dehydrogenase (ADH), pyruvate decarboxylase (PDC), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities and the malondialdehyde (MDA) content were significantly higher than the control. The increment of antioxidative enzyme activities under 10% O2 stress was significantly higher than that under 5% O2 stress, while the MDA content was higher under 5% O2 stress than under 10% O2 stress, suggesting that when the root-zone oxygen concentration was below 10%, the aerobic respiration of muskmelon at its fruit development stage was obviously inhibited while the anaerobic respiration was accelerated, and the root antioxidative enzymes induced defense reaction. With the increasing duration of hypoxic stress, the lipid peroxidation would be aggravated, resulting in the damages on muskmelon roots, inhibition of plant growth, and decrease of fruit yield and quality.

  20. The ADH7 Promoter of Saccharomyces cerevisiae is Vanillin-Inducible and Enables mRNA Translation Under Severe Vanillin Stress.

    PubMed

    Nguyen, Trinh T M; Iwaki, Aya; Izawa, Shingo

    2015-01-01

    Vanillin is one of the major phenolic aldehyde compounds derived from lignocellulosic biomass and acts as a potent fermentation inhibitor to repress the growth and fermentative ability of yeast. Vanillin can be reduced to its less toxic form, vanillyl alcohol, by the yeast NADPH-dependent medium chain alcohol dehydrogenases, Adh6 and Adh7. However, there is little information available regarding the regulation of their gene expression upon severe vanillin stress, which has been shown to repress the bulk translation activity in yeast cells. Therefore, in this study, we investigated expression patterns of the ADH6 and ADH7 genes in the presence of high concentrations of vanillin. We found that although both genes were transcriptionally upregulated by vanillin stress, they showed different protein expression patterns in response to vanillin. Expression of Adh6 was constitutive and gradually decreased under vanillin stress, whereas expression of Adh7 was inducible, and, importantly, occurred under severe vanillin stress. The null mutants of ADH6 or ADH7 genes were hypersensitive to vanillin and reduced vanillin less efficiently than the wild type, confirming the importance of Adh6 and Adh7 in vanillin detoxification. Additionally, we demonstrate that the ADH7 promoter is vanillin-inducible and enables effective protein synthesis even under severe vanillin stress, and it may be useful for the improvement of vanillin-tolerance and biofuel production efficiency via modification of yeast gene expression in the presence of high concentrations of vanillin.

  1. 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.

  2. Temperature and the catalytic activity of enzymes: a fresh understanding.

    PubMed

    Daniel, Roy M; Danson, Michael J

    2013-09-02

    The discovery of an additional step in the progression of an enzyme from the active to inactive state under the influence of temperature has led to a better match with experimental data for all enzymes that follow Michaelis-Menten kinetics, and to an increased understanding of the process. The new model of the process, the Equilibrium Model, describes an additional mechanism by which temperature affects the activity of enzymes, with implications for ecological, metabolic, structural, and applied studies of enzymes.

  3. The use of suicide substrates to select mutants of Escherichia coli lacking enzymes of alcohol fermentation.

    PubMed

    Cunningham, P R; Clark, D P

    1986-12-01

    Mutants of Escherichia coli resistant to chloroethanol or to chloroacetaldehyde were selected. Such mutants were found to lack the fermentative coenzyme A (CoA) linked acetaldehyde dehydrogenase activity. Most also lacked the associated fermentative enzyme alcohol dehydrogenase. Both types of mutants, those lacking acetaldehyde dehydrogenase alone or lacking both enzymes, mapped close to the regulatory adhC gene at 27 min on the E. coli genetic map. The previously described acd mutants which lack acetaldehyde dehydrogenase and which map at 63 min were shown to be pleiotropic, affecting respiration and growth on a variety of substrates. It therefore seems likely that the structural genes for both the acetaldehyde and alcohol dehydrogenases lie in the adhCE operon. This interpretation was confirmed by the isolation of temperature sensitive chloracetaldehyde-resistant mutants, some of which produced thermolabile acetaldehyde dehydrogenase and alcohol dehydrogenase and were also found to map at the adh locus. Reversion analysis indicated that mutants lacking one or both enzymes carried single mutations. The gene order in the adh region was determined by three point crosses to be trp-zch::Tn10-adh-galU-bglY-tyrT-chlC.

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

    PubMed

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

    2009-07-01

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

  5. Alcohol dehydrogenase AdhA plays a role in ethanol tolerance in model cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Vidal, Rebeca

    2017-02-03

    The protein AdhA from the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis) has been previously reported to show alcohol dehydrogenase activity towards ethanol and both NAD and NADP. This protein is currently being used in genetically modified strains of Synechocystis capable of synthesizing ethanol showing the highest ethanol productivities. In the present work, mutant strains of Synechocystis lacking AdhA have been constructed and tested for tolerance to ethanol. The lack of AdhA in the wild-type strain reduces survival to externally added ethanol at lethal concentration of 4% (v/v). On the other hand, the lack of AdhA in an ethanologenic strain diminishes tolerance of cells to internally produced ethanol. It is also shown that light-activated heterotrophic growth (LAHG) of the wild-type strain is impaired in the mutant strain lacking AdhA (∆adhA strain). Photoautotrophic, mixotrophic, and photoheterotrophic growth are not affected in the mutant strain. Based on phenotypic characterization of ∆adhA mutants, the possible physiological function of AdhA in Synechocystis is discussed.

  6. The Relationship between CmADHs and the Diversity of Volatile Organic Compounds of Three Aroma Types of Melon (Cucumis melo)

    PubMed Central

    Chen, Hao; Cao, Songxiao; Jin, Yazhong; Tang, Yufan; Qi, Hongyan

    2016-01-01

    Alcohol dehydrogenase (ADH) plays an important role in aroma volatile compounds synthesis of plants. In this paper, we tried to explore the relationship between CmADHs and the volatile organic compounds (VOCs) in oriental melon. Three different aroma types of melon were used as materials. The principle component analysis of three types of melon fruit was conducted. We also measured the CmADHs expression level and enzymatic activities of ADH and alcohol acyl-transferase (AAT) on different stages of fruit ripening. An incubation experiment was carried out to investigate the effect of substrates and inhibitor (4-MP, 4-methylpyrazole) on CmADHs expression, ADH activity, and the main compounds of oriental melon. The results illustrated that ethyl acetate, hexyl acetate (E,Z)-3,6-nonadien-1-ol and 2-ethyl-2hexen-1-ol were the four principal volatile compounds of these three types of melon. AAT activity was increasing with fruit ripening, and the AAT activity in CH were the highest, whereas ADH activity peaked on 32 DAP, 2 days before maturation, and the ADH activity in CB and CG were higher than that in CH. The expression pattern of 11 CmADH genes from 24 to 36 day after pollination (DAP) was found to vary in three melon varieties. CmADH4 was only expressed in CG and the expression levels of CmADH3 and CmADH12 in CH and CB were much higher than that in CG, and they both peaked 2 days before fruit ripening. Ethanol and 4-MP decreased the reductase activity of ADH, the expression of most CmADHs and ethyl acetate or hexyl acetate contents of CB, except for 0.1 mM 4-MP, while aldehyde improved the two acetate ester contents. In addition, we found a positive correlation between the expression of CmADH3 and CmADH12 and the key volatile compound of CB. The relationship between CmADHs and VOCs synthesis of oriental melon was discussed. PMID:27445845

  7. Functional relevance of human adh polymorphism.

    PubMed

    Eriksson, C J; Fukunaga, T; Sarkola, T; Chen, W J; Chen, C C; Ju, J M; Cheng, A T; Yamamoto, H; Kohlenberg-Müller, K; Kimura, M; Murayama, M; Matsushita, S; Kashima, H; Higuchi, S; Carr, L; Viljoen, D; Brooke, L; Stewart, T; Foroud, T; Su, J; Li, T K; Whitfield, J B

    2001-05-01

    This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were C. J. Peter Eriksson and Tatsushige Fukunaga. The presentations were (1) 4-Methylpyrazole as a tool in the investigation of the role of ADH in the actions of alcohol in humans, by Taisto Sarkola and C. J. Peter Eriksson; (2) ADH2 polymorphism and flushing in Asian populations, by Wei J. Chen, C. C. Chen, J. M. Ju, and Andrew T. A. Cheng; (3) Role of ADH3 genotypes in the acute effects of alcohol in a Finnish population, by Hidetaka Yamamoto, Kathrin Kohlenberg-Müller, and C. J. Peter Eriksson; (4) Clinical characteristics and disease course of alcoholics with different ADH2 genotypes, by Mitsuru Kimura, Masanobu Murayama, Sachio Matsushita, Haruo Kashima, and Susumu Higuchi; (5) ADH2 polymorphism, alcohol drinking, and birth defects, by Lucinda Carr, D. Viljoen, L. Brooke, T. Stewart, T. Foroud, J. Su, and Ting-Kai Li; and (6) ADH genotypes and alcohol use in Europeans, by John B. Whitfield.

  8. 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

  9. Cold-active enzymes studied by comparative molecular dynamics simulation.

    PubMed

    Spiwok, Vojtech; Lipovová, Petra; Skálová, Tereza; Dusková, Jarmila; Dohnálek, Jan; Hasek, Jindrich; Russell, Nicholas J; Králová, Blanka

    2007-04-01

    Enzymes from cold-adapted species are significantly more active at low temperatures, even those close to zero Celsius, but the rationale of this adaptation is complex and relatively poorly understood. It is commonly stated that there is a relationship between the flexibility of an enzyme and its catalytic activity at low temperature. This paper gives the results of a study using molecular dynamics simulations performed for five pairs of enzymes, each pair comprising a cold-active enzyme plus its mesophilic or thermophilic counterpart. The enzyme pairs included alpha-amylase, citrate synthase, malate dehydrogenase, alkaline protease and xylanase. Numerous sites with elevated flexibility were observed in all enzymes; however, differences in flexibilities were not striking. Nevertheless, amino acid residues common in both enzymes of a pair (not present in insertions of a structure alignment) are generally more flexible in the cold-active enzymes. The further application of principle component analysis to the protein dynamics revealed that there are differences in the rate and/or extent of opening and closing of the active sites. The results indicate that protein dynamics play an important role in catalytic processes where structural rearrangements, such as those required for active site access by substrate, are involved. They also support the notion that cold adaptation may have evolved by selective changes in regions of enzyme structure rather than in global change to the whole protein.

  10. 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.

  11. 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.

  12. 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.

  13. 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,…

  14. The Analysis of Polymorphism of Alcohol Dehydrogenase 3 (ADH3) Gene and Influence of Liver Function Status in Indonesia.

    PubMed

    Suhartini; Mustofa; Nurhantari, Yudha; Rianto, Bambang Udji Djoko

    2017-01-31

    Indonesian culture actually has no historical record of behaviors in consuming alcohol, but there are many recent reports of alcohol abuse among Asian people involving their traditional drink. In genotype studies, the damage of the liver caused by consuming alcohol is influenced by the presence of the polymorphism enzyme gene. The lack of study regarding such topic is a signal to further investigate ADH3 gene distribution and its effect on liver function status. The total of 197 research subjects of Javanese descent received alcohol dehydrogenase 3 (ADH3) genetic polymorphism and liver status tests in the city of Yogyakarta, Indonesian. An analytical study with a cross-sectional design was then conducted on the subjects, with the resulting isolated DNAs amplified through polymerase chain reaction (PCR). The genotype of ADH3 was determined by means of restriction fragment length polymorphism (RFLP) using Ssp1 restricting enzyme. Liver function status was assessed by measuring serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvate transaminase (SGPT) and gamma glutamyl transferase (GGT) using a photometric system. Gene types of ADH3*1 (2.1%), ADH3*2 (82.7%) and ADH3*1/3*2 (15.2%) on the subjects were concluded, finding that there is no difference between the gender. In conclusion most of the ADH3 gene polymorphism of the subjects were ADH3*2 (82.7%). The influence of genetic polymorphisms on the status of liver function in the subjects showed significant difference according to GGT measurement, but the same cannot be said on the other two values measuring SGOT and SGPT.

  15. Diffusional correlations among multiple active sites in a single enzyme.

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2014-04-07

    Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

  16. Toward cell-free biofuel production: Stable immobilization of oligomeric enzymes.

    PubMed

    Grimaldi, J; Collins, C H; Belfort, G

    2014-01-01

    To overcome the main challenges facing alcohol-based biofuel production, we propose an alternate simplified biofuel production scheme based on a cell-free immobilized enzyme system. In this paper, we measured the activity of two tetrameric enzymes, a control enzyme with a colorimetric assay, β-galactosidase, and an alcohol-producing enzyme, alcohol dehydrogenase, immobilized on multiple surface curvatures and chemistries. Several solid supports including silica nanoparticles (convex), mesopourous silica (concave), diatomaceous earth (concave), and methacrylate (concave) were examined. High conversion rates and low protein leaching was achieved by covalent immobilization of both enzymes on methacrylate resin. Alcohol dehydrogenase (ADH) exhibited long-term stability and over 80% conversion of aldehyde to alcohol over 16 days of batch cycles. The complete reaction scheme for the conversion of acid to aldehyde to alcohol was demonstrated in vitro by immobilizing ADH with keto-acid decarboxylase free in solution.

  17. 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 ...

  18. 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

  19. 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...

  20. 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.

  1. 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.

  2. Enzyme activity control by responsive redoxpolymers.

    PubMed

    Nagel, Birgit; Warsinke, Axel; Katterle, Martin

    2007-06-05

    A new thermoresponsive poly-N-isopropylacrylamide (PNIPAM)-ferrocene polymer was synthesized and characterized. PNIPAMFoxy bears additional oxirane groups which were used for attachment by a self-assembly process on a cysteamine-modified gold electrode to create a thin hydrophilic film. The new redox polymer enabled electrical communication between the cofactor pyrrolinoquinoline quinone (PQQ) of soluble glucose dehydrogenase (sGDH) and the electrode for sensitive detection of this enzyme as a prospective protein label. The temperature influence on the redox polymer/enzyme complex was investigated. An inverse temperature response behavior of surface bound PNIPAMFoxy compared to the soluble polymer was found and is discussed in detail. The highest efficiency of mediated electron transfer for the immobilized PNIPAMFoxy with sGDH was observed at 24 degrees C, which was twice as high as that of its soluble counterpart. A steady-state electrooxidation current densitiy of 4.5 microA.cm-2 was observed in the presence of 10 nM sGDH and 5 mM glucose. A detection limit of 0.5 nM of soluble PQQ-sGDH was obtained.

  3. 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

  4. 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.

  5. Rare ADH Variant Constellations are Specific for Alcohol Dependence

    PubMed Central

    Zuo, Lingjun; Zhang, Heping; Malison, Robert T.; Li, Chiang-Shan R.; Zhang, Xiang-Yang; Wang, Fei; Lu, Lingeng; Lu, Lin; Wang, Xiaoping; Krystal, John H.; Zhang, Fengyu; Deng, Hong-Wen; Luo, Xingguang

    2013-01-01

    Aims: Some of the well-known functional alcohol dehydrogenase (ADH) gene variants (e.g. ADH1B*2, ADH1B*3 and ADH1C*2) that significantly affect the risk of alcohol dependence are rare variants in most populations. In the present study, we comprehensively examined the associations between rare ADH variants [minor allele frequency (MAF) <0.05] and alcohol dependence, with several other neuropsychiatric and neurological disorders as reference. Methods: A total of 49,358 subjects in 22 independent cohorts with 11 different neuropsychiatric and neurological disorders were analyzed, including 3 cohorts with alcohol dependence. The entire ADH gene cluster (ADH7–ADH1C–ADH1B–ADH1A–ADH6–ADH4–ADH5 at Chr4) was imputed in all samples using the same reference panels that included whole-genome sequencing data. We stringently cleaned the phenotype and genotype data to obtain a total of 870 single nucleotide polymorphisms with 0< MAF <0.05 for association analysis. Results: We found that a rare variant constellation across the entire ADH gene cluster was significantly associated with alcohol dependence in European-Americans (Fp1: simulated global P = 0.045), European-Australians (Fp5: global P = 0.027; collapsing: P = 0.038) and African-Americans (Fp5: global P = 0.050; collapsing: P = 0.038), but not with any other neuropsychiatric disease. Association signals in this region came principally from ADH6, ADH7, ADH1B and ADH1C. In particular, a rare ADH6 variant constellation showed a replicable association with alcohol dependence across these three independent cohorts. No individual rare variants were statistically significantly associated with any disease examined after group- and region-wide correction for multiple comparisons. Conclusion: We conclude that rare ADH variants are specific for alcohol dependence. The ADH gene cluster may harbor a causal variant(s) for alcohol dependence. PMID:23019235

  6. 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.

  7. Effects of DNA on immunoglobulin production stimulating activity of alcohol dehydrogenase.

    PubMed

    Okamoto, T; Furutani, H; Sasaki, T; Sugahara, T

    1999-09-01

    Alcohol dehydrogenase-I (ADH-I) derived from horse liver stimulated IgM production by human-human hybridoma, HB4C5 cells and lymphocytes. The IPSF activity of ADH-I was suppressed by coexistence of short DNA whose chain length is less than 200 base pairs (bp) and fibrous DNA in a dose-dependent manner. These DNA preparations completely inhibited the IPSF activity at the concentration of 250 mug/ml and 1.0 mg/ml, respectively. DNA sample termed long DNA whose average chain length is 400-7000 bp slightly stimulated IPSF activity at 0.06 mug/ml. However, long DNA suppressed IPSF activity by half at 1.0 mg/ml. The laser confocal microscopic analysis had revealed that ADH-I was incorporated by HB4C5 cells. The uptake of ADH-I was strongly inhibited by short DNA and fibrous DNA. However, long DNA did not suppress the internalization of ADH-I into HB4C5 cells. These findings indicate that short DNA and fibrous DNA depress IPSF activity of ADH-I by inhibiting the internalization of this enzyme. According to the gel-filtration analysis using HPLC, ADH-I did not directly interact with short DNA. It is expected from these findings that short DNA influences HB4C5 cells to suppress the internalization of ADH-I. Moreover, these facts also strongly suggest that ADH-I acts as IPSF after internalization into the cell.

  8. Effect of early feed restriction and enzyme supplementation on digestive enzyme activities in broilers.

    PubMed

    Pinheiro, D F; Cruz, V C; Sartori, J R; Vicentini Paulino, M L M

    2004-09-01

    The effect of feed restriction and enzymatic supplementation on intestinal and pancreatic enzyme activities and weight gain was studied in broiler chickens. Quantitative feed restriction was applied to chickens from 7 to 14 d of age. An enzyme complex mainly consisting of protease and amylase was added to the chicken ration from hatching to the end of the experiment. Birds subjected to feed restriction whose diet was not supplemented showed an increase in sucrase, amylase, and lipase activities immediately after the restriction period. Amylase, lipase, and chymotrypsin activities were higher in chickens subjected to feed restriction and fed a supplemented diet than in those only subjected to feed restriction. Trypsin activity increased after feed restriction and after supplementation, but there was no interaction between these effects. Early feed restriction had no effect on enzyme activity in 42-d-old chickens. Chickens subjected to early restriction and fed the supplemented diet presented higher sucrase, maltase, and lipase activities than nonsupplemented ones (P < 0.05). There was no effect of early feed restriction or diet supplementation on weight gain to 42 d. Percentage weight gain from 14 to 42 d of age was equivalent in feed-restricted and ad libitum fed birds. Feed-restricted broilers fed a supplemented diet showed a higher percentage weight gain than nonsupplemented birds. We conclude that enzymatic supplementation potentiates the effect of feed restriction on digestive enzyme activity and on weight gain.

  9. 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

  10. SPINK1, ADH2, and ALDH2 gene variants and alcoholic chronic pancreatitis in Japan.

    PubMed

    Shimosegawa, Tooru; Kume, Kiyoshi; Masamune, Atsushi

    2008-03-01

    The serine protease inhibitor Kazal type 1 (SPINK1) is a potent antiprotease and an important inactivation factor of intrapancreatic trypsin activity. Loss of function by the SPINK1 mutations leads to decreased inhibitory capacity. The significance of SPINK1 mutations in alcoholic chronic pancreatitis (CP) in Japan and its functional role remain unclear. The aim of the present study was to clarify the incidence of SPINK1, alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) variants in CP patients in Japan. One hundred and 86 patients with CP, and 527 healthy volunteers were enrolled. Mutational analyses were performed by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. Serum pancreatic secretory trypsin inhibitor (PSTI) level was measured by radioimmunoassay. The frequencies of N34S and IVS3 + 2T > C in the SPINK1 gene were significantly higher in patients with non-alcoholic CP (12.9% and 8.6%, respectively) than in normal subjects (0.37% and 0%). In total, 18 of 93 (19.4%) patients with non-alcoholic CP had at least one SPINK1 mutation. Concerning alcoholic CP, we found IVS3 + 2T > C in a small number of patients (3.9%). Serum PSTI concentration was decreased in patients with the IVS3 + 2T > C mutation. The frequency of the ADH2*2 allele in the alcoholic CP group was significantly higher than that in alcoholics without pancreatitis. The frequency of the ALDH2*2 allele was significantly low in patients with alcoholic CP compared with healthy controls. In conclusion, SPINK1 mutations were associated with non-alcoholic CP. Furthermore, we revealed the amount of wild-type PSTI was decreased in patients with IVS3 + 2T > C mutation. Variants of alcohol-metabolizing enzymes appeared in the relation to alcoholic CP.

  11. ENZYMIC ACTIVITY IN FREEZE DRIED FOODS

    DTIC Science & Technology

    and bananas. Factors studied include, polyphenol oxidase , peroxidase, sucrase, alpha and beta amylase, pectinesterase and ascorbase activity as well...storage of freeze-dried and frozen peas at different moisture was studied. Lipase activity and production of free fatty acid was following during long term

  12. Inhibition of alcohol dehydrogenase after 2-propanol exposure in different geographic races of Drosophila mojavensis: lack of evidence for selection at the Adh-2 locus.

    PubMed

    Pfeiler, Edward; Reed, Laura K; Markow, Therese A

    2005-03-15

    High frequencies of the fast allele of alcohol dehydrogenase-2 (Adh-2F) are found in populations of Drosophila mojavensis that inhabit the Baja California peninsula (race BII) whereas the slow allele (Adh-2S) predominates at most other localities within the species' geographic range. Race BII flies utilize necrotic tissue of pitaya agria cactus (Stenocereus gummosus) which contains high levels of 2-propanol, whereas flies from most other localities utilize different cactus hosts in which 2-propanol levels are low. To test if 2-propanol acts as a selective force on Adh-2 genotype, or whether some other yet undetermined genetic factor is responsible, mature males of D. mojavensis lines derived from the Grand Canyon (race A) and Santa Catalina Island (race C), each with individuals homozygous for Adh-2F and Adh-2S, were exposed to 2-propanol for 24 h and ADH-2 specific activity was then determined on each genotype. Flies from five other localities homozygous for either the fast or slow allele also were examined. Results for all reported races of D. mojavensis were obtained. 2-propanol exposure inhibited ADH-2 specific activity in both genotypes from all localities, but inhibition was significantly less in two populations of race BII flies homozygous for Adh-2F. When F/F and S/S genotypes in flies from the same locality were compared, both genotypes showed high 2-propanol inhibition that was not statistically different, indicating that the F/F genotype alone does not provide a benefit against the inhibitory effects of 2-propanol. ADH-1 activity in female ovaries was inhibited less by 2-propanol than ADH-2. These results do not support the hypothesis that 2-propanol acts as a selective factor favoring the Adh-2F allele.

  13. Investigation of enzyme activity by SERRS using poly-functionalised benzotriazole derivatives as enzyme substrates.

    PubMed

    Ingram, Andrew M; Stirling, Kirsten; Faulds, Karen; Moore, Barry D; Graham, Duncan

    2006-08-07

    New methods of measuring biologically relevant concentrations of enzymes are necessary to allow greater understanding of biological systems. We have previously shown that aryl azo benzotriazolyl alkyl esters can act as enzyme substrates, with the progress of the reaction being monitored using SERRS (see Nat. Biotechnol., 2004, 22, 1133, ref. ). This is a wholly novel analytical application of SERRS, and the low detection levels of the technique allow for an ultra-sensitive enzyme assay. Masked enzyme substrates are used that are invisible to SERRS until enzymatic hydrolysis. Turnover of the substrate by the enzyme leads to the release of the surface-seeking dye necessary for SERRS, and intense signals are produced. Here we report an improved synthesis of 2H-benzotriazolyl alkyl esters via nucleophilic substitution of a chloromethyl ester by benzotriazolyl azo dyes, giving up to a ten-fold increase on previously reported yields. Introduction of electron-withdrawing groups to the benzotriazole ring allows control over the SERRS properties of the compounds. This is of great significance in expanding the synthetic flexibility and subsequently the fundamental use of these compounds as ultra-sensitive and selective reporters of enzyme activity.

  14. 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.

  15. Identification of putative active site residues of ACAT enzymes.

    PubMed

    Das, Akash; Davis, Matthew A; Rudel, Lawrence L

    2008-08-01

    In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

  16. 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.

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

    PubMed

    Winiarczyk, Krystyna; Gębura, Joanna

    2016-05-01

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

  18. Enzyme activities in mitochondria isolated from ripening tomato fruit.

    PubMed

    Jeffery, D; Goodenough, P W; Weitzman, P D

    1986-09-01

    Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.

  19. 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

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

    PubMed

    Mingarro, I; Abad, C; Braco, L

    1995-04-11

    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.

  1. Chimeric enzymes with improved cellulase activities

    DOEpatents

    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. 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

  3. Enzyme activation through the utilization of intrinsic dianion binding energy.

    PubMed

    Amyes, T L; Malabanan, M M; Zhai, X; Reyes, A C; Richard, J P

    2016-11-29

    We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol., 43: , 219-410]. The large ~12 kcal/mol intrinsic substrate phosphodianion binding energy for reactions catalyzed by triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase and glycerol-3-phosphate dehydrogenase is divided into 4-6 kcal/mol binding energy that is expressed on the formation of the Michaelis complex in anchoring substrates to the respective enzyme, and 6-8 kcal/mol binding energy that is specifically expressed at the transition state in activating the respective enzymes for catalysis. A structure-based mechanism is described where the dianion binding energy drives a conformational change that activates these enzymes for catalysis. Phosphite dianion plays the active role of holding TIM in a high-energy closed active form, but acts as passive spectator in showing no effect on transition-state structure. The result of studies on mutant enzymes is presented, which support the proposal that the dianion-driven enzyme conformational change plays a role in enhancing the basicity of side chain of E167, the catalytic base, by clamping the base between a pair of hydrophobic side chains. The insight these results provide into the architecture of enzyme active sites and the development of strategies for the de novo design of protein catalysts is discussed.

  4. 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

  5. [Activity of antioxidant enzymes in patients with liver cirrhosis].

    PubMed

    Czeczot, Hanna; Scibior, Dorota; Skrzycki, Michał; Podsiad, Małgorzata

    2006-01-01

    The aim of our studies was the estimation of activities of antioxidant enzymes in patients with liver cirrhosis. We investigated activities of superoxide dismutases (CuZnSOD, MnSOD), catalase (CAT), selenium dependent GSH peroxidase (Se-GSH-Px), selenium independent GSH peroxidase (non-Se-GSH-Px), GSH-S-transferase (GST), GSH reductase (GSHR) and the level ofreduced gutathione (GSH) in cirrhotic and healthy liver tissues. The activities of CuZnSOD, MnSOD, CAT and GSH-dependent enzymes (except GSHR) were found to be lower in cirrhotic tissue compared to healthy liver. Those changes were associated with decrease of GSH level in cirrhotic tissue compared with control liver tissue. Our results show that antioxidant barrier in liver cirrhosis is impaired. It is associated with decrease of glutathione level and changes of activities of antioxidant enzymes (SOD, CAT, GSHPx, GST, GSHR) in liver cirrhosis compared with healthy liver.

  6. Fluorogenic Peptide Substrate for Quantification of Bacterial Enzyme Activities

    PubMed Central

    Al-Abdullah, Ismail H.; Bagramyan, Karine; Bilbao, Shiela; Qi, Meirigeng; Kalkum, Markus

    2017-01-01

    A novel peptide substrate (A G G P L G P P G P G G) was developed for quantifying the activities of bacterial enzymes using a highly sensitive Fluorescence Resonance Energy Transfer (FRET) based assay. The peptide substrate was cleaved by collagenase class I, II, Liberase MTF C/T, collagenase NB1, and thermolysin/neutral protease, which was significantly enhanced in the presence of CaCl2. However, the activities of these enzymes were significantly decreased in the presence of ZnSO4 or ZnCl2. Collagenase I, II, Liberase MTF C/T, thermolysin/neutral protease share similar cleavage sites, L↓G and P↓G. However, collagenase NB1 cleaves the peptide substrate at G↓P and P↓L, in addition to P↓G. The enzyme activity is pH dependent, within a range of 6.8 to 7.5, but was significantly diminished at pH 8.0. Interestingly, the peptide substrate was not cleaved by endogenous pancreatic protease such as trypsin, chymotrypsin, and elastase. In conclusion, the novel peptide substrate is collagenase, thermolysin/neutral protease specific and can be applied to quantify enzyme activities from different microbes. Furthermore, the assay can be used for fine-tuning reaction mixtures of various agents to enhance the overall activity of a cocktail of multiple enzymes and achieve optimal organ/tissue digestion, while protecting the integrity of the target cells. PMID:28287171

  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. 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

  9. ADH single nucleotide polymorphism associations with alcohol metabolism in vivo

    PubMed Central

    Birley, Andrew J.; James, Michael R.; Dickson, Peter A.; Montgomery, Grant W.; Heath, Andrew C.; Martin, Nicholas G.; Whitfield, John B.

    2009-01-01

    We have previously found that variation in alcohol metabolism in Europeans is linked to the chromosome 4q region containing the ADH gene family. We have now typed 103 single nucleotide polymorphisms (SNPs) across this region to test for allelic associations with variation in blood and breath alcohol concentrations after an alcohol challenge. In vivo alcohol metabolism was modelled with three parameters that identified the absorption and rise of alcohol concentration following ingestion, and the rate of elimination. Alleles of ADH7 SNPs were associated with the early stages of alcohol metabolism, with additional effects in the ADH1A, ADH1B and ADH4 regions. Rate of elimination was associated with SNPs in the intragenic region between ADH7 and ADH1C, and across ADH1C and ADH1B. SNPs affecting alcohol metabolism did not correspond to those reported to affect alcohol dependence or alcohol-related disease. The combined SNP associations with early- and late-stage metabolism only account for approximately 20% of the total genetic variance linked to the ADH region, and most of the variance for in vivo alcohol metabolism linked to this region is yet to be explained. PMID:19193628

  10. 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.

  11. Does diet influence salivary enzyme activities in elephant species?

    PubMed

    Boehlke, Carolin; Pötschke, Sandra; Behringer, Verena; Hannig, Christian; Zierau, Oliver

    2017-01-01

    Asian elephants (Elephas maximus) and African elephants (Loxodonta africana) are herbivore generalists; however, Asian elephants might ingest a higher proportion of grasses than Africans. Although some studies have investigated nutrition-specific morphological adaptations of the two species, broader studies on salivary enzymes in both elephant species are lacking. This study focuses on the comparison of salivary enzymes activity profiles in the two elephant species; these enzymes are relevant for protective and digestive functions in humans. We aimed to determine whether salivary amylase (sAA), lysozyme (sLYS), and peroxidase (sPOD) activities have changed in a species-specific pattern during evolutionary separation of the elephant genera. Saliva samples of 14 Asian and eight African elephants were collected in three German zoos. Results show that sAA and sLYS are salivary components of both elephant species in an active conformation. In contrast, little to no sPOD activity was determined in any elephant sample. Furthermore, sAA activity was significantly higher in Asian compared with African elephants. sLYS and sPOD showed no species-specific differences. The time of food provision until sample collection affected only sAA activity. In summary, the results suggest several possible factors modulating the activity of the mammal-typical enzymes, such as sAA, sLYS, and sPOD, e.g., nutrition and sampling procedure, which have to be considered when analyzing differences in saliva composition of animal species.

  12. 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.

  13. Evolutionary transitions in enzyme activity of ant fungus gardens.

    PubMed

    De Fine Licht, Henrik H; Schiøtt, Morten; Mueller, Ulrich G; Boomsma, Jacobus J

    2010-07-01

    Fungus-growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher-attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower-attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.

  14. 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

  15. Inducible trehalase enzyme activity of Morchella conica Persoon mycelium.

    PubMed

    Czövek, Pálma; Király, I

    2011-03-01

    Morchella conica Pers. strains of the study were isolated from fruit bodies collected in ash-mixed forests. At first, the strains were cultured on potato dextrose agar (PDA), then on modified Murashige and Skoog (MS) solid agar media. A normal-growing strain was chosen for the trehalase induction experiments. During the trehalase induction treatment, mycelia were grown in liquid culture containing different concentrations of trehalose. After the induction period of trehalase enzymes, physiological state of the mycelium and the oxidative stress were monitored in the vegetative mycelia by measuring the change of the malondialdehyde content, superoxide dismutase enzyme activity, the fresh and dry weight. The examined Morchella conica strain utilized the trehalose properly. The rising amount of the trehalose triggered the increase of the mycelial trehalase enzyme activity. Our results clearly proved that both neutral and acidic trehalase isoenzyme activity of the Morchella conica mycelium are inducible and are playing important role in the utilization of external trehalose.

  16. Alcohol dehydrogenase activities and ethanol tolerance in Anastrepha (Diptera, Tephritidae) fruit-fly species and their hybrids

    PubMed Central

    2009-01-01

    The ADH (alcohol dehydrogenase) system is one of the earliest known models of molecular evolution, and is still the most studied in Drosophila. Herein, we studied this model in the genus Anastrepha (Diptera, Tephritidae). Due to the remarkable advantages it presents, it is possible to cross species with different Adh genotypes and with different phenotype traits related to ethanol tolerance. The two species studied here each have a different number of Adh gene copies, whereby crosses generate polymorphisms in gene number and in composition of the genetic background. We measured certain traits related to ethanol metabolism and tolerance. ADH specific enzyme activity presented gene by environment interactions, and the larval protein content showed an additive pattern of inheritance, whilst ADH enzyme activity per larva presented a complex behavior that may be explained by epistatic effects. Regression models suggest that there are heritable factors acting on ethanol tolerance, which may be related to enzymatic activity of the ADHs and to larval mass, although a pronounced environmental effect on ethanol tolerance was also observed. By using these data, we speculated on the mechanisms of ethanol tolerance and its inheritance as well as of associated traits. PMID:21637665

  17. 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.

  18. Regulation of eNOS enzyme activity by posttranslational modification.

    PubMed

    Heiss, Elke H; Dirsch, Verena M

    2014-01-01

    The regulation of endothelial NO synthase (eNOS) employs multiple different cellular control mechanisms impinging on level and activity of the enzyme. This review aims at summarizing the current knowledge on the posttranslational modifications of eNOS, including acylation, nitrosylation, phosphorylation, acetylation, glycosylation and glutathionylation. Sites, mediators and impact on enzyme localization and activity of the single modifications will be discussed. Moreover, interdependence, cooperativity and competition between the different posttranslational modifications will be elaborated with special emphasis on the susceptibility of eNOS to metabolic cues.

  19. Genome-wide responses to carbonyl electrophiles in Bacillus subtilis: control of the thiol-dependent formaldehyde dehydrogenase AdhA and cysteine proteinase YraA by the MerR-family regulator YraB (AdhR).

    PubMed

    Nguyen, Thi Thu Huyen; Eiamphungporn, Warawan; Mäder, Ulrike; Liebeke, Manuel; Lalk, Michael; Hecker, Michael; Helmann, John D; Antelmann, Haike

    2009-02-01

    Quinones and alpha,beta-unsaturated carbonyls are naturally occurring electrophiles that target cysteine residues via thiol-(S)-alkylation. We analysed the global expression profile of Bacillus subtilis to the toxic carbonyls methylglyoxal (MG) and formaldehyde (FA). Both carbonyl compounds cause a stress response characteristic for thiol-reactive electrophiles as revealed by the induction of the Spx, CtsR, CymR, PerR, ArsR, CzrA, CsoR and SigmaD regulons. MG and FA triggered also a SOS response which indicates DNA damage. Protection against FA is mediated by both the hxlAB operon, encoding the ribulose monophosphate pathway for FA fixation, and a thiol-dependent formaldehyde dehydrogenase (AdhA) and DJ-1/PfpI-family cysteine proteinase (YraA). The adhA-yraA operon and the yraC gene, encoding a gamma-carboxymuconolactone decarboxylase, are positively regulated by the MerR-family regulator, YraB(AdhR). AdhR binds specifically to its target promoters which contain a 7-4-7 inverted repeat (CTTAAAG-N4-CTTTAAG) between the -35 and -10 elements. Activation of adhA-yraA transcription by AdhR requires the conserved Cys52 residue in vivo. We speculate that AdhR is redox-regulated via thiol-(S)-alkylation by aldehydes and that AdhA and YraA are specifically involved in reduction of aldehydes and degradation or repair of damaged thiol-containing proteins respectively.

  20. Structural insights into substrate specificity and solvent tolerance in alcohol dehydrogenase ADH-'A' from Rhodococcus ruber DSM 44541.

    PubMed

    Karabec, Martin; Łyskowski, Andrzej; Tauber, Katharina C; Steinkellner, Georg; Kroutil, Wolfgang; Grogan, Gideon; Gruber, Karl

    2010-09-14

    The structure of the alcohol dehydrogenase ADH-'A' from Rhodococcus ruber reveals possible reasons for its remarkable tolerance to organic co-solvents and suggests new directions for structure-informed mutagenesis to produce enzymes of altered substrate specificity or improved selectivity.

  1. Protective effects of the alcohol dehydrogenase-ADH1B*3 allele on attention and behavior problems in adolescents exposed to alcohol during pregnancy.

    PubMed

    Dodge, Neil C; Jacobson, Joseph L; Jacobson, Sandra W

    2014-01-01

    Alcohol dehydrogenase is a critical enzyme in the metabolism of alcohol. Expression of three alleles at the ADH1B locus results in enzymes that differ in turnover rate and affinity for alcohol. The ADH1B*3 allele, which appears to be unique to individuals of African descent, is associated with more rapid alcohol metabolism than the more prevalent ADH1B*1 allele. It has been previously demonstrated that the presence of at least one maternal ADH1B*3 allele confers a protective effect against alcohol teratogenicity in infants and children. This study was conducted to determine whether the presence of the ADH1B*3 allele in the mother or child continues to be protective in alcohol-exposed individuals during adolescence. 186 adolescents and 167 mothers participating in a 14-year follow-up of the Detroit Longitudinal Cohort were genotyped for ADH1B alleles. Behavioral reports were obtained from classroom teachers. Frequencies of the ADH1B*3 allele were 17.6% in the mothers and 21.0% in the adolescents, which are consistent with the 15-20% expected for African Americans. Prenatal alcohol exposure was associated with increased attention problems and externalizing behaviors in adolescents born to mothers with two ADH1B*1 alleles but not in those whose mothers had at least one ADH1B*3 allele. A similar pattern was seen in relation to the presence or absence of an ADH1B*3 allele in the adolescent, which may have reflected the presence/absence of the maternal variant. This study is the first to demonstrate that the protective effects of the maternal ADH1B*3 allele continue to be evident during adolescence. These persistent individual differences in vulnerability of offspring to the behavioral effects of fetal alcohol exposure are likely attributable to more rapid metabolism of alcohol that the ADH1B*3 variant confers on the mother, leading to a reduction of the peak blood alcohol concentration to which the fetus is exposed during each drinking episode.

  2. 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

  3. 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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. 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.

  6. [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.

  7. 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.

  8. The active site structure and mechanism of phosphoenolpyruvate utilizing enzymes

    SciTech Connect

    Cheng, K.C.

    1989-01-01

    Arginine specific reagents showed irreversible inhibition of avian liver mitochondrial phosphoenolpyruvate carboxykinase. Potent protection against modification was elicited by CO{sub 2} or CO{sub 2} in the presence of other substrates. Labeling of enzyme with (7-{sup 14}C) phenylglyoxal showed that 1 or 2 arginines are involved in CO{sub 2} binding and activation. Peptide map studies showed this active site arginine residues is located at position 289. Histidine specific reagents showed pseudo first order inhibition of avian mitochondrial phosphoenolpyruvate carboxykinase activity. The best protection against modification was elicited by IDP or IDP and Mn{sup +2}. One histidine residue is at or near the phosphoenolpyruvate binding site as demonstrated in the increased absorbance at 240 nm and proton relaxation rate studies. Circular dichroism studies reveal that enzyme structure was perturbed by diethylpyrocarbonate modification. Metal binding studies suggest that this enzyme has only one metal binding site. The putative binding sites from several GTP and phosphoenolpyruvate utilizing enzymes are observed in P-enolpyruvate carboxykinase from different species.

  9. 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

  10. 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.

  11. Functionally diverse biotin-dependent enzymes with oxaloacetate decarboxylase activity.

    PubMed

    Lietzan, Adam D; St Maurice, Martin

    2014-02-15

    Biotin-dependent enzymes catalyze carboxylation, decarboxylation and transcarboxylation reactions that participate in the primary metabolism of a wide range of organisms. In all cases, the overall reaction proceeds via two half reactions that take place in physically distinct active sites. In the first half-reaction, a carboxyl group is transferred to the 1-N' of a covalently tethered biotin cofactor. The tethered carboxybiotin intermediate subsequently translocates to a second active site where the carboxyl group is either transferred to an acceptor substrate or, in some bacteria and archaea, is decarboxylated to biotin and CO2 in order to power the export of sodium ions from the cytoplasm. A homologous carboxyltransferase domain is found in three enzymes that catalyze diverse overall reactions: carbon fixation by pyruvate carboxylase, decarboxylation and sodium transport by the biotin-dependent oxaloacetate decarboxylase complex, and transcarboxylation by transcarboxylase from Propionibacterium shermanii. Over the past several years, structural data have emerged which have greatly advanced the mechanistic description of these enzymes. This review assembles a uniform description of the carboxyltransferase domain structure and catalytic mechanism from recent studies of pyruvate carboxylase, oxaloacetate decarboxylase and transcarboxylase, three enzymes that utilize an analogous carboxyltransferase domain to catalyze the biotin-dependent decarboxylation of oxaloacetate.

  12. A generic rate law for surface-active enzymes.

    PubMed

    Kartal, Onder; Ebenhöh, Oliver

    2013-09-02

    Many biochemical reactions are confined to interfaces, such as membranes or cell walls. Despite their importance, no canonical rate laws describing the kinetics of surface-active enzymes exist. Combining the approach chosen by Michaelis and Menten 100 years ago with concepts from surface chemical physics, we here present an approach to derive generic rate laws of enzymatic processes at surfaces. We illustrate this by a simple reversible conversion on a surface to stress key differences to the classical case in solution. The available area function, a concept from surface physics which enters the rate law, covers different models of adsorption and presents a unifying perspective on saturation effects and competition between enzymes. A remarkable implication is the direct dependence of the rate of a given enzyme on all other enzymatic species able to bind at the surface. The generic approach highlights general principles of the kinetics of surface-active enzymes and allows to build consistent mathematical models of more complex pathways involving reactions at interfaces.

  13. 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

  14. Antioxidant enzyme activities in maize plants colonized with Piriformospora indica.

    PubMed

    Kumar, Manoj; Yadav, Vikas; Tuteja, Narendra; Johri, Atul Kumar

    2009-03-01

    The bioprotection performance of Piriformospora indica against the root parasite Fusarium verticillioides was studied. We found that maize plants first grown with F. verticillioides and at day 10 inoculated with P. indica showed improvements in biomass, and root length and number as compared with plants grown with F. verticillioides alone. To validate our finding that inoculation with P. indica suppresses colonization by F. verticillioides, we performed PCR analyses using P. indica- and F. verticillioides-specific primers. Our results showed that inoculation with P. indica suppresses further colonization by F. verticillioides. We hypothesized that as the colonization by P. indica increases, the presence of/colonization by F. verticillioides decreases. In roots, catalase (CAT), glutathione reductase (GR), glutathione S-transferase (GST) and superoxide dismutase (SOD) activities were found to be higher in F. verticillioides-colonized plants than in non-colonized plants. Increased activity of antioxidant enzymes minimizes the chances of oxidative burst (excessive production of reactive oxygen species), and therefore F. verticillioides might be protected from the oxidative defence system during colonization. We also observed decreased antioxidant enzyme activities in plants first inoculated with F. verticillioides and at day 10 inoculated with P. indica as compared with plants inoculated with F. verticillioides alone. These decreased antioxidant enzyme activities due to the presence of P. indica help the plant to overcome the disease load of F. verticillioides. We propose that P. indica can be used as a bioprotection agent against the root parasite F. verticillioides.

  15. 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.

  16. 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.

  17. Activity-Based Screening of Metagenomic Libraries for Hydrogenase Enzymes.

    PubMed

    Adam, Nicole; Perner, Mirjam

    2017-01-01

    Here we outline how to identify hydrogenase enzymes from metagenomic libraries through an activity-based screening approach. A metagenomic fosmid library is constructed in E. coli and the fosmids are transferred into a hydrogenase deletion mutant of Shewanella oneidensis (ΔhyaB) via triparental mating. If a fosmid exhibits hydrogen uptake activity, S. oneidensis' phenotype is restored and hydrogenase activity is indicated by a color change of the medium from yellow to colorless. This new method enables screening of 48 metagenomic fosmid clones in parallel.

  18. Digestive enzymes activity in larvae of Cameraria ohridella (Lepidoptera: Gracillariidae).

    PubMed

    Stygar, Dominika; Dolezych, Bogdan; Nakonieczny, Mirosław; Migula, Pawel; Michalczyk, Katarzyna; Zaak, Maria

    2010-10-01

    This article presents the activity of carbohydratases and proteases in the midgut of Cameraria ohridella larvae--an oligophagous pest whose preferred feeding is horse chestnuts leaves. Optimal media pH of the assayed enzymes were similar to those of other Lepidopterans. Relatively high amylase activity, as well as maltase and sucrase activities, indicates that starch and sucrose are the main digested saccharides. Trehalase activity was similar to that described in other Lepidopterans. Activities of glycosidases were significantly lower than those of disaccharidases what suggests that neither cellulose nor glycosides are important for C. ohridella. Trypsin is the main endoprotease of this pest. Like in other leaf-eaters carboxypeptidase activity was higher than that of aminopeptidase. The activity of the majority of examined enzymes increased in the following successive pest generations, which could be explained by the decreased nutritional value of older leaves. Probably this phenomenon in hydrolases activity in Cameraria is a nonspecific mechanism present at this stage of co-evolution of the horse chestnut and its pest.

  19. Hemodynamic and ADH responses to central blood volume shifts in cardiac-denervated humans

    NASA Technical Reports Server (NTRS)

    Convertino, V. A.; Thompson, C. A.; Benjamin, B. A.; Keil, L. C.; Savin, W. M.; Gordon, E. P.; Haskell, W. L.; Schroeder, J. S.; Sandler, H.

    1990-01-01

    Hemodynamic responses and antidiuretic hormone (ADH) were measured during body position changes designed to induce blood volume shifts in ten cardiac transplant recipients to assess the contribution of cardiac and vascular volume receptors in the control of ADH secretion. Each subject underwent 15 min of a control period in the seated posture, then assumed a lying posture for 30 min at 6 deg head down tilt (HDT) followed by 20 min of seated recovery. Venous blood samples and cardiac dimensions (echocardiography) were taken at 0 and 15 min before HDT, 5, 15, and 30 min of HDT, and 5, 15, and 30 min of seated recovery. Blood samples were analyzed for hematocrit, plasma osmolality, plasma renin activity (PRA), and ADH. Resting plasma volume (PV) was measured by Evans blue dye and percent changes in PV during posture changes were calculated from changes in hematocrit. Heart rate (HR) and blood pressure (BP) were recorded every 2 min. Results indicate that cardiac volume receptors are not the only mechanism for the control of ADH release during acute blood volume shifts in man.

  20. Evaluation of the Saccharomyces cerevisiae ADH2 promoter for protein synthesis.

    PubMed

    Lee, K Michael; DaSilva, Nancy A

    2005-04-30

    The Saccharomyces cerevisiae ADH2 promoter (P(ADH2)) is repressed several hundred-fold in the presence of glucose; transcription is initiated once the glucose in the medium is exhausted. The promoter can thus be utilized for effective regulation of recombinant gene expression in S. cerevisiae without the addition of an inducer. To evaluate this promoter in the absence of plasmid copy number and stability variations, the P(ADH2)-lacZ cassette was integrated into the yeast chromosomes. The effects of medium composition, glucose concentration and cultivation time on promoter derepression and expression level were investigated. Maximum protein activity was obtained after 48 h of growth in complex YPD medium containing 1% glucose. The widely used S. cerevisiae GAL1 and CUP1 promoters both require the addition of an inducer [galactose and copper(II) ion, respectively] before regulated genes will be expressed. The strengths of these three different promoters were compared for cells containing one copy of an integrated lacZ gene under their control. The ADH2 promoter was superior for all induction strategies investigated.

  1. [Lysosomal enzyme activity in white blood cells in leukemias].

    PubMed

    Rybakova, L P; Kharchenko, M F

    1996-01-01

    Total enzyme activity of acidic hydrolases and total neutral proteinase were compared in the post-nuclear fraction of leukocytes from healthy subjects and leukemia patients. The levels of acidic phosphotase and neutral proteinase in lymphoid cells of healthy donors were 11 and 7 times lower than those in myeloid cells, respectively. Patients suffering chronic myeloid leukemia revealed enhanced levels of beta-glucuronidase and neutral proteinases whereas B-chronic lymphoid leukemia involved acidic hydrolase concentrations lower than normal. As chronic myeloid leukemia advanced, neutral proteinase activity dropped dramatically (2.5 times); an aggressive course of B-chronic lymphoid leukemia was accompanied by a 3-fold decrease in acidic hydrolase level. The results may be used as indirect evidence of differences in the role of lysosomal enzymes in the mechanism of protein processing involved in myeloid and lymphoid proliferative pathologies.

  2. Activity of anandamide (AEA) metabolic enzymes in rat placental bed.

    PubMed

    Fonseca, B M; Battista, N; Correia-da-Silva, G; Rapino, C; Maccarrone, M; Teixeira, N A

    2014-11-01

    Endocannabinoids are endogenous lipid mediators, with anandamide (AEA) being the first member identified. It is now widely accepted that AEA influences early pregnancy events and its levels, which primarily depend on its synthesis by an N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and degradation by a fatty acid amide hydrolase (FAAH), must be tightly regulated. Previous studies demonstrated that AEA levels require in situ regulation of these respective metabolic enzymes, and thus, any disturbance in AEA levels may impact maternal remodeling processes occurring during placental development. In this study, the activities of the AEA-metabolic enzymes that result in the establishment of proper local AEA levels during rat gestation were examined. Here, we demonstrate that during placentation NAPE-PLD and FAAH activities change in a temporal manner. Our findings suggest that NAPE-PLD and FAAH create the appropriate AEA levels required for tissue remodeling in the placental bed, a process essential to pregnancy maintenance.

  3. 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.

  4. 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.

  5. 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.

  6. Evidence for a role for AtMYB2 in the induction of the Arabidopsis alcohol dehydrogenase gene (ADH1) by low oxygen.

    PubMed Central

    Hoeren, F U; Dolferus, R; Wu, Y; Peacock, W J; Dennis, E S

    1998-01-01

    The transcription factor AtMYB2 binds to two sequence motifs in the promoter of the Arabidopsis ADH1 gene. The binding to the GT-motif (5'-TGGTTT-3') is essential for induction of ADH1 by low oxygen, while binding to the second motif, MBS-2, is not essential for induction. We show that AtMYB2 is induced by hypoxia with kinetics compatible with a role in the regulation of ADH1. Like ADH1, AtMYB2 has root-limited expression. When driven by a constitutive promoter, AtMYB2 is able to transactivate ADH1 expression in transient assays in both Arabidopsis and Nicotiana plumbaginifolia protoplasts, and in particle bombardment of Pisum sativum leaves. Mutation of the GT-motif abolished binding of AtMYB2 and caused loss of activity of the ADH1 promoter in both transient assays and transgenic Arabidopsis plants. These results are consistent with AtMYB2 being a key regulatory factor in the induction of the ADH1 promoter by low oxygen. PMID:9611167

  7. 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

    2015-09-08

    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

  8. 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.

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

    PubMed

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

    2009-02-01

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

  10. Development of a synchronous enzyme-reaction system for a highly sensitive enzyme immunoassay.

    PubMed

    Inouye, Kuniyo; Ueno, Iori; Yokoyama, Shin-ichi; Sakaki, Toshiyuki

    2002-01-01

    A synchronous enzyme-reaction system using water-soluble formazan and a non-enzymatic electron mediator was developed and applied to an enzyme immunoassay (EIA). The reaction system consists of four steps: (I) dephosphorylation of NADP(+) to produce NAD(+) by alkaline phosphatase (ALP), (II) reduction of NAD(+) to produce NADH with oxidation of ethanol to yield acetaldehyde by alcohol dehydrogenase (ADH), (III) reduction of water-soluble tetrazolium salt (WST-1) to produce formazan by NADH via 1-methoxy-5-methyl-phenazinium methyl sulfate (PMS), and (IV) re-reduction of NAD(+) to produce NADH by ADH. During each cycle, one molecule of tetrazolium is converted to one molecule of formazan. The concentration of formazan during the reaction was given by second-order polynomials of the reaction time. Kinetic studies strongly suggested that the synchronous enzyme-reaction system had the potential to detect an analyte at the attomole level in EIA. On the basis of the kinetic studies, optimal conditions for EIA incorporating the synchronous system were examined. NADP(+) was purified thoroughly to remove minor traces of NAD(+) in the preparation, and an ADH preparation contaminated with the lowest level of ALP activity was used. When the synchronous system was applied to a sandwich-type EIA for human C-reactive protein, the protein was detected with a sensitivity of 50 attomole per well of a micro-titer plate (0.1 ml) in a 1-h reaction. In addition, EIA with water-soluble formazan showed a more quantitative and sensitive result than that with insoluble formazan. These findings indicated that the (WST-1)-PMS system introduced in this study has a great potential for highly sensitive enzyme immunoassay.

  11. 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.

  12. An Extended Polyanion Activation Surface in Insulin Degrading Enzyme

    PubMed Central

    Song, Eun Suk; Ozbil, Mehmet; Zhang, Tingting; Sheetz, Michael; Lee, David; Tran, Danny; Li, Sheng; Prabhakar, Rajeev; Hersh, Louis B.; Rodgers, David W.

    2015-01-01

    Insulin degrading enzyme (IDE) is believed to be the major enzyme that metabolizes insulin and has been implicated in the degradation of a number of other bioactive peptides, including amyloid beta peptide (Aβ), glucagon, amylin, and atrial natriuretic peptide. IDE is activated toward some substrates by both peptides and polyanions/anions, possibly representing an important control mechanism and a potential therapeutic target. A binding site for the polyanion ATP has previously been defined crystallographically, but mutagenesis studies suggest that other polyanion binding modes likely exist on the same extended surface that forms one wall of the substrate-binding chamber. Here we use a computational approach to define three potential ATP binding sites and mutagenesis and kinetic studies to confirm the relevance of these sites. Mutations were made at four positively charged residues (Arg 429, Arg 431, Arg 847, Lys 898) within the polyanion-binding region, converting them to polar or hydrophobic residues. We find that mutations in all three ATP binding sites strongly decrease the degree of activation by ATP and can lower basal activity and cooperativity. Computational analysis suggests conformational changes that result from polyanion binding as well as from mutating residues involved in polyanion binding. These findings indicate the presence of multiple polyanion binding modes and suggest the anion-binding surface plays an important conformational role in controlling IDE activity. PMID:26186535

  13. 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.

  14. [Changes in kidney function and the cortisol and ADH levels after peritoneal dialysis with 5% glucose in dogs].

    PubMed

    Nachev, N; Bratanova, Ts; Pavlov, D

    1975-01-01

    The authors made peritoneal dialisis with 5% of glucose (7 ml/kg of body weight) in 11 dogs under the conditions of an acute experiments. They examined cortiosl and ADH activity, hematocrite and plasma protein in the samples of blood, obtained on the 20th and 50th minute. ADH was titrated biologicaly by a new method, proposed by Nacev. The results were compared with the changes in the circulatory and renal indices, obtained at the same procedure in the preceding investigations. There was an increase in the cortisol and ADH activity, which could be explained by the total hypovolemia, induced by peritoneal dialisis. The increase of the cortisol level is described as a separate link in a more complex mechanism, assuring metabolic homeostasis.

  15. Is ADH1C genotype relevant for the cardioprotective effect of alcohol?

    PubMed

    Høiseth, Gudrun; Magnus, Per; Knudsen, Gun Peggy; Jansen, Mona Dverdal; Næss, Oyvind; Tambs, Kristian; Mørland, Jørg

    2013-03-01

    The cardioprotective effect of ethanol has been suggested to be linked to one of the ethanol metabolizing enzymes (ADH1C), which constitutes a high V(max) and a low V(max) variant. This has been demonstrated in some studies, while others have not been able to replicate the findings. The aim of the present study was to investigate the relation between the different ADH1C genotypes, death from coronary heart disease (CHD) and alcohol in a material larger than the previously published studies. Eight hundred CHD deaths as well as 1303 controls were genotyped for the high V(max) (γ1) and the low V(max) (γ2) ADH1C variant. Information of alcohol use was available for all subjects. Multiple logistic regression analyses was used to study if the decreased risk of death from CHD in alcohol consuming subjects was more pronounced in subjects homozygous for the γ2 allele (γ2γ2 subjects) compared to γ1γ1 and γ1γ2 subjects. The odds ratio (OR) for death from CHD in alcohol consumers compared to abstainers was similar in the genotype groups, i.e., 0.62 (95% CI: 0.43-0.88) in γ1γ1 subjects and 0.62 (95% CI: 0.42-0.91) in γ2γ2 subjects. Also when stratifying the results by gender and when dividing alcohol consumers into different alcohol consumption groups, there was no difference in the OR between the different genotype groups. This study, which included the largest study group published so far, failed to find any link between the ADH1C genotype and the cardioprotective effects of alcohol.

  16. Polymorphisms in Alcohol Metabolism Genes ADH1B and ALDH2, Alcohol Consumption and Colorectal Cancer

    PubMed Central

    Crous-Bou, Marta; Rennert, Gad; Cuadras, Daniel; Salazar, Ramon; Cordero, David; Saltz Rennert, Hedy; Lejbkowicz, Flavio; Kopelovich, Levy; Monroe Lipkin, Steven; Bernard Gruber, Stephen; Moreno, Victor

    2013-01-01

    Background Colorectal cancer (CRC) is a leading cause of cancer death worldwide. Epidemiological risk factors for CRC included alcohol intake, which is mainly metabolized to acetaldehyde by alcohol dehydrogenase and further oxidized to acetate by aldehyde dehydrogenase; consequently, the role of genes in the alcohol metabolism pathways is of particular interest. The aim of this study is to analyze the association between SNPs in ADH1B and ALDH2 genes and CRC risk, and also the main effect of alcohol consumption on CRC risk in the study population. Methodology/Principal Findings SNPs from ADH1B and ALDH2 genes, included in alcohol metabolism pathway, were genotyped in 1694 CRC cases and 1851 matched controls from the Molecular Epidemiology of Colorectal Cancer study. Information on clinicopathological characteristics, lifestyle and dietary habits were also obtained. Logistic regression and association analysis were conducted. A positive association between alcohol consumption and CRC risk was observed in male participants from the Molecular Epidemiology of Colorectal Cancer study (MECC) study (OR = 1.47; 95%CI = 1.18-1.81). Moreover, the SNPs rs1229984 in ADH1B gene was found to be associated with CRC risk: under the recessive model, the OR was 1.75 for A/A genotype (95%CI = 1.21-2.52; p-value = 0.0025). A path analysis based on structural equation modeling showed a direct effect of ADH1B gene polymorphisms on colorectal carcinogenesis and also an indirect effect mediated through alcohol consumption. Conclusions/Significance Genetic polymorphisms in the alcohol metabolism pathways have a potential role in colorectal carcinogenesis, probably due to the differences in the ethanol metabolism and acetaldehyde oxidation of these enzyme variants. PMID:24282520

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

    PubMed Central

    Mande, Sharmila S.

    2015-01-01

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

  18. 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.

  19. Role of cardiac volume receptors in the control of ADH release during acute simulated weightlessness in man

    NASA Technical Reports Server (NTRS)

    Convertino, V. A.; Benjamin, B. A.; Keil, L. C.; Sandler, H.

    1984-01-01

    Hemodynamic responses and antidiuretic hormone (ADH) were measured during body position changes, designed to induce central blood volume shifts in ten cardiac and one heart-lung transplant recipients, to assess the contribution of cardiac volume receptors in the control of ADH release during the initial acute phase of exposure to weightlessness. Each subject underwent 15 min of a sitting-control period (C) followed by 30 min of 6 deg headdown tilt (T) and 30 min of resumed sitting (S). Venous blood samples and cardiac dimensions were taken at 0 and 15 min of C; 5, 15, and 30 min of T; and 5, 15, and 30 min of S. Blood samples were analyzed for hematocrit, plasma osmolality, plasma renin activity (PRA), and ADH. Heart rate and blood pressure were recorded every two min. Plasma osmolality was not altered by posture changes. Mean left ventricular end-diastolic volume increased (P less than 0.05) from 90 ml in C to 106 ml in T and returned to 87 ml in S. Plasma ADH was reduced by 20 percent (P less than 0.05) with T, and returned to control levels with S. These responses were similar in six normal cardiac-innervated control subjects. These data may suggest that cardiac volume receptors are not the primary mechanism for the control of ADH release during acute central volume shifts in man.

  20. Protein stability and enzyme activity at extreme biological temperatures.

    PubMed

    Feller, Georges

    2010-08-18

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

  1. Serum angiotensin converting enzyme activity in chronic beryllium disease.

    PubMed

    Newman, L S; Orton, R; Kreiss, K

    1992-07-01

    Serum angiotensin converting enzyme (SACE) activity is used as a marker of sarcoidosis activity and severity, but in chronic beryllium disease (CBD) the studies of SACE give conflicting results. We examined SACE activity in 23 CBD patients, five patients with beryllium sensitization, and 25 beryllium-exposed control subjects. CBD patients underwent complete clinical evaluation, including physical examination, pulmonary function testing, exercise physiology testing, chest radiography, and bronchoscopy with bronchoalveolar lavage and biopsy. CBD SACE activity was systematically compared with these clinical markers of disease severity. Of CBD patients, 22% had elevated SACE activity. The test did not discriminate CBD patients from those in the beryllium-sensitized or beryllium-exposed groups. However, SACE activity in CBD correlated with the extent of pulmonary granulomatous inflammation as reflected by the symptom of breathlessness, the number of white cells in bronchoalveolar lavage (r = 0.44), the number of lavage lymphocytes (r = 0.58), the lavage lymphocyte percentage (r = 0.55), and the profusion of small opacities on chest radiograph (r = 0.41). The test-retest reliability of the assay was high (r = 0.84), as was the agreement between fresh and -70 degrees C frozen sera (r = 0.93). We conclude that SACE activity levels may reflect the extent of pulmonary granulomatous inflammation in CBD but that the test does not help discriminate disease from nondisease.

  2. 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.

  3. 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.

  4. 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.

  5. Characterization and chillproofing activity of two enzymes from Streptomyces species.

    PubMed

    Etok, C A; Eka, O U

    1996-01-01

    Two enzymes, amylase and protease of Streptomyces species were purified by a combination of ion exchange chromatography and gel filtration and characterized. The amylase had an exoaction on starch yielding maltose as a major end product and was identified as beta-amylase. The purified amylase had a molecular weight of 48,000 and was maximally active at 35 degrees C and at pH 6.0. On the other hand, protease had a molecular weight of 21,000 and was most active at pH 10.0 and at a temperature of 30 degrees C. The Km or MICHAELIS constant of amylase for maize starch was 0.333 mg/ml while that of protease for casein was 2.5 mg/ml. The feasibility of using the purified protease for various industrial application especially in the chillproofing of beer is discussed.

  6. 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.

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

    PubMed

    Chu, Wen-Ting; Wang, Jin

    2016-06-14

    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.

  8. 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.

  9. 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

  10. Comparative insecticide susceptibility and detoxification enzyme activities among pestiferous blattodea.

    PubMed

    Valles, S M; Koehler, P G; Brenner, R J

    1999-11-01

    Topical bioassays using propoxur, chlorpyrifos, and lambda-cyhalothrin were conducted on eight cockroach species. Based on lethal dose values, the relative toxicities of the insecticide classes were generally pyrethroid > carbamate > organophosphorous. Lambda-Cyhalothrin and propoxur were more toxic toward the Blattidae as compared with the Blattellidae. The order of lambda-cyhalothrin toxicity was Periplaneta americana > Periplaneta brunnea = Periplaneta australasiae = Periplaneta fuliginosa = Blatta orientalis > Blattella asahinai = Blattella germanica > Blattella vaga. The order of propoxur toxicity was B. orientalis > P. americana > P. brunnea = P. australasiae > B. asahinai > P. fuliginosa = B. germanica > B. vaga. The order of chlorpyrifos toxicity was P. americana > B. asahinai = B. vaga > B. orientalis = P. australasiae = P. brunnea > B. germanica = P. fuliginosa. Detoxification enzyme activities for each species also were measured and compared with insecticide toxicity. Propoxur LD50 was significantly (P = 0.01; r = 0.81) correlated with glutathione S-transferase activity. Lambda-Cyhalothrin LD50 correlated with methoxyresorufin O-demethylase activity (P = 0.01; r = 0.81), carboxylesterase activity (P = 0.03; r = - 0.75), general esterase activity (P = 0.02; r = - 0.79), and cockroach weight (P = 0.01; r = -0.95).

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

    PubMed Central

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

    2014-01-01

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

  12. Sequence variation of alcohol dehydrogenase (Adh) paralogs in cactophilic Drosophila.

    PubMed Central

    Matzkin, Luciano M; Eanes, Walter F

    2003-01-01

    This study focuses on the population genetics of alcohol dehydrogenase (Adh) in cactophilic Drosophila. Drosophila mojavensis and D. arizonae utilize cactus hosts, and each host contains a characteristic mixture of alcohol compounds. In these Drosophila species there are two functional Adh loci, an adult form (Adh-2) and a larval and ovarian form (Adh-1). Overall, the greater level of variation segregating in D. arizonae than in D. mojavensis suggests a larger population size for D. arizonae. There are markedly different patterns of variation between the paralogs across both species. A 16-bp intron haplotype segregates in both species at Adh-2, apparently the product of an ancient gene conversion event between the paralogs, which suggests that there is selection for the maintenance of the intron structure possibly for the maintenance of pre-mRNA structure. We observe a pattern of variation consistent with adaptive protein evolution in the D. mojavensis lineage at Adh-1, suggesting that the cactus host shift that occurred in the divergence of D. mojavensis from D. arizonae had an effect on the evolution of the larval expressed paralog. Contrary to previous work we estimate a recent time for both the divergence of D. mojavensis and D. arizonae (2.4 +/- 0.7 MY) and the age of the gene duplication (3.95 +/- 0.45 MY). PMID:12586706

  13. Study on optimization of process parameters for enhancing the multi-hydrolytic enzyme activity in garbage enzyme produced from preconsumer organic waste.

    PubMed

    Arun, C; Sivashanmugam, P

    2017-02-01

    The garbage enzymes produced from preconsumer organic waste containing multi hydrolytic enzyme activity which helps to solubilize the waste activated sludge. The continuous production of garbage enzyme and its scaling up process need a globe optimized condition. In present study the effect of fruit peel composition and sonication time on enzyme activity were investigated. Garbage enzyme produced from 6g pineapple peels: 4g citrus peels pre-treated with ultrasound for 20min shows higher hydrolytic enzymes activity. Simultaneously statistical optimization tools were used to model garbage enzyme production with higher activity of amylase, lipase and protease. The maximum activity of amylase, lipase and protease were predicted to be 56.409, 44.039, 74.990U/ml respectively at optimal conditions (pH (6), temperature (37°C), agitation (218 RPM) and fermentation duration (3days)). These optimized conditions can be successfully used for large scale production of garbage enzyme with higher hydrolytic enzyme activity.

  14. 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.

  15. 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

  16. 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.

  17. Endothelin-converting enzyme 2 differentially regulates opioid receptor activity

    PubMed Central

    Gupta, A; Fujita, W; Gomes, I; Bobeck, E; Devi, L A

    2015-01-01

    BACKGROUND AND PURPOSE Opioid receptor function is modulated by post-activation events such as receptor endocytosis, recycling and/or degradation. While it is generally understood that the peptide ligand gets co-endocytosed with the receptor, relatively few studies have investigated the role of the endocytosed peptide and peptide processing enzymes in regulating receptor function. In this study, we focused on endothelin-converting enzyme 2 (ECE2), a member of the neprilysin family of metallopeptidases that exhibits an acidic pH optimum, localizes to an intracellular compartment and selectively processes neuropeptides including opioid peptides in vitro, and examined its role in modulating μ receptor recycling and resensitization. EXPERIMENTAL APPROACH The effect of ECE2 inhibition on hydrolysis of the endocytosed peptide was examined using thin-layer chromatography and on μ opioid receptor trafficking using either elisa or microscopy. The effect of ECE2 inhibition on receptor signalling was measured using a cAMP assay and, in vivo, on antinociception induced by intrathecally administered opioids by the tail-flick assay. KEY RESULTS The highly selective ECE2 inhibitor, S136492, significantly impaired μ receptor recycling and signalling by only those ligands that are ECE2 substrates and this was seen both in heterologous cells and in cells endogenously co-expressing μ receptors with ECE2. We also found that ECE2 inhibition attenuated antinociception mediated only by opioid peptides that are ECE2 substrates. CONCLUSIONS AND IMPLICATIONS These results suggest that ECE2, by selectively processing endogenous opioid peptides in the endocytic compartment, plays a role in modulating opioid receptor activity. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24990314

  18. Cerebrospinal fluid enzymes in acute brain injury. 1. Dynamics of changes in CSF enzyme activity after acute experimental brain injury.

    PubMed Central

    Maas, A I

    1977-01-01

    Changes in CSF enzyme activity were studied after brain trauma for their prognostic value. Raised values of CPK and HBDH were demonstrated in the CSF of patients with severe brain injuries. Standardised cold lesions of the brain were induced in cats. The activities of the enzymes CPK, HBDH, LDH, GOT, GPT, and pseudocholinesterase were studied at half hour intervals in the cerebrospinal fluid and at hourly intervals in the serum. A statistically highly significant increase of all enzymes studied developed in the CSF. The greatest changes occurred within four hours of freezing. Large increases could occur in half an hour. Isoenzyme studies demonstrated that CPK and LDH were of cerebral origin. No consistently significant changes could be shown in the serum enzyme activity. It is concluded that after brain injuries, enzymes are released into the extracellular fluid of the brain and transported to the CSF. The limited value of a single enzyme estimation is emphasised. The results described seem to provide indirect evidence for transependymal flow of extracellular fluid in brain oedema. Images PMID:915509

  19. 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

  20. 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...

  1. Influence of dietary nutritional composition on caterpillar salivary enzyme activity.

    PubMed

    Babic, Branislav; Poisson, Alexandre; Darwish, Shireef; Lacasse, Jean; Merkx-Jacques, Magali; Despland, Emma; Bede, Jacqueline C

    2008-01-01

    Caterpillars are faced with nutritional challenges when feeding on plants. In addition to harmful secondary metabolites and protein- and water-limitations, tissues may be carbohydrate-rich which may attenuate optimal caterpillar performance. Therefore, caterpillars have multiple strategies to cope with surplus carbohydrates. In this study, we raise the possibility of a pre-ingestive mechanism to metabolically deal with excess dietary sugars. Many Noctuid caterpillars secrete the labial salivary enzyme glucose oxidase (GOX), which oxidizes glucose to hydrogen peroxide and gluconate, a nutritionally unavailable carbohydrate to the insect. Beet armyworm, Spodoptera exigua, larvae were restricted to diets varying in protein to digestible carbohydrate (P:C) ratio (42p:21c; 33p:30c; 21p:42c) and total nutrient concentration (42% and 63%). High mortality and longer developmental time were observed when caterpillars were reared on the C-biased, P-poor diet (21p:42c). As the carbohydrate content of the diet increased, caterpillars egested excess glucose and a diet-dependent difference in assimilated carbohydrates and pupal biomass was not observed, even though caterpillars restricted to the C-biased diet (21p:42c) accumulated greater pupal lipid reserves. Larval labial salivary GOX activity was also diet-dependent and gluconate, the product of GOX activity, was detected in the frass. Unexpectedly, GOX activity was strongly and positively correlated with dietary protein content.

  2. Flavonoid inhibition of aromatase enzyme activity in human preadipocytes.

    PubMed

    Campbell, D R; Kurzer, M S

    1993-09-01

    Eleven flavonoid compounds were compared with aminoglutethimide (AG), a pharmaceutical aromatase inhibitor, for their abilities to inhibit aromatase enzyme activity in a human preadipocyte cell culture system. Flavonoids exerting no effect on aromatase activity were catechin, daidzein, equol, genistein, beta-naphthoflavone (BNF), quercetin and rutin. The synthetic flavonoid, alpha-naphthoflavone (ANF), was the most potent aromatase inhibitor, with an I50 value of 0.5 microM. Three naturally-occurring flavonoids, chrysin, flavone, and genistein 4'-methyl ether (Biochanin A) showed I50 values of 4.6, 68, and 113 microM, respectively, while AG showed an I50 value of 7.4 microM. Kinetic analyses showed that both AG and the flavonoids acted as competitive inhibitors of aromatase. The Ki values, indicating the effectiveness of inhibition, were 0.2, 2.4, 2.4, 22, and 49 microM, for ANF, AG, chrysin, flavone, and Biochanin A, respectively. Chrysin, the most potent of the naturally-occurring flavonoids, was similar in potency and effectiveness to AG, a pharmaceutical aromatase inhibitor used clinically in cases of estrogen-dependent carcinoma. These data suggest that flavonoid inhibition of peripheral aromatase activity may contribute to the observed cancer-preventive hormonal effects of plant-based diets.

  3. 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

  4. Phosphate-Modified Nucleotides for Monitoring Enzyme Activity.

    PubMed

    Ermert, Susanne; Marx, Andreas; Hacker, Stephan M

    2017-04-01

    Nucleotides modified at the terminal phosphate position have been proven to be interesting entities to study the activity of a variety of different protein classes. In this chapter, we present various types of modifications that were attached as reporter molecules to the phosphate chain of nucleotides and briefly describe the chemical reactions that are frequently used to synthesize them. Furthermore, we discuss a variety of applications of these molecules. Kinase activity, for instance, was studied by transfer of a phosphate modified with a reporter group to the target proteins. This allows not only studying the activity of kinases, but also identifying their target proteins. Moreover, kinases can also be directly labeled with a reporter at a conserved lysine using acyl-phosphate probes. Another important application for phosphate-modified nucleotides is the study of RNA and DNA polymerases. In this context, single-molecule sequencing is made possible using detection in zero-mode waveguides, nanopores or by a Förster resonance energy transfer (FRET)-based mechanism between the polymerase and a fluorophore-labeled nucleotide. Additionally, fluorogenic nucleotides that utilize an intramolecular interaction between a fluorophore and the nucleobase or an intramolecular FRET effect have been successfully developed to study a variety of different enzymes. Finally, also some novel techniques applying electron paramagnetic resonance (EPR)-based detection of nucleotide cleavage or the detection of the cleavage of fluorophosphates are discussed. Taken together, nucleotides modified at the terminal phosphate position have been applied to study the activity of a large diversity of proteins and are valuable tools to enhance the knowledge of biological systems.

  5. Effects of ADH2 overexpression in Saccharomyces bayanus during alcoholic fermentation.

    PubMed

    Maestre, Oscar; García-Martínez, Teresa; Peinado, Rafael A; Mauricio, Juan C

    2008-02-01

    The effect of overexpression of the gene ADH2 on metabolic and biological activity in Saccharomyces bayanus V5 during alcoholic fermentation has been evaluated. This gene is known to encode alcohol dehydrogenase II (ADH II). During the biological aging of sherry wines, where yeasts have to grow on ethanol owing to the absence of glucose, this isoenzyme plays a prominent role by converting the ethanol into acetaldehyde and producing NADH in the process. Overexpression of the gene ADH2 during alcoholic fermentation has no effect on the proteomic profile or the net production of some metabolites associated with glycolysis and alcoholic fermentation such as ethanol, acetaldehyde, and glycerol. However, it affects indirectly glucose and ammonium uptakes, cell growth, and intracellular redox potential, which lead to an altered metabolome. The increased contents in acetoin, acetic acid, and L-proline present in the fermentation medium under these conditions can be ascribed to detoxification by removal of excess acetaldehyde and the need to restore and maintain the intracellular redox potential balance.

  6. 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.

  7. Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial isozyme of alcohol dehydrogenase in Saccharomyces cerevisiae.

    PubMed Central

    Young, E T; Pilgrim, D

    1985-01-01

    The Saccharomyces cerevisiae nuclear gene, ADH3, that encodes the mitochondrial alcohol dehydrogenase isozyme ADH III was cloned by virtue of its nucleotide homology to ADH1 and ADH2. Both chromosomal and plasmid-encoded ADH III isozymes were repressed by glucose and migrated heterogeneously on nondenaturing gels. Nucleotide sequence analysis indicated 73 and 74% identity for ADH3 with ADH1 and ADH2, respectively. The amino acid identity between the predicted ADH III polypeptide and ADH I and ADH II was 79 and 80%, respectively. The open reading frame encoding ADH III has a highly basic 27-amino-acid amino-terminal extension relative to ADH I and ADH II. The nucleotide sequence of the presumed leader peptide has a high degree of identity with the untranslated leader regions of ADH1 and ADH2 mRNAs. A strain containing a null allele of ADH3 did not have a detectably altered phenotype. The cloned gene integrated at the ADH3 locus, indicating that this is the structural gene for ADH III. Images PMID:2943982

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

    PubMed

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

    2016-05-03

    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.

  9. 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.

  10. 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

  11. 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.

  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. 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.

  14. 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

  15. 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.

  16. Substrate supply, fine roots, and temperature control proteolytic enzyme activity in temperate forest soils.

    PubMed

    Brzostek, Edward R; Finzi, Adrien C

    2011-04-01

    Temperature and substrate availability constrain the activity of the extracellular enzymes that decompose and release nutrients from soil organic matter (SOM). Proteolytic enzymes are the primary class of enzymes involved in the depolymerization of nitrogen (N) from proteinaceous components of SOM, and their activity affects the rate of N cycling in forest soils. The objectives of this study were to determine whether and how temperature and substrate availability affect the activity of proteolytic enzymes in temperate forest soils, and whether the activity of proteolytic enzymes and other enzymes involved in the acquisition of N (i.e., chitinolytic and ligninolytic enzymes) differs between trees species that form associations with either ectomycorrhizal or arbuscular mycorrhizal fungi. Temperature limitation of proteolytic enzyme activity was observed only early in the growing season when soil temperatures in the field were near 4 degrees C. Substrate limitation to proteolytic activity persisted well into the growing season. Ligninolytic enzyme activity was higher in soils dominated by ectomycorrhizal associated tree species. In contrast, the activity of proteolytic and chitinolytic enzymes did not differ, but there were differences between mycorrhizal association in the control of roots on enzyme activity. Roots of ectomycorrhizal species but not those of arbuscular mycorrhizal species exerted significant control over proteolytic, chitinolytic, and ligninolytic enzyme activity; the absence of ectomycorrhizal fine roots reduced the activity of all three enzymes. These results suggest that climate warming in the absence of increases in substrate availability may have a modest effect on soil-N cycling, and that global changes that alter belowground carbon allocation by trees are likely to have a larger effect on nitrogen cycling in stands dominated by ectomycorrhizal fungi.

  17. 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.

  18. 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.

  19. Acute and chronic ethanol exposure differentially alters alcohol dehydrogenase and aldehyde dehydrogenase activity in the zebrafish liver.

    PubMed

    Tran, Steven; Nowicki, Magda; Chatterjee, Diptendu; Gerlai, Robert

    2015-01-02

    Chronic ethanol exposure paradigms have been successfully used in the past to induce behavioral and central nervous system related changes in zebrafish. However, it is currently unknown whether chronic ethanol exposure alters ethanol metabolism in adult zebrafish. In the current study we examine the effect of acute ethanol exposure on adult zebrafish behavioral responses, as well as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) activity in the liver. We then examine how two different chronic ethanol exposure paradigms (continuous and repeated ethanol exposure) alter behavioral responses and liver enzyme activity during a subsequent acute ethanol challenge. Acute ethanol exposure increased locomotor activity in a dose-dependent manner. ADH activity was shown to exhibit an inverted U-shaped curve and ALDH activity was decreased by ethanol exposure at all doses. During the acute ethanol challenge, animals that were continuously housed in ethanol exhibited a significantly reduced locomotor response and increased ADH activity, however, ALDH activity did not change. Zebrafish that were repeatedly exposed to ethanol demonstrated a small but significant attenuation of the locomotor response during the acute ethanol challenge but ADH and ALDH activity was similar to controls. Overall, we identified two different chronic ethanol exposure paradigms that differentially alter behavioral and physiological responses in zebrafish. We speculate that these two paradigms may allow dissociation of central nervous system-related and liver enzyme-dependent ethanol induced changes in zebrafish.

  20. 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.

  1. Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP).

    PubMed

    Ma, Hongyan; Delafield, Daniel G; Wang, Zhe; You, Jianlan; Wu, Si

    2017-04-01

    The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion. Graphical Abstract ᅟ.

  2. Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

    NASA Astrophysics Data System (ADS)

    Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

    2017-01-01

    The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

  3. Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity.

    PubMed Central

    Valero, E; Varón, R; García-Carmona, F

    1995-01-01

    A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response. PMID:7619054

  4. 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.

  5. Structures of benzylsuccinate synthase elucidate roles of accessory subunits in glycyl radical enzyme activation and activity

    PubMed Central

    Funk, Michael A.; Judd, Evan T.; Marsh, E. Neil G.; Elliott, Sean J.; Drennan, Catherine L.

    2014-01-01

    Anaerobic degradation of the environmental pollutant toluene is initiated by the glycyl radical enzyme benzylsuccinate synthase (BSS), which catalyzes the radical addition of toluene to fumarate, forming benzylsuccinate. We have determined crystal structures of the catalytic α-subunit of BSS with its accessory subunits β and γ, which both bind a [4Fe-4S] cluster and are essential for BSS activity in vivo. We find that BSSα has the common glycyl radical enzyme fold, a 10-stranded β/α-barrel that surrounds the glycyl radical cofactor and active site. Both accessory subunits β and γ display folds related to high potential iron–sulfur proteins but differ substantially from each other in how they interact with the α-subunit. BSSγ binds distally to the active site, burying a hydrophobic region of BSSα, whereas BSSβ binds to a hydrophilic surface of BSSα that is proximal to the active site. To further investigate the function of BSSβ, we determined the structure of a BSSαγ complex. Remarkably, we find that the barrel partially opens, allowing the C-terminal region of BSSα that houses the glycyl radical to shift within the barrel toward an exit pathway. The structural changes that we observe in the BSSαγ complex center around the crucial glycyl radical domain, thus suggesting a role for BSSβ in modulating the conformational dynamics required for enzyme activity. Accompanying proteolysis experiments support these structural observations. PMID:24982148

  6. Guanidinylated Neomycin Mediates Heparan Sulfate–dependent Transport of Active Enzymes to Lysosomes

    PubMed Central

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

    2010-01-01

    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, β--glucuronidase (GUS) and α--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. PMID:20442709

  7. Genetic and biochemical analysis of anaerobically-induced enzymes during seed germination of Echinochloa crus-galli varieties tolerant and intolerant of anoxia.

    PubMed

    Fukao, Takeshi; Kennedy, Robert A; Yamasue, Yuji; Rumpho, Mary E

    2003-05-01

    To compare the regulation of anaerobic metabolism during germination in anoxia-tolerant and intolerant plants, enzymes associated with anaerobic metabolism such as sucrose synthase, aldolase, enolase, pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH) were assayed in two varieties of Echinochloa crus-galli, formosensis (tolerant) and praticola (intolerant). The initial and intervening enzymes of the pathway (sucrose synthase and aldolase) and enzymes in the last part of the pathway (PDC, ADH and ALDH) revealed similar changing patterns in activities during germination. This implies that each group of enzymes may be controlled by an identical regulatory mechanism. During anoxia, activities of all enzymes increased 1.5-30-fold in both varieties compared to their activities under aerobic conditions. Activities of sucrose synthase, enolase and ADH exhibited the same induction patterns under anoxia in formosensis and praticola. However, the activities of aldolase, ALDH and PDC were more strongly induced in formosensis under anoxia (1.2-2-fold) than in praticola. These enzymes were also assayed in F(3) families which varied in their anaerobic germinability. For PDC, activities under anoxia in anoxia-tolerant families were similar to those of an anoxia-intolerant family during the whole period although the family did not exhibit anaerobic germinability. This suggests that there is no correlation between PDC activity and anaerobic germinability. For ALDH, activities were more strongly induced under anoxia in anoxia-tolerant families than in anoxia-intolerant families, a trend also exhibited by the parents. This indicates that ALDH may play a role in detoxifying acetaldehyde formed through alcoholic fermentation during anaerobic germination.

  8. Copper-induced changes in tissue enzyme activity in a freshwater mussel.

    PubMed

    Rajalakshmi, S; Mohandas, A

    2005-09-01

    Changes in enzyme activity levels are of great diagnostic value. Lysosomal membrane is often the target of injury by xenobiotics, resulting in destabilization. Variations in the activity of acid phosphatase (ACP) a marker enzyme, in gills and hepatopancreas of the freshwater mussel Lamellidens corrianus (Lea) exposed to different concentrations of copper for 24, 120, and 168 h are discussed. The aim was to determine if the metal caused any variation in enzyme activity in the two tissues studied and, if so, whether the length of exposure had any influence on enzyme activity. ACP activity was determined as described in Sigma Technical Bulletin No. 104 and expressed as micromoles of p-nitrophenol liberated per milligram of protein per hour. Both concentration of the metal and length of exposure were found to influence enzyme activity. Higher concentrations of metals are assumed to induce stress proteins like metallothioneins.

  9. Spinach Thylakoid Polyphenol Oxidase : ISOLATION, ACTIVATION, AND PROPERTIES OF THE NATIVE CHLOROPLAST ENZYME.

    PubMed

    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. The higher molecular weight enzyme is the predominant form in freshly isolated preparations but on aging or further purification, the amount of lower molecular weight enzyme increases at the expense of the higher.Sonication releases polyphenol oxidase from the membrane largely in the latent state. C(18) fatty acids, especially linolenic acid, are potent activators of the enzymic activity. 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. The K(m) values for 3,4-dihydroxyphenylalanine and O(2) are 6.5 and 0.065 millimolar, respectively. 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(m) A large seasonal variation in polyphenol oxidase activity may result from a decrease in enzyme content rather than inhibition of the enzyme present.

  10. 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.

  11. Group III alcohol dehydrogenase from Pectobacterium atrosepticum: insights into enzymatic activity and organization of the metal ion-containing region.

    PubMed

    Elleuche, Skander; Fodor, Krisztian; von der Heyde, Amélie; Klippel, Barbara; Wilmanns, Matthias; Antranikian, Garabed

    2014-05-01

    NAD(P)(+)-dependent alcohol dehydrogenases (ADH) are widely distributed in all phyla. These proteins can be assigned to three nonhomologous groups of isozymes, with group III being highly diverse with regards to catalytic activity and primary structure. Members of group III ADHs share a conserved stretch of amino acid residues important for cofactor binding and metal ion coordination, while sequence identities for complete proteins are highly diverse (<20 to >90 %). A putative group III ADH PaYqhD has been identified in BLAST analysis from the plant pathogenic enterobacterium Pectobacterium atrosepticum. The PaYqhD gene was expressed in the heterologous host Escherichia coli, and the recombinant protein was purified in a two-step purification procedure to homogeneity indicating an obligate dimerization of monomers. Four conserved amino acid residues involved in metal ion coordination were substituted with alanine, and their importance for catalytic activity was confirmed by circular dichroism spectrum determination, in vitro, and growth experiments. PaYqhD exhibits optimal activity at 40 °C with short carbon chain aldehyde compounds and NADPH as cofactor indicating the enzyme to be an aldehyde reductase. No oxidative activities towards alcoholic compounds were detectable. EDTA completely inhibited catalytic activity and was fully restored by the addition of Co(2+). Activity measurements together with sequence alignments and structure analysis confirmed that PaYqhD belongs to the butanol dehydrogenase-like enzymes within group III of ADHs.

  12. 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

  13. Coexpression of Lactobacillus brevis ADH with GDH or G6PDH in Arxula adeninivorans for the synthesis of 1-(R)-phenylethanol.

    PubMed

    Rauter, Marion; Prokoph, Alexandra; Kasprzak, Jakub; Becker, Karin; Baronian, Keith; Bode, Rüdiger; Kunze, Gotthard; Vorbrodt, H- Matthias

    2015-06-01

    The yeast Arxula adeninivorans was used for the overexpression of an ADH gene of Lactobacillus brevis coding for (R)-specific alcohol dehydrogenase (LbADH) to synthesise enantiomerically pure 1-(R)-phenylethanol. Glucose dehydrogenase gene from Bacillus megaterium (BmGDH) or glucose 6-phosphate dehydrogenase of Bacillus pumilus (BpG6PDH) were coexpressed in Arxula to regenerate the cofactor NADPH by oxidising glucose or glucose 6-phosphate. The yeast strain expressing LbADH and BpG6PDH produced 5200 U l(-1) ADH and 370 U l(-1) G6PDH activity, whereas the strain expressing LbADH and BmGDH produced 2700 U l(-1) ADH and 170 U l(-1) GDH activity. However, the crude extract of both strains reduced 40 mM acetophenone to pure 1-(R)-phenylethanol with an enantiomeric excess (ee) of >99 % in 60 min without detectable by-products. An increase in yield was achieved using immobilised crude extracts (IEs), Triton X-100 permeabilised cells (PCs) and permeabilised immobilised cells (PICs) with PICs being most stable with GDH regeneration over 52 cycles. Even though the activity and synthesis rate of 1-(R)-phenylethanol with the BpG6PDH and LbADH coexpressing strain was higher, the BmGDH-LbADH strain was more stable over successive reaction cycles. This, combined with its higher total turnover number (TTN) of 391 mol product per mole NADP(+), makes it the preferred strain for continuous reaction systems. The initial non-optimised semi-continuous reaction produced 9.74 g l(-1) day(-1) or 406 g kg(-1) dry cell weight (dcw) day(-1) isolated 1-(R)-phenylethanol with an ee of 100 % and a TTN of 206 mol product per mole NADP(+). In conclusion, A. adeninivorans is a promising host for LbADH and BpG6PDH or BmGDH production and offers a simple method for the production of enantiomerically pure alcohols.

  14. [Effect Of Polyelectrolytes on Catalytic Activity of Alcohol Dehydrogenase].

    PubMed

    Dubrovsky, A V; Musina, E V; Kim, A L; Tikhonenko, S A

    2016-01-01

    Fluorescent and optical spectroscopy were used to study the interaction of alcohol dehydrogenase (ADH) with negatively charged polystyrene sulfonate (PSS) and dextran sulfate (DS), as well as positively charged poly(diallyldimethylammonium) (PDADMA). As found, DS and PDADMA did not affect the structural and catalytic enzyme properties. In contrast, PSS slightly decreased the protein self-fluorescence over 1 h of incubation, which is associated with partial destruction of its quaternary (globular) structure. Investigation of the ADH activity with and without PSS showed its dependency on the incubation time and the PSS presence. Sodium chloride (2.0 M and 0.2 M) or ammonium sulfate (0.1 M) added to the reaction mixture did not completely protect the enzyme quaternary structure from the PSS action. However ammonium sulfate or 0.2 M sodium chloride stabilized the enzyme and partially inhibited the negative PSS effect.

  15. Queuine promotes antioxidant defence system by activating cellular antioxidant enzyme activities in cancer.

    PubMed

    Pathak, Chandramani; Jaiswal, Yogesh K; Vinayak, Manjula

    2008-04-01

    Constant generation of Reactive oxygen species (ROS) during normal cellular metabolism of an organism is generally balanced by similar rate of consumption by antioxidants. Imbalance between ROS production and antioxidant defense results in increased level of ROS causing oxidative stress which leads to promotion of malignancy. Queuine is a hyper modified base analogue of guanine, found at first anti-codon position of Q- family of tRNAs. These tRNAs are completely modified with respect to queuosine in terminally differentiated somatic cells, however hypomodification of Q-tRNAs is close association with cell proliferation. Q-tRNA modification is essential for normal development, differentiation and cellular functions. Queuine is a nutrient factor to eukaryotes. It is found to promote cellular antioxidant defense system and inhibit tumorigenesis. The activities of antioxidant enzymes like catalase, SOD, glutathione peroxidase and glutathione reductase are found to be low in Dalton's lymphoma ascites transplanted (DLAT) mouse liver compared to normal. However, exogenous administration of queuine to DLAT mouse improves the activities of antioxidant enzymes. The results suggest that queuine promotes antioxidant defense system by increasing antioxidant enzyme activities and in turn inhibits oxidative stress and tumorigenesis.

  16. 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

  17. 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

    2015-04-03

    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.

  18. 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

  19. New hydroxamate inhibitors of neurotensin-degrading enzymes. Synthesis and enzyme active-site recognition.

    PubMed

    Bourdel, E; Doulut, S; Jarretou, G; Labbe-Jullie, C; Fehrentz, J A; Doumbia, O; Kitabgi, P; Martinez, J

    1996-08-01

    Selective and mixed inhibitors of the three zinc metallopeptidases that degrade neurotensin (NT), e.g. endopeptidase 24-16 (EC 3.4.24.16), endopeptidase 24-11 (EC 3.4.24.11 or neutral endopeptidase, NEP) and endopeptidase 24-15 (EC 3.4.24.15), and leucine-aminopeptidase (type IV-S), that degrades the NT-related peptides, Neuromedin N (NN), are of great interest. On the structural basis of compound JMV 390-1 (N-[3-[(hydroxyamino)carbonyl]-1-oxo-2(R)-benzylpropyl]-L- isoleucyl-L-leucine), which was a full inhibitor of the major NT degrading enzymes, several hydroxamate inhibitors corresponding to the general formula HONHCO-CH2-CH(CH2-C6H5)CO-X-Y-OH (with X-Y = dipeptide) have been synthesized. Compound 7a (X-Y = Ile-Ala) was nearly 40-times more potent in inhibiting EC 24-16 than NEP and more than 800-times more potent than EC 24-15, with an IC50 (12 nM) almost equivalent to that of compound JMV 390-1. Therefore, this compound is an interesting selective inhibitor of EC 24-16, and should be an interesting probe to explore the physiological involvement of EC 24-16 in the metabolism of neurotensin.

  20. The involvement of several enzymes in methanol detoxification in Drosophila melanogaster adults.

    PubMed

    Wang, Shu-Ping; Hu, Xing-Xing; Meng, Qing-Wei; Muhammad, Shahid Arain; Chen, Rui-Rui; Li, Fei; Li, Guo-Qing

    2013-09-01

    Methanol is among the most common short-chain alcohols in fermenting fruits, the natural food and oviposition sites of the fruit fly Drosophila melanogaster. Our previous results showed that cytochrome P450 monooxygenases (CYPs) were associated with methanol detoxification in the larvae. Catalases, alcohol dehydrogenases (ADHs), esterases (ESTs) and glutathione S-transferases (GSTs) were specifically inhibited by 3-amino-1,2,4-triazole (3-AT), 4-methylpyrazole (4-MP), triphenyl phosphate (TPP) and diethylmeleate (DEM), respectively. CYPs were inhibited by piperonyl butoxide (PBO) and 1-aminobenzotriazole (1-ABT). In the present paper, the involvements of these enzymes in methanol metabolism were investigated in female and male adults by determining the combination indices of methanol and their corresponding inhibitors. When PBO, 1-ABT, 3-AT, 4-MP and TPP were individually mixed with methanol, they exhibited significant synergism to the mortality of the adults after 72h of dietary exposure. In contrast, the DEM and methanol mixture showed additive effects. Moreover, methanol exposure dramatically increased CYP activity and up-regulated mRNA expression levels of several Cyp genes. Bioassays using different strains revealed that the variation in ADH activity and RNAi-mediated knockdown of α-Est7 significantly changed LC50 values for methanol. These results suggest that CYPs, catalases, ADHs and ESTs are partially responsible for methanol elimination in adults. It seems that there are some differences in methanol metabolism between larvae and adults, but not between female and male adults.

  1. 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

  2. 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

  3. Expression, purification and characterization of recombinant human choline acetyltransferase: phosphorylation of the enzyme regulates catalytic activity.

    PubMed Central

    Dobransky, T; Davis, W L; Xiao, G H; Rylett, R J

    2000-01-01

    Choline acetyltransferase synthesizes acetylcholine in cholinergic neurons and, in humans, may be produced in 82- and 69-kDa forms. In this study, recombinant choline acetyltransferase from baculovirus and bacterial expression systems was used to identify protein isoforms by two-dimensional SDS/PAGE and as substrate for protein kinases. Whereas hexa-histidine-tagged 82- and 69-kDa enzymes did not resolve as individual isoforms on two-dimensional gels, separation of wild-type choline acetyltransferase expressed in insect cells revealed at least nine isoforms for the 69-kDa enzyme and at least six isoforms for the 82-kDa enzyme. Non-phosphorylated wild-type choline acetyltransferase expressed in Escherichia coli yielded six (69 kDa) and four isoforms (82 kDa) respectively. Immunofluorescent labelling of insect cells expressing enzyme showed differential subcellular localization with the 69-kDa enzyme localized adjacent to plasma membrane and the 82-kDa enzyme being cytoplasmic at 24 h. By 64 h, the 69-kDa form was in cytoplasm and the 82-kDa form was only present in nucleus. Studies in vitro showed that recombinant 69-kDa enzyme was a substrate for protein kinase C (PKC), casein kinase II (CK2) and alpha-calcium/calmodulin-dependent protein kinase II (alpha-CaM kinase), but not for cAMP-dependent protein kinase (PKA); phosphorylation by PKC and CK2 enhanced enzyme activity. The 82-kDa enzyme was a substrate for PKC and CK2 but not for PKA or alpha-CaM kinase, with only PKC yielding increased enzyme activity. Dephosphorylation of both forms of enzyme by alkaline phosphatase decreased enzymic activity. These studies are of functional significance as they report for the first time that phosphorylation enhances choline acetyltransferase catalytic activity. PMID:10861222

  4. High-throughput fluorometric measurement of potential soil extracellular enzyme activities.

    PubMed

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

    2013-11-15

    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

  5. 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.

  6. The Enterococcus hirae Mur-2 enzyme displays N-acetylglucosaminidase activity

    PubMed Central

    Eckert, Catherine; Magnet, Sophie; Mesnage, Stéphane

    2007-01-01

    Enterococcus hirae produces two autolytic enzymes named Mur-1 and Mur-2, both previously described as N-acetylmuramidases. We used tandem mass spectrometry to show that Mur-2 in fact displays N-acetylglucosaminidase activity. This result reveals that Mur-2 and its counterparts studied to date, which are members of glycosyl hydrolase family 73 from the CAZy (Carbohydrate-Active enZyme) database, display the same catalytic activity. PMID:17258207

  7. A study on the stability and enzymatic activity of yeast alcohol dehydrogenase in presence of the self-assembling block copolymer Poloxamer 407.

    PubMed

    Pucciarelli, Stefania; Bonacucina, Giulia; Bernabucci, Franco; Cespi, Marco; Mencarelli, Giovanna; De Fronzo, Giusi Serena; Natalini, Paolo; Palmieri, Giovanni Filippo

    2012-05-01

    Yeast alcohol dehydrogenase (ADH) is an enzyme widely studied for biotechnological applications due to its involvement in fermentation industry, and various attempts to improve its catalytic properties and its thermal stability have been carried out. In this paper, the influence of a block copolymer (Poloxamer 407) on ADH enzymatic activity and thermal behaviour has been studied in order to get new insights about the use of poloxamers in formulation of sustained release systems for therapeutic proteins. Poloxamer 407 has the ability to form micelles and gel due to its self-assembling and thermoresponsive properties. The effect of the copolymer towards thermal stress and pH changes, which often reduce enzymes activity it has been investigated by means of enzymatic assays and differential scanning calorimetry. Results showed that at pH 9.1 and 7.3, the Poloxamer in the form of unimeric, micellar and gel state is able to effectively preserve the enzyme from thermoinactivation. In addition by calorimetric data Poloxamer 407 has showed an effect in preserving ADH from aggregation at pH 7.3. In conclusion, Poloxamer 407 seems to be very effective in protecting ADH from stress related events, like alkaline inactivation and aggregation.

  8. 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...

  9. Temperature-responsive enzyme-polymer nanoconjugates with enhanced catalytic activities in organic media.

    PubMed

    Zhu, Jingying; Zhang, Yifei; Lu, Diannan; Zare, Richard N; Ge, Jun; Liu, Zheng

    2013-07-11

    A general approach for preparing enzyme-polymer nanoconjugates that respond to temperature in organic media is presented. These nanoconjugates readily dissolve in organic solvents for homogenous catalysis at 40 °C and showed greatly enhanced apparent catalytic activities. The recovery of the soluble enzyme-polymer nanoconjugates is accomplished by temperature-induced precipitation.

  10. 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...

  11. 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)

  12. Mechanism of allopurinol-mediated increase in enzyme activity in man

    PubMed Central

    Beardmore, Thomas D.; Cashman, Jay S.; Kelley, William N.

    1972-01-01

    Allopurinol therapy in man interferes with pyrimidine biosynthesis de novo by inhibition of one or both of the two enzymes, orotate phosphoribosyltransferase (OPRT) and orotidylic decarboxylase (ODC), responsible for the conversion of orotic acid to uridine-5′-monophosphate. Inhibition of this pathway in vivo is followed in 1-3 wk by an increase in the activity of both of these enzymes in erythrocytes and of ODC in circulating leukocytes. This drug-mediated increase in enzyme activity in erythrocytes could not be attributed to enzyme stabilization or induction in vivo but appeared to be due to enzymeactivation.” “Activation” of the OPRT enzyme was directly demonstrated in erythrocytes studied in vitro after incubation with oxipurinol, and to a lesser extent, with allopurinol. No evidence for “activation” of the ODC enzyme was demonstrated in vitro. This response to allopurinol therapy provides an excellent model for examining the mechanism of increased enzyme activity in response to drug administration. PMID:5032526

  13. Genome-level and biochemical diversity of the acyl-activating enzyme superfamily in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In higher plants, the superfamily of carboxyl-CoA ligases and related proteins, collectively called acyl activating enzymes (AAEs), has evolved to provide enzymes for many pathways of primary and secondary metabolism and for the conjugation of hormones to amino acids. Across the superfamily there is...

  14. 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.

  15. 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...

  16. 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…

  17. 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).

  18. A new regulatory element mediates ethanol repression of KlADH3, a Kluyveromyces lactis gene coding for a mitochondrial alcohol dehydrogenase.

    PubMed

    Saliola, Michele; Getuli, Claudia; Mazzoni, Cristina; Fantozzi, Ivana; Falcone, Claudio

    2007-08-01

    KlADH3 is a Kluyveromyces lactis alcohol dehydrogenase gene induced in the presence of all respiratory carbon sources except ethanol, which specifically represses this gene. Deletion analysis of the KlADH3 promoter revealed the presence of both positive and negative elements. However, by site-directed mutagenesis and gel retardation experiments, we identified a 15-bp element responsible for the transcriptional repression of this gene by ethanol. In particular, this element showed putative sites required for the sequential binding of ethanol-induced factors responsible for the repressed conditions, and the binding of additional factors relieved repression. In addition, we showed that the ethanol element was required for in vivo repression of KlAdh3 activity.

  19. Modification of thiamine pyrophosphate dependent enzyme activity by oxythiamine in Saccharomyces cerevisiae cells.

    PubMed

    Tylicki, Adam; Czerniecki, Jan; Dobrzyn, Pawel; Matanowska, Agnieszka; Olechno, Anna; Strumilo, Slawomir

    2005-10-01

    Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phosphate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS-PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger inhibition of mitochondrial pyruvate dehydrogenase than of cytosolic enzymes.

  20. 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.

  1. [Effect of space flight on the Kosmos-1129 biosatellite on enzyme activity of the rat liver].

    PubMed

    Nemeth, S; Tigranian, R A

    1983-01-01

    After the 18.5 day Cosmos-1129 flight the activity of 7 glucocorticoid-stimulated enzymes of the rat liver was measured. Immediately postflight the activity of tyrosine aminotransferase, tryptophan pyrolase and serine dehydrogenase increased. These enzymes rapidly (within several hours) react to increased glucocorticoids. The activity of aspartate and alanine aminotransferases also increased. These enzymes require many days of a continuous effect of glucocorticoids. The glycogen concentration in the rat liver also grew. At R + 6 the activity of tryptophan pyrolase and serine dehydrogenase decreased and that of the other enzymes returned to normal. The immobilization stress applied postflight led to an increased activity of tyrosine aminotransferase and tryptophan pyrolase. This study gives evidence that after space flight rats are in an acute stress state, evidently, produced by the biosatellite recovery.

  2. 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.

  3. Array-on-Array Strategy For Activity-Based Enzyme Profiling.

    PubMed

    Sieow, Brendan Fu-Long; Uttamchandani, Mahesh

    2017-01-01

    We describe a novel array on array strategy intended to enhance the throughput of enzymatic activity screening using microarrays. This strategy consists of spotting a first array with large droplets of enzymes with varying concentrations and subsequently spotting a second array with small droplets of fluorogenic substrate on top of the enzyme array. By varying the array on array spotting patterns of different classes of enzyme (e.g., proteases, phosphatases, kinases) and their corresponding fluorogenic substrates, we have the unprecedented ability for testing enzymes and mixed samples in a multiplexed fashion within a single microarray slide. This new approach enables rapid enzyme characterization building upon a one enzyme on one slide droplet-based screening concept previously established.

  4. 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

  5. Lectin-directed enzyme activated prodrug therapy (LEAPT): Synthesis and evaluation of rhamnose-capped prodrugs.

    PubMed

    Garnier, Philippe; Wang, Xiang-Tao; Robinson, Mark A; van Kasteren, Sander; Perkins, Alan C; Frier, Malcolm; Fairbanks, Antony J; Davis, Benjamin G

    2010-12-01

    The lectin-directed enzyme activated prodrug therapy (LEAPT) bipartite drug delivery system utilizes glycosylated enzyme, localized according to its sugar pattern, and capped prodrugs released by that enzyme. In this way, the sugar coat of a synthetic enzyme determines the site of release of a given drug. Here, prodrugs of doxorubicin and 5-fluorouracil capped by the nonmammalian l-rhamnosyl sugar unit have been efficiently synthesized and evaluated for use in the LEAPT system. Both are stable in blood, released by synthetically d-galactosylated rhamnosidase enzyme, and do not inhibit the uptake of the synthetic enzyme to its liver target. These results are consistent with their proposed mode of action and efficacy in models of liver cancer, and confirm modular flexibility in the drugs that may be used in LEAPT.

  6. Aerobic and anaerobic bioprocessing of activated sludge: floc disintegration by enzymes.

    PubMed

    Ayol, Azize; Filibeli, Ayse; Sir, Diclehan; Kuzyaka, Ersan

    2008-11-01

    Hydrolytic enzymes such as glucosidases, lipases, and proteases have an imperative function at the hydrolysis stage of complex organic structures in the degradation of biodegradable particulate organic matter. As a key factor, extracellular polymeric substances (EPS) control the extracellular hydrolytic enzymes in this degradation mechanism. A flocculated matrix of EPS bridging with bacteria holds back the dewaterability properties of the bioprocessed sludges. Disruption of the flocculated matrix leads to improved solubilization of sludge solids by attacking the hydrolytic enzymes to polymeric substances forming enzyme-substrate complexes. To determine the floc disintegration mechanisms by enzymes during aerobic and anaerobic bioprocessing of sludges, experimental data obtained from three aerobic digesters and three anaerobic digesters were evaluated. As part of a broader project examining the overall fate and effects of hydrolytic enzymes in biological sludge stabilization, this paper compares the performances of aerobic and anaerobic reactors used in this study and reports significant improvements in enzymatic treatment of activated sludge.

  7. 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

  8. Activation of a Covalent Enzyme-Substrate Bond by Noncovalent Interaction with an Effector

    PubMed Central

    Malhotra, O. P.; Bernhard, Sidney A.

    1973-01-01

    The absorption spectrum of an activesite specific chromophoric acyl enzyme, sturgeon β-(2-furyl)-acryloyl-glyceraldehyde-3-phosphate dehydrogenase, is reported. This acyl enzyme undergoes all of the catalyzed reactions characteristic of the intermediate of the physiological acyl enzyme, 3-phospho-D-glyceroyl-glyceraldehyde-3-phosphate dehydrogenease. The rates of reactions of both these acyl enzymes depend strongly on the extent of interaction of the acyl enzyme with the oxidized coenzyme, NAD+, even where the “redox” properties of the coenzyme are not required. Likewise, the spectral properties of chromophoric acyl enzyme are affected by the extent of bound NAD. Under the pseudophysiological conditions reported herein, there is a stoichiometric limitation of two furylacryloyl-acyl groups per enzyme molecule containing four covalently-equivalent subunits. The binding of NAD both to the apoenzyme and to the diacyl enzyme is heterogeneous: at low extents of NAD occupancy, NAD binding is stronger. The binding to acyl enzyme can be quantitatively described by an enzyme model involving a tetramer with 2-fold symmetry, and consequently containing equal numbers of two classes of sites. NAD binding to difurylacryloyl-enzyme occurs virtually discretely, first to the two unmodified (tight-binding) sites, followed by looser binding to the two acyl-sites. NAD occupancy at these latter sites transforms the chromophoric acyl spectrum from that characteristic of a model furylacryloyl-thiol ester in H2O to a highly perturbed furylacryloyl spectrum characteristic of monomeric native “active-thiol” furylacryloyl-enzymes. Likewise the acyl reactivity towards arsenolysis depends on the extent of NAD bound to the loose sites. Elimination of the tight binding of NAD to the difurylacryloyl enzyme tetramer by alkylation of the remaining two free SH groups with iodoacetate has no apparent influence on the NAD-dependent furylacryloyl-spectral perturbation at the “two equivalent

  9. 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.

  10. 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.

  11. Role of enzyme-substrate flexibility in catalytic activity: an evolutionary perspective.

    PubMed

    Demetrius, L

    1998-09-21

    Site-directed mutagenesis has proved an effective experimental technique to investigate catalytic mechanisms and to determine relations between enzyme structure and function. This article invokes an analytical model based on evolution by mutation and natural selection-Nature's analogue of site-directed mutagenesis-to derive a set of general rules relating enzyme structure and activity. The catalysts are described in terms of the structural parameters, rigidity and flexibility, and the functional variables, reaction rate and substrate specificity. The evolutionary model predicts the following structure-activity relations: (a) rigid enzyme-flexible substrate: large variation in reaction rates, broad substrate specificity; (b) rigid enzyme-rigid substrate: diffusion controlled rates, absolute specificity; (c) flexible enzyme-rigid substrate: intermediate reaction rates, group specificity; (d) flexible enzyme-flexible substrate: slow rates, absolute specificity. Spectroscopic methods and X-ray crystallography now yield important characteristics of enzyme-substrate complexes such as molecular flexibility. The evolutionary analysis we have exploited provides general principles for inferring catalytic activity from structural studies of enzyme-substrate complexes.

  12. Dosage Compensation in Drosophila: Nadp-Enzyme Activities and Cross-Reacting Material

    PubMed Central

    Williamson, John H.; Bentley, Michael M.

    1983-01-01

    The relationships between gene dosage, enzyme activities and CRM levels have been determined for G6PD and 6PGD. Enzyme activities and CRM levels were directly proportional and increased in genotypes carrying duplications of the respective structural genes. When a duplication consisting of the distal 45% of the X chromosome was used to duplicate Pgd+, 6PGD activity and CRM increased and G6PD activity decreased. When the proximal 55% of the X chromosome was duplicated, G6PD activity and CRM increased whereas 6PGD activity and CRM levels decreased. These observations support the model of dosage compensation of X-linked genes that invokes an autosomal activator in limited concentrations for which X-linked loci compete. The distal 45% of the X chromosome, when duplicated, caused a significant increase in NADP-malic enzyme activity and CRM levels, as if a structural gene for NADP-ME is sex-linked. PMID:6406296

  13. 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.

  14. Mesoporous silica-encapsulated gold nanoparticles as artificial enzymes for self-activated cascade catalysis.

    PubMed

    Lin, Youhui; Li, Zhenhua; Chen, Zhaowei; Ren, Jinsong; Qu, Xiaogang

    2013-04-01

    A significant challenge in chemistry is to create synthetic structures that mimic the complexity and function of natural systems. Here, a self-activated, enzyme-mimetic catalytic cascade has been realized by utilizing expanded mesoporous silica-encapsulated gold nanoparticles (EMSN-AuNPs) as both glucose oxidase- and peroxidase-like artificial enzymes. Specifically, EMSN helps the formation of a high degree of very small and well-dispersed AuNPs, which exhibit an extraordinarily stability and dual enzyme-like activities. Inspired by these unique and attractive properties, we further piece them together into a self-organized artificial cascade reaction, which is usually completed by the oxidase-peroxidase coupled enzyme system. Our finding may pave the way to use matrix as the structural component for the design and development of biomimetic catalysts and to apply enzyme mimics for realizing higher functions.

  15. 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

  16. Seasonal variation in the temperature sensitivity of proteolytic enzyme activity in temperate forest soils

    NASA Astrophysics Data System (ADS)

    Brzostek, Edward R.; Finzi, Adrien C.

    2012-03-01

    Increasing soil temperature has the potential to alter the activity of the extracellular enzymes that mobilize nitrogen (N) from soil organic matter (SOM) and ultimately the availability of N for primary production. Proteolytic enzymes depolymerize N from proteinaceous components of SOM into amino acids, and their activity is a principal driver of the within-system cycle of soil N. The objectives of this study were to investigate whether the soils of temperate forest tree species differ in the temperature sensitivity of proteolytic enzyme activity over the growing season and the role of substrate limitation in regulating temperature sensitivity. Across species and sampling dates, proteolytic enzyme activity had relatively low sensitivity to temperature with a mean activation energy (Ea) of 33.5 kJ mol-1. Ea declined in white ash, American beech, and eastern hemlock soils across the growing season as soils warmed. By contrast, Eain sugar maple soil increased across the growing season. We used these data to develop a species-specific empirical model of proteolytic enzyme activity for the 2009 calendar year and studied the interactive effects of soil temperature (ambient or +5°C) and substrate limitation (ambient or elevated protein) on enzyme activity. Declines in substrate limitation had a larger single-factor effect on proteolytic enzyme activity than temperature, particularly in the spring. There was, however, a large synergistic effect of increasing temperature and substrate supply on proteolytic enzyme activity. Our results suggest limited increases in N availability with climate warming unless there is a parallel increase in the availability of protein substrates.

  17. 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 %.

  18. Detection of Extracellular Enzyme Activity in Penicillium using Chromogenic Media.

    PubMed

    Yoon, Ji Hwan; Hong, Seung Beom; Ko, Seung Ju; Kim, Seong Hwan

    2007-09-01

    A total of 106 Penicillium species were tested to examine their ability of degrading cellobiose, pectin and xylan. The activity of β-glucosidase was generally strong in all the Penicillium species tested. P. citrinum, P. charlesii, P. manginii and P. aurantiacum showed the higher ability of producing β-glucosidase than other tested species. Pectinase activity was detected in 24 Penicillium species. P. paracanescens, P. sizovae, P. sartoryi, P. chrysogenum, and P. claviforme showed strong pectinase activity. In xylanase assay, 84 Penicillium species showed activity. Strong xylanase activity was detected from P. megasporum, P. sartoryi, P. chrysogenum, P. glandicola, P. discolor, and P. coprophilum. Overall, most of the Penicillium species tested showed strong β-glucosidase activity. The degree of pectinase and xylanase activity varied depending on Penicillium species.

  19. 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

  20. 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.

  1. Defying the activity-stability trade-off in enzymes: taking advantage of entropy to enhance activity and thermostability.

    PubMed

    Siddiqui, Khawar Sohail

    2017-05-01

    The biotechnological applications of enzymes are limited due to the activity-stability trade-off, which implies that an increase in activity is accompanied by a concomitant decrease in protein stability. This premise is based on thermally adapted homologous enzymes where cold-adapted enzymes show high intrinsic activity linked to enhanced thermolability. In contrast, thermophilic enzymes show low activity around ambient temperatures. Nevertheless, genetically and chemically modified enzymes are beginning to show that the activity-stability trade-off can be overcome. In this review, the origin of the activity-stability trade-off, the thermodynamic basis for enhanced activity and stability, and various approaches for escaping the activity-stability trade-off are discussed. The role of entropy in enhancing both the activity and the stability of enzymes is highlighted with a special emphasis placed on the involvement of solvent water molecules. This review is concluded with suggestions for further research, which underscores the implications of these findings in the context of productivity curves, the Daniel-Danson equilibrium model, catalytic antibodies, and life on cold planets.

  2. The importance of myeloperoxidase enzyme activity in the pathogenesis of Crimean-Congo haemorrhagic fever.

    PubMed

    Guven, F M K; Aydin, H; Yildiz, G; Engin, A; Celik, V K; Bakir, D; Deveci, K

    2013-03-01

    Crimean-Congo haemorrhagic fever (CCHF) is a disease with a severe course including acute viral haemorrhagic fever, ecchymosis, thrombocytopenia, hepatic function disorder and high mortality. Myeloperoxidase (MPO) is an enzyme located in neutrophil granulocytes and plays an important role in the destruction of phagocytosed micro-organisms. The aim of this study was to analyse MPO enzyme activity in CCHF cases compared with a control group. A total of 47 randomly selected CCHF patients admitted to the Department of Infectious Diseases of Cumhuriyet University Hospital in Sivas, Turkey, were studied, and as a control group, 41 age- and sex-matched individuals without any systemic disease were included in this study. MPO enzyme activity was measured in plasma and leukocytes for both groups by the ELISA method. MPO plasma and MPO leukocyte values were calculated as 57.62 ± 8.85 and 44.84 ± 9.71 in CCHF patients, and 0.79 ± 0.29 and 0.49 ± 0.11 in the controls, respectively. MPO enzyme activity was statistically significantly higher in patients with CCHF when compared to the control group. In conclusion, MPO enzyme activity is directly related to the activation of phagocytic leukocytes, and increases in both the plasma and leukocytes in CCHF patients. The increase of the MPO enzyme activity in leukocytes due to viral load leads to the destruction of the leukocyte. It is thought that MPO enzyme activity in plasma was higher in CCHF patients due to the destruction of leukocytes. MPO enzyme activity may be important in terms of the prognosis in patients with CCHF; however, more extensive studies are required on this subject.

  3. Enzyme assays.

    PubMed

    Reymond, Jean-Louis; Fluxà, Viviana S; Maillard, Noélie

    2009-01-07

    Enzyme assays are analytical tools to visualize enzyme activities. In recent years a large variety of enzyme assays have been developed to assist the discovery and optimization of industrial enzymes, in particular for "white biotechnology" where selective enzymes are used with great success for economically viable, mild and environmentally benign production processes. The present article highlights the aspects of fluorogenic and chromogenic substrates, sensors, and enzyme fingerprinting, which are our particular areas of interest.

  4. 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

  5. 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.

  6. 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.

  7. 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

    2012-03-06

    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.

  8. Associations between PON1 enzyme activities in human ovarian follicular fluid and serum specimens

    PubMed Central

    Kim, Keewan; Fujimoto, Victor Y.; Browne, Richard W.

    2017-01-01

    The importance of high-density lipoprotein (HDL) particle components to reproduction is increasingly recognized, including the constituent paraoxonase 1 (PON1). However, the reliability characteristics of PON1 enzymes in ovarian follicular fluid (FF) as biomarkers for clinical and epidemiologic studies have not been described. Therefore, we characterized PON1 enzymes in FF and serum and assessed the impact of the PON1 Q192R polymorphism on associations between enzyme activities in two compartments. We also evaluated associations between HDL particle size and enzyme activities. We collected FF and serum from 171 women undergoing in vitro fertilization. PON1 activities were measured as paraoxonase and arylesterase activities, and HDL particle size was determined by 1H NMR spectrometry. Reliability indices for PON1 activities were characterized and we evaluated HDL particle sizes as predictors of PON1 enzyme activities. We found that PON1 enzyme activities were correlated between compartments, but higher in serum than in FF. For FF, the index of individuality (II) was low and the coefficient of variation (CV%) was high for paraoxonase activity overall (0.12 and 11.51%, respectively). However, IIs increased (0.33–1.30) and CV%s decreased (5.58%-8.52%) when stratified by PON1 Q192R phenotype. The intraclass correlation coefficient (ICC) for FF paraoxonase activity was high overall (0.89) but decreased when stratified by PON1 Q192R phenotype (0.43–0.75). We found similar, although more modest, patterns for FF arylesterase activity. For enzyme activities in serum, ICCs were close to 1.00 across all phenotypes. Additionally, different HDL particle sizes predicted PON1 enzyme activities according to PON1 Q192R phenotype. Overall, stratification by PON1 Q192R phenotype improved the reliability characteristics of FF PON1 enzymes as biomarkers for use in clinical investigations but diminished usefulness for epidemiologic studies. Thus, we recommend stratification by PON1 Q

  9. [Soil enzyme activities under two forest types as affected by different levels of nitrogen deposition].

    PubMed

    Zhao, Yu-tao; Li, Xue-feng; Han, Shi-jie; Hu, Yan-ling

    2008-12-01

    A simulation test was conducted to study the change trends of soil cellulase, polyphenol oxidase, and sucrase activities under natural broadleaf-Korean pine (Pinus koraiensis) and secondary poplar (Populus davidiana) -birch (Betula platyphylla) mixed forests as affected by 0, 25, and 50 kg x hm(-2) x a(-1) of N deposition. The results showed that the effects of elevated N deposition on test enzyme activities varied with forest type, and short-term nitrogen addition could significantly affect the test enzyme activities. High N deposition decreased soil polyphyneol oxidase activity, and correspondingly, soil cellulase and sucrase activities also had a trend of decrease.

  10. Control of butanol formation in Clostridium acetobutylicum by transcriptional activation.

    PubMed

    Thormann, Kai; Feustel, Lothar; Lorenz, Karin; Nakotte, Stephan; Dürre, Peter

    2002-04-01

    The sol operon of Clostridium acetobutylicum is the essential transcription unit for formation of the solvents butanol and acetone. The recent proposal that transcriptional regulation of this operon is controlled by the repressor Orf5/SolR (R. V. Nair, E. M. Green, D. E. Watson, G. N. Bennett, and E. T. Papoutsakis, J. Bacteriol. 181:319-330, 1999) was found to be incorrect. Instead, regulation depends on activation, most probably by the multivalent transcription factor Spo0A. The operon is transcribed from a single promoter. A second signal identified in primer extension studies results from mRNA processing and can be observed only in the natural host, not in a heterologous host. The first structural gene in the operon (adhE, encoding a bifunctional butyraldehyde/butanol dehydrogenase) is translated into two different proteins, the mature AdhE enzyme and the separate butanol dehydrogenase domain. The promoter of the sol operon is preceded by three imperfect repeats and a putative Spo0A-binding motif, which partially overlaps with repeat 3 (R3). Reporter gene analysis performed with the lacZ gene of Thermoanaerobacterium thermosulfurigenes and targeted mutations of the regulatory region revealed that the putative Spo0A-binding motif, R3, and R1 are essential for control. The data obtained also indicate that an additional activator protein is involved.

  11. 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.

  12. 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.

  13. Effects of age, species difference, antibiotics and toxicants on intestinal enzyme activity and genotoxicity

    SciTech Connect

    Chadwick, R.W.; George, S.E.; Kohan, M.J.; Allison, J.C. . Health Effects Research Lab.); Chang, J.; Long, J.E.; Duffy, M.C. ); Dekker, J.P.; Forehand, L.R. )

    1993-08-01

    Altered intestinal enzyme activity significantly affects the biotransformation and toxicity of many xenobiotics. This article summarizes research, supported by the US Air Force Bioenvironmental Hazards Research Program, that employs a novel gas-liquid chromatographic assay to investigate the effects of age, species difference, antibiotics, and environmental chemicals on enzyme activity in various regions of the intestinal tract. Significant research findings include the following: (a) age-dependent alterations in enzyme activity in the gastrointestinal (GI) tract of the developing animal that suggest a changing susceptibility to toxicants during this period; (b) discovery of previously unreported mucosal enzymes in the small intestine that are present in germ-free rats and are not susceptible to antibiotics; (c) markedly greater intestinal nitroreductase activity and significantly higher bioactivation of the procarcinogen 2,6-dinitrotoluene (DNT) in CD-1 mice than in Fischer 344 rats; (d) significantly altered intestinal enzyme activity in rats pretreated with lindane, pentachlorophenol, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), or Aroclor 1254; (e) potentiated DNT genotoxicity by Aroclor 1254 and pentachlorophenol pretreatment; and (f) a transient antagonism of DNT genotoxicity by 2,4,5-T pretreatment. Enzyme activity in the small intestine may have greater toxicological importance than previously thought in the biotransformation of environmental chemicals and as an indicator of change in the microbial flora.

  14. Purine enzyme activities in recent onset rheumatoid arthritis: are there differences between patients and healthy controls?

    PubMed Central

    Stolk, J N; Boerbooms, A M; De Abreu, R A; Kerstens, P J; de Koning, D G; de Graaf, R; Mulder, J; van de Putte, L B

    1996-01-01

    OBJECTIVE: Purine enzyme activities may predict the effectiveness of azathioprine treatment and be associated with increased deaths from infectious diseases. In rheumatoid arthritis, patients show variable responses to azathioprine and a higher percentage of death is caused by infections. The aim of the study was to investigate possible rheumatoid arthritis associated abnormalities of purine enzyme activities by measuring several of these enzymes in patients with recent onset rheumatoid arthritis before treatment with disease modifying antirheumatic drugs or prednisone. METHODS: 23 patients with recent onset rheumatoid arthritis and 28 healthy controls were studied. Activities of the enzymes 5'-nucleotidase, purine nucleoside phosphorylase (PNP), hypoxanthine guanine phosphoribosyltransferase (HGPRT), and thiopurine methyltransferase (TPMT) were measured. Assessment of disease activity and blood sampling for routine measurements and HLA typing were done simultaneously. RESULTS: Purine enzyme activities did not differ between patients and healthy controls. Enzyme activities had no significant relations with indices of disease activity or rheumatoid factor titre or with the rheumatoid arthritis associated HLA types. Activity of 5'nucleotidase decreased with age (P < or = 0.05) and was lower by about 27% (P = 0.007) in males than in females. CONCLUSIONS: In rheumatoid arthritis patients, neither the variability in azathioprine effectiveness nor the increased death rate from infections can be explained by pre-existing abnormalities in the activities of the purine enzymes 5'-nucleotidase, PNP, HGPRT, or TPMT at an early stage of the disease, before disease modifying antirheumatic drugs or prednisone treatment. Besides adjustment for age, results of studies involving purine 5' nucleotidase activity should also be adjusted for sex. PMID:8984938

  15. Active-site zinc ligands and activated H2O of zinc enzymes.

    PubMed Central

    Vallee, B L; Auld, D S

    1990-01-01

    The x-ray crystallographic structures of 12 zinc enzymes have been chosen as standards of reference to identify the ligands to the catalytic and structural zinc atoms of other members of their respective enzyme families. Universally, H2O is a ligand and critical component of the catalytically active zinc sites. In addition, three protein side chains bind to the catalytic zinc atom, whereas four protein ligands bind to the structural zinc atom. The geometry and coordination number of zinc can vary greatly to accommodate particular ligands. Zinc forms complexes with nitrogen and oxygen just as readily as with sulfur, and this is reflected in catalytic zinc sites having a binding frequency of His much greater than Glu greater than Asp = Cys, three of which bind to the metal atom. The systematic spacing between the ligands is striking. For all catalytic zinc sites except the coenzyme-dependent alcohol dehydrogenase, the first two ligands are separated by a "short-spacer" consisting of 1 to 3 amino acids. These ligands are separated from the third ligand by a "long spacer" of approximately 20 to approximately 120 amino acids. The spacer enables formation of a primary bidentate zinc complex, whereas the long spacer contributes flexibility to the coordination sphere, which can poise the zinc for catalysis as well as bring other catalytic and substrate binding groups into apposition with the active site. The H2O is activated by ionization, polarization, or poised for displacement. Collectively, the data imply that the preferred mechanistic pathway for activating the water--e.g., zinc hydroxide or Lewis acid catalysis--will be determined by the identity of the other three ligands and their spacing. Images PMID:2104979

  16. 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

  17. Alcohol oxidizing enzymes and ethanol-induced cytotoxicity in rat pancreatic acinar AR42J cells.

    PubMed

    Bhopale, Kamlesh K; Falzon, Miriam; Ansari, G A S; Kaphalia, Bhupendra S

    2014-04-01

    Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disease causing significant morbidity and mortality. Due to lack of a suitable animal model, the underlying mechanism of ACP is poorly understood. Chronic alcohol abuse inhibits alcohol dehydrogenase (ADH) and facilitates nonoxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs) in the pancreas frequently damaged during chronic ethanol abuse. Earlier, we reported a concentration-dependent formation of FAEEs and cytotoxicity in ethanol-treated rat pancreatic tumor (AR42J) cells, which express high FAEE synthase activity as compared to ADH and cytochrome P450 2E1. Therefore, the present study was undertaken to investigate the role of various ethanol oxidizing enzymes in ethanol-induced pancreatic acinar cell injury. Confluent AR42J cells were pre-treated with inhibitors of ADH class I and II [4-methylpyrazole (MP)] or class I, II, and III [1,10-phenanthroline (PT)], cytochrome P450 2E1 (trans-1,2-dichloroethylene) or catalase (sodium azide) followed by incubation with 800 mg% ethanol at 37°C for 6 h. Ethanol metabolism, cell viability, cytotoxicity (apoptosis and necrosis), cell proliferation status, and formation of FAEEs in AR42J cells were measured. The cell viability and cell proliferation rate were significantly reduced in cells pretreated with 1,10-PT + ethanol followed by those with 4-MP + ethanol. In situ formation of FAEEs was twofold greater in cells incubated with 1,10-PT + ethanol and ∼1.5-fold in those treated with 4-MP + ethanol vs. respective controls. However, cells treated with inhibitors of cytochrome P450 2E1 or catalase in combination of ethanol showed no significant changes either for FAEE formation, cell death or proliferation rate. Therefore, an impaired ADH class I-III catalyzed oxidation of ethanol appears to be a key contributing factor in ethanol-induced pancreatic injury via formation of nonoxidative metabolites of ethanol.

  18. 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.

  19. A new understanding of how temperature affects the catalytic activity of enzymes.

    PubMed

    Daniel, Roy M; Danson, Michael J

    2010-10-01

    The two established thermal properties of enzymes are their activation energy and their thermal stability, but experimental data do not match the expectations of these two properties. The recently proposed Equilibrium Model (EM) provides a quantitative explanation of enzyme thermal behaviour under reaction conditions by introducing an inactive (but not denatured) intermediate in rapid equilibrium with the active form. It was formulated as a mathematical model, and fits the known experimental data. Importantly, the EM gives rise to a number of new insights into the molecular basis of the temperature control of enzymes and their environmental adaptation and evolution, it is consistent with active site properties, and it has fundamental implications for enzyme engineering and other areas of biotechnology.

  20. [Influence of ionizing radiation on activity of enzymes of antioxidant defense of Paecilomyces lilaclvus (Thom) Samson].

    PubMed

    Tuhaĭ, T I

    2011-01-01

    The level of activity of antioxidant protection enzymes (superoxide dismutase, catalase and peroxidase) under exposure to ionizing radiation and without it in strain Paecilomyces lilacinus, showing radioadaptive properties, and in control one has been investigated. It has been established that the researched strains are characterized by the high level activity of superoxide dismutase (200-800 AU/mg protein), extracellular and intracellular catalase (0.02-40 mmol min(-1) mg(-1) protein) and peroxidase (0.2-4 mmol min(-1) mg(-1) protein). Ionizing radiation was the inducer of significant changes in antioxidant enzyme activity of the control strain (from the lack of influence to the change of activity by an order) and showed considerably less influence on their activity in the strain, showing radioadaptive properties (the activity changes by 40-50%). The complex response of antioxidant enzymes in investigated strains under the exposure to ionizing radiation has been revealed.

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

    PubMed

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

    2005-03-09

    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.

  2. Laccase activity in soils: considerations for the measurement of enzyme activity.

    PubMed

    Eichlerová, Ivana; Šnajdr, Jaroslav; Baldrian, Petr

    2012-08-01

    Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are copper-containing enzymes that catalyze the oxidative conversion of a variety of chemicals, such as mono-, oligo-, and polyphenols and aromatic amines. Laccases have been proposed to participate in the transformation of organic matter and xenobiotics as well as microbial interactions. Several laccase assays have been proposed and used in soils. Here, we show that the optimal pH conditions for the laccase substrates 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS, pH 3-5), 2,6-dimethoxyphenol (4-5.5), L-3,4-dihydroxyphenylalanine (DOPA; 4-6), guaiacol (3.5-5), 4-methylcatechol (3.5-5), and syringaldazine (5.5-7.0) are similar between purified laccases from Trametes versicolor and Pyricularia sp. and soil extracts; the substrate affinities of purified enzymes (K(M)) and soil extracts were also similar. The laccase assays showed specificity overlap with tyrosinase and ligninolytic peroxidases when hydrogen peroxide is present. The ABTS oxidation assay is able to reliably detect the presence of 13.5 pg mL(-1) or 0.199×10(-12) mol mL(-1) of T. versicolor laccase, which is three times more sensitive than the 2,6-dimethoxyphenol-based assay and more than 40 times more sensitive than any of the other assays. The low molecular mass soil-derived compounds and the isolated fulvic and humic acids influence the laccase assays and should be removed from the soil extracts before measurements of the enzyme activity are performed.

  3. Direct solubilization of enzyme aggregates with enhanced activity in nonaqueous media.

    PubMed

    Akbar, Umar; Aschenbrenner, Carl D; Harper, Michael R; Johnson, Harvey R; Dordick, Jonathan S; Clark, Douglas S

    2007-04-15

    A protein solubilization method has been developed to directly solubilize protein clusters into organic solvents containing small quantities of surfactant and trace amounts of water. Termed "direct solubilization," this technique was shown to solubilize three distinct proteins - subtilisin Carlsberg, lipase B from Candida antarctica, and soybean peroxidase - with much greater efficiencies than extraction of the protein from aqueous solution into surfactant-containing organic solvents (referred to as extraction). More significant, however, was the dramatic increase in directly solubilized enzyme activity relative to extracted enzyme activity, particularly for subtilisin and lipase in polar organic solvents. For example, in THF the initial rate towards bergenin transesterification was ca. 70 times higher for directly solubilized subtilisin than for the extracted enzyme. Furthermore, unlike their extracted counterparts, the directly solubilized enzymes yielded high product conversions across a spectrum of non-polar and polar solvents. Structural characterization of the solubilized enzymes via light scattering and atomic force microscopy revealed soluble proteins consisting of active enzyme aggregates containing approximately 60 and 100 protein molecules, respectively, for subtilisin and lipase. Formation of such clusters appears to provide a microenvironment conducive to catalysis and, in polar organic solvents at least, may protect the enzyme from solvent-induced inactivation.

  4. Genetic and Cytogenetic Analysis of the ADH Region in DROSOPHILA MELANOGASTER

    PubMed Central

    O'Donnell, Janis; Mandel, Howard C.; Krauss, Marc; Sofer, William

    1977-01-01

    Eighteen Adh-negative mutations were selected with 1-pentyn-3-ol after feeding of formaldehyde. Twelve of the 18 were shown by cytological and genetic analysis to be deletions. Cytological examination of the deletions allowed us to localize the Adh gene to a region including bands 35B3–5 on the left arm of chromosome 2. The deletions were also used to order known visible loci located near Adh.—The vital loci near Adh were also investigated. A total of 109 lethal mutations were generated with EMS and 33 of these, localized within a region defined by the overlap of two of the deletions, were found to belong to 13 complementation groups. If one includes three other loci known to belong there (el, Adh and Sco), a total of 16 complementation groups have been identified in the region close to Adh. PMID:408228

  5. [Effect of colchicine and Triton X-100 on expression of the enzyme-encoding genes in nongerminating seeds of sugarbeet (Beta vulgaris L.)].

    PubMed

    Kirikovich, S S; Levites, E V

    2011-01-01

    The expression of the enzyme-coding genes, controlling glucose-phosphate isomerase (GPI), malate dehydrogenase (MDH), and alcohol dehydrogenase (ADH), was examined in nongerminating seeds of sugarbeet after Triton X-100 (TX-100) and colchicine treatment. Two types of changes revealed included modification of the enzymatic loci expression (change of the isozyme electrophoretic mobility) and inactivation of standard profiles. In the MDH and GPI systems, these processes were found to be associated. Complete isozyme modification was accompanied with the disappearance of standard profiles. In the ADH system, the treatment with TX-100 and colchicine gave rise to two independent processes, including silencing of the Adh1 locus and the appearance of the ADH isozymes with abnormal electrophoretic mobility, which were probably the products of the Adh2 locus. It was suggested that the effect of TX-100 and colchicine on the expression of the enzyme-encoding genes examined depended on the intracellular localization of the encoded enzymes.

  6. A comparison of maximal bioenergetic enzyme activities obtained with commonly used homogenization techniques.

    PubMed

    Grace, M; Fletcher, L; Powers, S K; Hughes, M; Coombes, J

    1996-12-01

    Homogenization of tissue for analysis of bioenergetic enzyme activities is a common practice in studies examining metabolic properties of skeletal muscle adaptation to disease, aging, inactivity or exercise. While numerous homogenization techniques are in use today, limited information exists concerning the efficacy of specific homogenization protocols. Therefore, the purpose of this study was to compare the efficacy of four commonly used approaches to homogenizing skeletal muscle for analysis of bioenergetic enzyme activity. The maximal enzyme activity (Vmax) of citrate synthase (CS) and lactate dehydrogenase (LDH) were measured from homogenous muscle samples (N = 48 per homogenization technique) and used as indicators to determine which protocol had the highest efficacy. The homogenization techniques were: (1) glass-on-glass pestle; (2) a combination of a mechanical blender and a teflon pestle (Potter-Elvehjem); (3) a combination of the mechanical blender and a biological detergent; and (4) the combined use of a mechanical blender and a sonicator. The glass-on-glass pestle homogenization protocol produced significantly higher (P < 0.05) enzyme activities compared to all other protocols for both enzymes. Of the four protocols examined, the data demonstrate that the glass-on-glass pestle homogenization protocol is the technique of choice for studying bioenergetic enzyme activity in skeletal muscle.

  7. Theoretical analysis of the relationship between positive/negative cooperativity and enzyme activation/inhibition.

    PubMed

    Ge, Hao; Qian, Min

    2009-09-01

    Cooperativity is one of the "paradigms" in enzyme kinetics and molecular biology. But the classical textbook treatment of enzyme kinetics always indeed separates the concepts of positive/negative cooperativity from enzyme activation/inhibition, at least partially. Few theoretical analysis of their relationship has been discussed, although its experimental investigations might date back at least to 1970s. In the present paper, we try to apply the change of free energy as a connective parameter for investigating the relationship between positive/negative cooperativity and enzyme activation/inhibition through several classic equilibrium binding models. It is explicitly shown that the terms of positive/negative cooperativity could be equivalently regarded as enzyme activation/inhibition of the saturation function induced by the substrate molecule itself rather than any other additional effectors. Moreover, both the degree of cooperativity phenomenon and the degree of enzyme activation/inhibition monotonically increase with the change of free energy. Note that this result is quite different from the idea of relating cooperativity to the concepts of "substrate activation/inhibition", which is identified when at high substrate concentrations the reaction rate decreases instead of tending towards the maximum velocity, since it always needs a second substrate molecule.

  8. 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.

  9. 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.

  10. Localization of the active site of an enzyme, bacterial luciferase, using two-quantum affinity modification

    NASA Astrophysics Data System (ADS)

    Benimetskaya, L. Z.; Gitelzon, I. I.; Kozionov, Andrew L.; Novozhilov, S. Y.; Petushkov, V. N.; Rodionova, N. S.; Stockman, Mark I.

    1991-11-01

    For the first time the method of two-quantum affinity modification has been employed to probe the structure of an enzyme, bacterial luciferase. Position of the flavin-binding site of this enzyme, which was previously unknown, has been established. The obtained data indicate that the flavin site is positioned on the (alpha) -subunit. The closest contact of the protein chain of the enzyme with the chromophoric group of the flavin takes place near 80 +/- 10 and 120 +/- 10 amino acid residues; the regions 50 +/- 10 and 215 +/- 10 are also close to the flavin. The established localization does not contradict suggestions on positions of the flavin and phosphate sites of the bacterial luciferase, which had earlier been made from the data on evolutionary stability of various luciferases. The present method can, in principle, be applied to a great number of enzymes, including all flavin-dependent enzymes. Enzymatic catalysis has high speed and specificity. Creation of a method of determination of the elements of the primary structure of a protein, making up the active site (in which substratum conversion occurs), could be a significant advance in clearing up mechanisms of enzymatic catalysis. It was proposed to localize active sites of the enzymes, whose substrata are chromophores, using this method of two-quantum affinity modification. An enzyme- substratum complex is irradiated with laser light of sufficiently long wavelength ((lambda) 300 nm) which is not directly absorbed by the enzyme. Two-quantum quasiresonant excitation of the substratum activates it to the state with energy 5-7 eV, which is then radiativelessly transferred to neighboring protein groups. This energy exceeds the energy of activation of peptide bond breakage. Therefore, the enzyme will be disrupted in the vicinity of its active site. In the present paper the above approach has been implemented for the first time. Information has been obtained about the position of the flavin-binding site of bacterial

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. [1-butanol synthesis by Escherichia coli cells through butyryl-CoA formation by heterologous enzymes of clostridia and native enzymes of fatty acid beta-oxidation].

    PubMed

    Gulevich, A Iu; Skorokhodova, A Iu; Morzhakova, A A; Antonova, S V; Sukhozhenko, A V; Shakulov, R S; Debabov, V G

    2012-01-01

    Anaerobic biosynthesis of 1-butanol from glucose is investigated in recombinant Escherichia coli strains which form butyryl-CoA using the heterologous enzyme complex of clostridia or as a result of a reversal in the action of native enzymes of the fatty acid beta-oxidation pathway. It was revealed that when the basic pathways of acetic and lactic acid formation are inactivated due to deletions in the ackA, pta, poxB, and ldhA genes, the efficiency of butyryl-CoA biosynthesis and its reduced product, i.e., 1-butanol, by two types of recombinant stains is comparable. The limiting factor for 1-butanol production by the obtained strains is the low substrate specificity of the basic CoA-dependent alcohol/aldehyde AdhE dehydrogenase from E. coli to butyryl-CoA. It was concluded that, in order to construct an efficient 1-butanol producer based on a model strain synthesizing butyryl-CoA as a result of a reversal in fatty acid beta-oxidation enzymes, it is necessary to provide intensive formation of acetyl-CoA and enhanced activity of alternative alcohol and aldehyde dehydrogenases in the cells of a strain.

  18. 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

  19. 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

  20. Creation of catalytically active particles from enzymes crosslinked with a natural bifunctional agent--homocysteine thiolactone.

    PubMed

    Stroylova, Yulia Y; Semenyuk, Pavel I; Asriyantz, Regina A; Gaillard, Cedric; Haertlé, Thomas; Muronetz, Vladimir I

    2014-09-01

    The current study describes an approach to creation of catalytically active particles with increased stability from enzymes by N-homocysteinylation, a naturally presented protein modification. Enzymatic activities and properties of two globular tetrameric enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) were studied before and after N-homocysteinylation. Modification of these proteins concerns the accessible lysine residues and introduces an average of 2-2,5 homocysteine residues per protein monomer. Formation of a range of aggregates was observed for both enzymes, which assemble via formation of intermolecular noncovalent bonds and by disulfide bonds. It was demonstrated that both studied enzymes retain their catalytic activities on modification and the subsequent formation of oligomeric forms. At low concentrations of homocysteine thiolactone, modification of GAPDH leads not only to prevention of spontaneous inactivation but also increases thermal stability of this enzyme on heating to 80°C. A moderate reduction of the activity of GAPDH observed in case of its crosslinking with 50-fold excess of homocysteine thiolactone per lysine is probably caused by hindered substrate diffusion. Spherical particles of 100 nm and larger diameters were observed by transmission electron microscopy and atomic force microscope techniques after modification of GAPDH with different homocysteine thiolactone concentrations. In case of LDH, branched fibril-like aggregates were observed under the same conditions. Interestingly, crosslinked samples of both proteins were found to have reversible thermal denaturation profiles, indicating that modification with homocysteine thiolactone stabilizes the spatial structure of these enzymes.

  1. 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.

  2. 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.

  3. 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...

  4. 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.

  5. A genetic analysis of Adh1 regulation. Progress report, June 1991--February 1992

    SciTech Connect

    Freeling, M.

    1992-03-01

    The overall goal of our research proposal is to understand the meaning of the various cis-acting sites responsible for AdH1 expression in the entire maize plant. Progress is reported in the following areas: Studies on the TATA box and analysis of revertants of the Adh1-3F1124 allele; screening for more different mutants that affect Adh1 expression differentially; studies on cis-acting sequences required for root-specific Adh1 expression; refinement of the use of the particle gun; and functional analysis of a non- glycolytic anaerobic protein.

  6. 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.

  7. Overexpression of ADH1 and HXT1 genes in the yeast Saccharomyces cerevisiae improves the fermentative efficiency during tequila elaboration.

    PubMed

    Gutiérrez-Lomelí, Melesio; Torres-Guzmán, Juan Carlos; González-Hernández, Gloria Angélica; Cira-Chávez, Luis Alberto; Pelayo-Ortiz, Carlos; Ramírez-Córdova, Jose de Jesús

    2008-05-01

    This work assessed the effect of the overexpression of ADH1 and HXT1 genes in the Saccharomyces cerevisiae AR5 strain during fermentation of Agave tequilana Weber blue variety must. Both genes were cloned individually and simultaneously into a yeast centromere plasmid. Two transformant strains overexpressing ADH1 and HXT1 individually and one strain overexpressing both genes were randomly selected and named A1, A3 and A5 respectively. Overexpression effect on growth and ethanol production of the A1, A3 and A5 strains was evaluated in fermentative conditions in A. tequilana Weber blue variety must and YPD medium. During growth in YPD and Agave media, all the recombinant strains showed lower cell mass formation than the wild type AR5 strain. Adh enzymatic activity in the recombinant strains A1 and A5 cultivated in A. tequilana and YPD medium was higher than in the wild type. The overexpression of both genes individually and simultaneously had no significant effect on ethanol formation; however, the fermentative efficiency of the A5 strain increased from 80.33% to 84.57% and 89.40% to 94.29% in YPD and Agave medium respectively.

  8. 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.

  9. 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

  10. [Effects of simulated warming on soil enzyme activities in two subalpine coniferous forests in west Sichuan].

    PubMed

    Xu, Zhen-feng; Tang, Zheng; Wan, Chuan; Xiong, Pei; Cao, Gang; Liu, Qing

    2010-11-01

    With open top chamber (OTC), this paper studied the effects of simulated warming on the activities of soil invertase, urease, catalase, polyphenol oxidase in two contrasting subalpine coniferous forests (a dragon spruce plantation and a natural conifer forest) in west Sichuan. The dynamic changes of soil temperature and soil moisture were monitored synchronously. In the whole growth season, simulated warming enhanced the daily mean temperature at soil depth 5 cm by 0.61 degrees C in the plantation, and by 0.55 degrees C in the natural forest. Conversely, the volumetric moisture at soil depth 10 cm was declined by 4.10% and 2.55%, respectively. Simulated warming also increased soil invertase, urease, catalase, and polyphenol oxidase activities. The interactive effect of warming and forest type was significant on soil urease and catalase, but not significant on soil invertase and polyphenol oxidase. The warming effect on soil catalase depended, to some extent, on season change. In all treatments, the soil enzyme activities in the natural forest were significantly higher than those in the plantation. The seasonal changes of test soil enzyme activities were highly correlated with soil temperature, but less correlated with soil moisture. This study indicated that warming could enhance soil enzyme activities, and the effect had definite correlations with forest type, enzyme category, and season change. The soil enzyme activities in the subalpine coniferous forests were mainly controlled by soil temperature rather than soil moisture.

  11. 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.

  12. [Effects of copper pollution on Trifolium repens growth and soil enzyme activities].

    PubMed

    Chu, Ling; Wang, Youbao; Ding, Jiahong; Li, Zheng; Liu, Dengyi

    2005-12-01

    The study with pot experiment showed that with increasing Cu concentration, soil urease, invertase, catalase and polyphenol oxidase activities decreased gradually. There was a significant correlation between Cu concentration and soil enzyme activities, with the correlated degree followed the order of invertase > polyphenol oxidase > urease > catalase. Under a fixed Cu concentration, soil enzyme activities changed with time, and the changes were different between high and low Cu concentrations, being increased slightly under low Cu concentration (< 500 mg x kg(-1)), but decreased gradually as Cu concentration increased (500-3000 mg x kg(-1)). Statistical analysis indicated that within the range of test Cu concentrations, the activities of test soil enzymes were significantly different among different Cu concentration (P < 0.01), which was accorded with the seedlings growth status. Soil pH was decreased, while electric conductivity was increased with increasing Cu concentration (500-3000 mg x kg(-1)), but they were increased with time under a fixed Cu concentration, with significant difference among different Cu concentration (P < 0.01) . Soil pH and electric conductivity were highly related to soil enzyme activities, with the order of polyphenol oxidase > invertase > catalase > urease. The test soil enzyme activities could be used as the indices of soil environment quality.

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

    PubMed

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

    2016-10-17

    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 Ce(3+)/Ce(4+) 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 Ce(3+)/Ce(4+) 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.

  14. 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.

  15. 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

  16. Immobilization of enzymes using non-ionic colloidal liquid aphrons (CLAs): Activity kinetics, conformation, and energetics.

    PubMed

    Ward, Keeran; Xi, Jingshu; Stuckey, David C

    2016-05-01

    This study seeks to examine the ability of non-ionic/non-polar Colloidial Liquid Aphrons (CLAs) to preserve enzyme functionality upon immobilization and release. CLAs consisting of micron-sized oil droplets surrounded by a thin aqueous layer stabilized by a mixture of surfactants, were formulated by direct addition (pre-manufacture addition) using 1% Tween 80/mineral oil and 1% Tween 20 and the enzymes lipase, aprotinin and α-chymotrypsin. The results of activity assays for both lipase and α-chymotrypsin showed that kinetic activity increased upon immobilization by factors of 7 and 5.5, respectively, while aprotinin retained approximately 85% of its native activity. The conformation of the enzymes released through desorption showed no significant alterations compared to their native state. Changes in pH and temperature showed that optimum conditions did not change after immobilization, while analysis of activation energy for the immobilized enzyme showed an increase in activity at higher temperatures. Furthermore, the effect of bound water within the aphron structure allowed for some degree of enzyme hydration, and this hydration was needed for an active conformation with results showing a decrease in ΔH* for the immobilized system compared to its native counterpart.

  17. Characterization of AmiBA2446, a Novel Bacteriolytic Enzyme Active against Bacillus Species

    PubMed Central

    Mehta, Krunal K.; Paskaleva, Elena E.; Azizi-Ghannad, Saba; Ley, Daniel J.; Page, Martin A.

    2013-01-01

    There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis, the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls. In this work, we performed in silico analysis of the genome of Bacillus anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446. This enzyme exists as a homodimer, as determined by size exclusion studies. It possesses N-acetylmuramoyl-l-alanine amidase activity, as determined from liquid chromatography-mass spectrometry (LC-MS) analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 was an autolysin of bacterial origin. We characterized the effects of enzyme concentration and phase of bacterial growth on bactericidal activity and observed close to a 5-log reduction in the viability of cells of Bacillus cereus 4342, a surrogate for B. anthracis. We further tested the bactericidal activity of AmiBA2446 against various Bacillus species and demonstrated significant activity against B. anthracis and B. cereus strains. We also demonstrated activity against B. anthracis spores after pretreatment with germinants. AmiBA2446 enzyme was also stable in solution, retaining its activity after 4 months of storage at room temperature. PMID:23872558

  18. The influence of age and gender on antioxidant enzyme activities in humans and laboratory animals.

    PubMed

    Giergiel, Marta; Lopucki, Maciej; Stachowicz, Norbert; Kankofer, Marta

    2012-12-01

    Antioxidative/oxidative balance is one of the important factors for homeostasis. Antioxidative systems which protect from peroxidative damage are supposed to be under the influence of steroid hormones. The implications of this influence are age and gender as well as tissue dependent alterations in antioxidative enzyme activities. Apart from hormonal influence, antioxidative enzymes require the presence of microelements in their active centers as well as concerted action of non enzymatic antioxidants which support enzymes in their scavenging action. The aim of this review is to analyze and compare existing knowledge about the changes in activity of antioxidant enzymes in human and animal females and males of different age. Evidence as regards participation of oxidative stress in senescence are specific diseases which, to some extent, are gender dependent and appear more frequently in males or females. Several experiments in laboratory animals revealed that changes in enzyme activities are reflected in histopathological pictures of cells. The alterations observed during perimenopausal period provide with additional evidence of the participation of steroid hormones in the regulation of antioxidative system activity. Moreover, estrogens themselves exhibit antioxidative activity which is receptor independent. In conclusion, apart from genetic-related influences, also diet and style of life may have an impact on the antioxidative system which requires appropriate supplementation in microelements and vitamins for its effective function of scavenging excess of free radicals.

  19. Cryoenzymology in mixed solvents without cosolvent effects on enzyme specific activity.

    PubMed Central

    Douzou, P; Balny, C

    1977-01-01

    Water-soluble polyelectrolytes in interaction with proteins are described. These polyelectrolytes make it possible to investigate enzyme-catalyzed reactions in cooled mixed solvents without the usual effects of their organic solvent component on enzyme specific activity. The applicability of techniques developed is illustrated by results obtained on several systems. The possibility of an electrostatic "sorting out" of solvents and its potentialities in cryoenzymology are discussed. PMID:18734

  20. Effects of electrokinetic treatment of a heavy metal contaminated soil on soil enzyme activities.

    PubMed

    Cang, Long; Zhou, Dong-Mei; Wang, Quan-Ying; Wu, Dan-Ya

    2009-12-30

    There is a growing concern on the potential application of a direct current (DC) electric field to soil for removing contaminants, but little is known about its impact on soil enzyme activities. This study investigated the change of enzyme activities of a heavy metal contaminated soil before and after electrokinetic (EK) treatments at lab-scale and the mechanisms of EK treatment to affect soil enzyme activities were explored. After treatments with 1-3 V cm(-1) of voltage gradient for 420 h, soil pH, electrical conductivity (EC), soil organic carbon, dissolved organic carbon (DOC), soil heavy metal concentration and enzyme activities were analyzed. The results showed that the average removal efficiencies of soil copper were about 65% and 83% without and with pH control of catholyte, respectively, and all the removal efficiencies of cadmium were above 90%. The soil invertase and catalase activities increased and the highest invertase activity was as 170 times as the initial one. The activities of soil urease and acidic phosphatase were lower than the initial ones. Bivariate correlation analyses indicated that the soil invertase and acidic phosphatase activities were significantly correlated with soil pH, EC, and DOC at P<0.05, but the soil urease activities had no correlation with the soil properties. On the other hand, the effects of DC electric current on solution invertase and catalase enzyme protein activities indicated that it had negative effect on solution catalase activity and little effect on solution invertase activity. From the change of invertase and catalase activities in soil and solution, the conclusion can be drawn that the dominant effect mechanism is the change of soil properties by EK treatments.

  1. Enzyme activities of D-glucose metabolism in the fission yeast Schizosaccharomyces pombe.

    PubMed

    Tsai, C S; Shi, J L; Beehler, B W; Beck, B

    1992-12-01

    The activities of key enzymes that are members of D-glucose metabolic pathways in Schizosaccharomyces pombe undergoing respirative, respirofermentative, and fermentative metabolisms are monitored. The steady-state activities of glycolytic enzymes, except phosphofructokinase, decrease with a reduced efficiency in D-glucose utilization by yeast continuous culture. On the other hand, the enzymic activities of pentose monophosphate pathway reach the maximum when the cell mass production of the cultures is optimum. Enzymes of tricarboxylate cycle exhibit the maximum activities at approximately the washout rate. The steady-state activity of pyruvate dehydrogenase complex increases rapidly when D-glucose is efficiently utilized. By comparison, the activity of pyruvate decarboxylase begins to increase only when ethanol production occurs. Depletion of dissolved oxygen suppresses the activity of pyruvate dehydrogenase complex but facilitates that of pyruvate decarboxylase. Acetate greatly enhances the acetyl CoA synthetase activity. Similarly, ethanol stimulates alcohol dehydrogenase and aldehyde dehydrogenase activities. Evidence for the existence of alcohol dehydrogenase isozymes in the fission yeast is presented.

  2. Purification and characterization of a trehalase-invertase enzyme with dual activity from Candida utilis.

    PubMed

    Lahiri, Sagar; Basu, Arghya; Sengupta, Shinjinee; Banerjee, Shakri; Dutta, Trina; Soren, Dhananjay; Chattopadhyay, Krishnananda; Ghosh, Anil K

    2012-06-15

    Trehalose and sucrose, two important anti-stress non-reducing natural disaccharides, are catabolized by two enzymes, namely trehalase and invertase respectively. In this study, a 175 kDa enzyme protein active against both substrates was purified from wild type Candida utilis and characterized in detail. Substrate specificity assay and activity staining revealed the enzyme to be specific for both sucrose and trehalose. The ratio between trehalase and invertase activity was found to be constant at 1:3.5 throughout the entire study. Almost 40-fold purification and 30% yield for both activities were achieved at the final step of purification. The presence of common enzyme inhibitors, thermal and pH stress had analogous effects on its trehalase and invertase activity. Km values for two activities were similar while Vmax and Kcat also differed by a factor of 3.5. Competition plot for both substrates revealed the two activities to be occurring at the single active site. N-terminal sequencing and MALDI-TOF data analysis revealed higher similarity of the purified protein to previously known neutral trehalases. While earlier workers mentioned independent purification of neutral trehalase or invertase from different sources, the present study reports the purification of a single protein showing dual activity.

  3. Structural and biochemical studies of the distinct activity profiles of Rai1 enzymes

    PubMed Central

    Wang, Vivien Ya-Fan; Jiao, Xinfu; Kiledjian, Megerditch; Tong, Liang

    2015-01-01

    Recent studies showed that Rai1 and its homologs are a crucial component of the mRNA 5′-end capping quality control mechanism. They can possess RNA 5′-end pyrophosphohydrolase (PPH), decapping, and 5′-3′ exonuclease (toward 5′ monophosphate RNA) activities, which help to degrade mRNAs with incomplete 5′-end capping. A single active site in the enzyme supports these apparently distinct activities. However, each Rai1 protein studied so far has a unique set of activities, and the molecular basis for these differences are not known. Here, we have characterized the highly diverse activity profiles of Rai1 homologs from a collection of fungal organisms and identified a new activity for these enzymes, 5′-end triphosphonucleotide hydrolase (TPH) instead of PPH activity. Crystal structures of two of these enzymes bound to RNA oligonucleotides reveal differences in the RNA binding modes. Structure-based mutations of these enzymes, changing residues that contact the RNA but are poorly conserved, have substantial effects on their activity, providing a framework to begin to understand the molecular basis for the different activity profiles. PMID:26101253

  4. 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.

  5. Improvement of activated sludge resistance to shock loading by fungal enzyme addition during textile wastewater treatment.

    PubMed

    Manai, Imène; Miladi, Baligh; El Mselmi, Abdellatif; Hamdi, Moktar; Bouallagui, Hassib

    2017-04-01

    The effects of the additions of the fungal enzymatic extract were investigated in relation to the treatment of real textile wastewater (RTW) by the activated sludge process (ASP). The used enzyme cocktail was produced by a new isolated fungal Chaetomium globosum IMA1. The system that was operated with enzyme addition showed a better chemical oxygen demand (COD) removal efficiency (95%) compared to the control system (75%). In addition, the improvement of color removal (OD620) efficiencies was around 15%, when the newly consortium fungal enzymes was added. As the organic loading rate (OLR) increased from 0.33 g to 0.66 g COD L(-1) d(-1), a decrease in the performance of the two reactors was observed by monitoring the quality of treated effluents. However, the ASP working with enzyme addition showed a strong resistance to shock loadings and restored after few days compared to the control system, which was strongly inhibited. In fact, the enzyme addition improved the sludge volume index (SVI) and the activity of microorganisms. A high activity of laccase (300 U.L(-1)) enzyme was observed throughout the decolorization process in the improved system.

  6. Selection of commercial hydrolytic enzymes with potential antifouling activity in marine environments.

    PubMed

    Zanaroli, Giulio; Negroni, Andrea; Calisti, Cecilia; Ruzzi, Maurizio; Fava, Fabio

    2011-12-10

    In this work, the marine antifouling potential of some commercially available hydrolytic enzymes acting on the main constituents of extracellular polymeric substances (EPS) involved in bacterial biofilm formation was determined. The selected protease (i.e., alpha-chymotrypsin from bovine pancreas), carbohydrase (i.e., alpha-amylase from porcine pancreas) and lipase (from porcine pancreas) exhibited remarkable hydrolytic activities towards target macromolecules typically composing EPS under a wide range of pHs (6.5-9.0 for alpha-chymotrysin and alpha-amylase; 7.0-8.5 for the lipase) and temperatures (from 10 °C to 30 °C), as well as relevant half-lives (from about 2 weeks to about 2 months), in a marine synthetic water. The activity displayed by each enzyme was poorly affected by the co-presence of the other enzymes, thus indicating their suitability to be employed in combination. None of the enzymes was able to inhibit the formation of biofilm by an actual site marine microbial community when applied singly. However, a mixture of the same enzymes reduced biofilm formation by about 90% without affecting planktonic growth of the same microbial community. This indicates that multiple hydrolytic activities are required to efficiently prevent biofilm formation by complex microbial communities, and that the mixture of enzymes selected in this study has the potential to be employed as an environmental friendly antifouling agent in marine antifouling coatings.

  7. 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.

  8. 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.

  9. 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.

  10. Regulation of aflatoxin biosynthesis: effect of glucose on activities of various glycolytic enzymes.

    PubMed Central

    Buchanan, R L; Lewis, D F

    1984-01-01

    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. PMID:6091545

  11. UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme.

    PubMed Central

    McGrath, J P; Jentsch, S; Varshavsky, A

    1991-01-01

    All known functions of ubiquitin are mediated through its covalent attachment to other proteins. The post-translational formation of ubiquitin--protein conjugates is preceded by an ATP-requiring step in which the carboxyl terminus of ubiquitin is adenylated and subsequently joined, through a thiolester bond, to a cysteine residue in the ubiquitin-activating enzyme, also known as E1. We report the isolation and functional analysis of the gene (UBA1) for the ubiquitin-activating enzyme of the yeast Saccharomyces cerevisiae. UBA1 encodes a 114 kd protein whose amino acid sequence contains motifs characteristic of nucleotide-binding sites. Expression of catalytically active UBA1 protein in E. coli, which lacks the ubiquitin system, confirmed that the yeast UBA1 gene encodes a ubiquitin-activating enzyme. Deletion of the UBA1 gene is lethal, demonstrating that the formation of ubiquitin--protein conjugates is essential for cell viability. Images PMID:1989885

  12. [Effect of heavy metals on wheat seedlings; activation of antioxidant enzymes].

    PubMed

    Murzaeva, S V

    2004-01-01

    Accumulation of heavy metals in wheat grain exposed to multicomponent pollutants (industrial waste-water) was studied. The absolute content of metals (Zn, Cd, Cu, Cr, Ni, Pb, and Mn) was found to be determined by the extent of purification of wastewater. An increase in the degree of grain contamination with heavy metals was accompanied by activation of antioxidant enzymes (superoxide dismutase, EC 1.15.1; catalase, EC 1.11.1.6; and peroxidase, EC 1.11.1.7) in leaves and activation of superoxide dismutase and peroxidase in roots. The ratio of activity of membrane enzymes to activity of cytosol enzymes was demonstrated to be high. It was concluded that the membrane-tropic effect of multicomponent contaminants was due to accumulation of heavy metals capable of inducing the antioxidant protection in the next generation of wheat seedlings.

  13. Effects of Cu on metabolisms and enzyme activities of microbial communities in the process of composting.

    PubMed

    Guo, Xingliang; Gu, Jie; Gao, Hua; Qin, Qingjun; Chen, Zhixue; Shao, Li; Chen, Lin; Li, Hailong; Zhang, Weijuan; Chen, Shengnan; Liu, Jiang

    2012-03-01

    With the compost matrix of pig manure, wheat straw, and spent mushroom substrate, and then inoculated with the Compound Microbe Preparation, the study investigated the effects of the heavy metal Cu on the process of composting. Biolog EcoPlate™ test revealed that at a low content, Cu could improve the capacities of microbial communities to transform and exploit carbon sources in the form of polymer, thus speeding up the decomposition of agricultural wastes, and at a high content, Cu presented inhibiting effect on microbial communities to exploit complex macromolecular carbon sources, thus extending the decomposition of agricultural wastes. Enzyme activity testing showed that at a low content, Cu presented enzyme activity-activating effect at the early period of composting and inhibiting effect in the late period of composting, and at a high content, Cu presented enzyme activity-inhibiting effects through the process of composting.

  14. 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.

  15. Effect of acute vs chronic H2O2-induced oxidative stress on antioxidant enzyme activities.

    PubMed

    Miguel, Fernanda; Augusto, Amanda C; Gurgueira, Sonia A

    2009-04-01

    H2O2 can freely crosses membranes and in the presence of Fe2+ (or Cu+) it is prone to participate in Fenton reaction. This study evaluated the concentration and time-dependent effects of H2O2-induced oxidative stress on MnSOD, Se:GPx and catalase and on aconitase. Acute and chronic H2O2 treatments were able to induce oxidative stress in HeLa cells as they significantly decreased aconitase activity and also caused a very significant decrease on antioxidant enzyme activities. The inhibition of enzyme activities was time- and concentration-dependent. Chronic treatment with 5 microM H2O2/h after 24 h was able to decrease all enzyme activities almost at the same level as the acute treatment. Acute and chronic treatments on antioxidant enzyme activities were prevented by cell treatment with ascorbic acid or N-acetylcysteine. These results indicate that antioxidant enzymes can also be affected by the same ROS they produce or neutralize if the time of exposure is long enough.

  16. Genome-wide investigation of schizophrenia associated plasma Ndel1 enzyme activity.

    PubMed

    Gadelha, Ary; Coleman, Jonathan; Breen, Gerome; Mazzoti, Diego Robles; Yonamine, Camila M; Pellegrino, Renata; Ota, Vanessa Kiyomi; Belangero, Sintia Iole; Glessner, Joseph; Sleiman, Patrick; Hakonarson, Hakon; Hayashi, Mirian A F; Bressan, Rodrigo A

    2016-04-01

    Ndel1 is a DISC1-interacting oligopeptidase that cleaves in vitro neuropeptides as neurotensin and bradykinin, and which has been associated with both neuronal migration and neurite outgrowth. We previously reported that plasma Ndel1 enzyme activity is lower in patients with schizophrenia (SCZ) compared to healthy controls (HCs). To our knowledge, no previous study has investigated the genetic factors associated with the plasma Ndel1 enzyme activity. In the current analyses, samples from 83 SCZ patients and 92 control subjects that were assayed for plasma Ndel1 enzyme activity were genotyped on Illumina Omni Express arrays. A genetic relationship matrix using genome-wide information was then used for ancestry correction, and association statistics were calculated genome-wide. Ndel1 enzyme activity was significantly lower in patients with SCZ (t=4.9; p<0.001) and was found to be associated with CAMK1D, MAGI2, CCDC25, and GABGR3, at a level of suggestive significance (p<10(-6)), independent of the clinical status. Then, we performed a model to investigate the observed differences for case/control measures. 2 SNPs at region 1p22.2 reached the p<10(-7) level. ZFPM2 and MAD1L1 were the only two genes with more than one hit at 10(-6) order of p value. Therefore, Ndel1 enzyme activity is a complex trait influenced by many different genetic variants that may contribute to SCZ physiopathology.

  17. Activity of key enzymes in glucose catabolism during the growth and metacyclogenesis of Leishmania infantum.

    PubMed

    Louassini, M; Foulquié, M R; Benítez, R; Adroher, F J

    1999-04-01

    This paper follows the development in the activity of the key enzymes of glycolysis and dehydrogenases of the pentose phosphate shunt throughout the in vitro growth and metacyclogenesis of two human strains of Leishmania infantum - one visceral (VL) and the other cutaneous (CL) - together with changes in the glucose, ammonium, and proton concentrations in the culture medium. In the first stage, ammonium was generated and no glucose was consumed. Later on, all the glucose was consumed and, finally, ammonium was generated again. The ammonium concentration increased 16- and 21-fold in cultures of VL and CL strains, respectively. The activities of the glycosomal enzymes hexokinase and phosphofructokinase differed in each strain, always being higher in CL than in VL and increasing throughout the culture period in CL while decreasing in VL. This probably indicates a different capability to adapt to the culture medium conditions. The activities of the pentose phosphate shunt enzymes examined indicate that 6-phosphogluconate dehydrogenase is possibly a rate-limiting enzyme for this pathway. Pyruvate kinase is a cytosolic control enzyme of glycolysis in trypanosomatids, and its activity decreased throughout the growth and differentiation of both strains of L. infantum, as occurs in other trypanosomatids. It was also observed that glucose catabolism was more active in the cutaneous strain than in the visceral one.

  18. Characterization of aldehyde oxidase enzyme activity in cryopreserved human hepatocytes.

    PubMed

    Hutzler, J Matthew; Yang, Young-Sun; Albaugh, Daniel; Fullenwider, Cody L; Schmenk, Jennifer; Fisher, Michael B

    2012-02-01

    Substrates of aldehyde oxidase (AO), for which human clinical pharmacokinetics are reported, were selected and evaluated in pooled mixed-gender cryopreserved human hepatocytes in an effort to quantitatively characterize AO activity. Estimated hepatic clearance (Cl(h)) for BIBX1382, carbazeran, O⁶-benzylguanine, zaleplon, and XK-469 using cryopreserved hepatocytes was 18, 17, 12, <4.3, and <4.3 ml · min⁻¹ · kg⁻¹, respectively. The observed metabolic clearance in cryopreserved hepatocytes was confirmed to be a result of AO-mediated metabolism via two approaches. Metabolite identification after incubations in the presence of H₂¹⁸O confirmed that the predominant oxidative metabolite was generated by AO, as expected isotope patterns in mass spectra were observed after analysis by high-resolution mass spectrometry. Second, clearance values were efficiently attenuated upon coincubation with hydralazine, an inhibitor of AO. The low exposure after oral doses of BIBX1382 and carbazeran (∼5% F) would have been fairly well predicted using simple hepatic extraction (f(h)) values derived from cryopreserved hepatocytes. In addition, the estimated hepatic clearance value for O⁶-benzylguanine was within ∼80% of the observed total clearance in humans after intravenous administration (15 ml · min⁻¹ · kg⁻¹), indicating a reasonable level of quantitative activity from this in vitro system. However, a 3.5-fold underprediction of total clearance was observed for zaleplon, despite the 5-oxo metabolite being clearly observed. These data taken together suggest that the use of cryopreserved hepatocytes may be a practical approach for assessing AO-mediated metabolism in discovery and potentially useful for predicting hepatic clearance of AO substrates.

  19. 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

    2011-05-09

    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.

  20. Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers.

    PubMed

    Yuan, Lin; Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

    2017-01-01

    Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

  1. Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers

    PubMed Central

    Wang, Mingfa; Zhang, Xiaotu; Wang, Zhixiang

    2017-01-01

    Three hundred one-day-old male broiler chickens (Ross-308) were fed corn-soybean basal diets containing non-starch polysaccharide (NSP) enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI) and average daily gain (ADG) were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05). Feed-to-gain ratio (FGR) was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05). Apparent digestibility of crude protein (ADCP) was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05). Cholecystokinin (CCK) level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05), but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05), respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05). However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05). The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05). Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

  2. Recombinant L-asparaginase 1 from Saccharomyces cerevisiae: an allosteric enzyme with antineoplastic activity

    PubMed Central

    Costa, Iris Munhoz; Schultz, Leonardo; de Araujo Bianchi Pedra, Beatriz; Leite, Mariana Silva Moreira; Farsky, Sandra H. P.; de Oliveira, Marcos Antonio; Pessoa, Adalberto; Monteiro, Gisele

    2016-01-01

    L-asparaginase (L-ASNase) (EC 3.5.1.1) is an important enzyme for the treatment of acute lymphoblastic leukaemia. Currently, the enzyme is obtained from bacteria, Escherichia coli and Erwinia chrysanthemi. The bacterial enzymes family is subdivided in type I and type II; nevertheless, only type II have been employed in therapeutic proceedings. However, bacterial enzymes are susceptible to induce immune responses, leading to a high incidence of adverse effects compromising the effectiveness of the treatment. Therefore, alternative sources of L-ASNase may be useful to reduce toxicity and enhance efficacy. The yeast Saccharomyces cerevisiae has the ASP1 gene responsible for encoding L-asparaginase 1 (ScASNase1), an enzyme predicted as type II, like bacterial therapeutic isoforms, but it has been poorly studied. Here we characterised ScASNase1 using a recombinant enzyme purified by affinity chromatography. ScASNase1 has specific activity of 196.2 U/mg and allosteric behaviour, like type I enzymes, but with a low K0.5 = 75 μM like therapeutic type II. We showed through site-directed mutagenesis that the T64-Y78-T141-K215 residues are involved in catalysis. Furthermore, ScASNase1 showed cytotoxicity for the MOLT-4 leukemic cell lineage. Our data show that ScASNase1 has characteristics described for the two subfamilies of l-asparaginase, types I and II, and may have promising antineoplastic properties. PMID:27824095

  3. 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.

  4. 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.

  5. Aptamer-mediated universal enzyme assay based on target-triggered DNA polymerase activity.

    PubMed

    Park, Ki Soo; Lee, Chang Yeol; Kang, Kyoung Suk; Park, Hyun Gyu

    2017-02-15

    We herein describe an innovative method for a universal fluorescence turn-on enzyme assay, which relies on the target enzyme-triggered DNA polymerase activity. In the first target recognition step, the target enzyme is designed to destabilize detection probe derived from an aptamer specific to DNA polymerase containing the overhang sequence and the complementary blocker DNA, which consequently leads to the recovery of DNA polymerase activity inhibited by the detection probe. This target-triggered polymerase activity is monitored in the second signal transduction step based on primer extension reaction coupled with TaqMan probe. Utilizing this design principle, we have successfully detected the activities of two model enzymes, exonuclease I and uracil DNA glycosylase with high sensitivity and selectivity. Since this strategy is composed of separated target recognition and signal transduction modules, it could be universally employed for the sensitive determination of numerous different target enzymes by simply redesigning the overhang sequence of detection probe, while keeping TaqMan probe-based signal transduction module as a universal signaling tool.

  6. 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.

  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.

  8. 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

  9. Antioxidant enzyme activities of human peripheral blood mononuclear cells exposed to trace elements.

    PubMed

    Kuppusamy, U R; Dharmani, M; Kanthimathi, M S; Indran, M

    2005-07-01

    The trace elements copper, zinc, and selenium are important immune modulators and essential cofactors of the antioxidant enzymes. In the present study, the proliferative effect of human peripheral mononuclear cells (PBMCs) that have been exposed to copper, zinc, and selenium and the corresponding activities of antioxidant enzymes, namely superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, were determined. Zinc and copper stimulated the PBMC proliferation in a dose-dependent manner within the dose range 25-200 micromol/L. SOD and GPx activities in PBMCs exposed to zinc were inhibited, whereas catalase activity was unaffected. All the three antioxidant enzymes in the cells exposed to copper were inhibited. Selenium exerted more potent inhibition of the cell proliferation while causing stimulation of the antioxidant enzymes at the lowest dose (25 micromol/L) than at the highest dose (200 micromol/L) tested. A significant negative correlation was observed between proliferation and antioxidant enzyme (SOD and GPx) activities in trace-element-exposed PBMC. The present findings substantiate the importance of trace elements as immune modulators and the involvement of enzymatic antioxidant system in the immune cell regulation.

  10. Biobehavioral and neuroendocrine correlates of antioxidant enzyme activity in ovarian carcinoma.

    PubMed

    Bayer, Jennifer L; Spitz, Douglas R; Christensen, Desire; McCormick, Michael L; Farley, Donna; DeGeest, Koen; Damoush, Laila; Aust, Samantha; Sood, Anil K; Lutgendorf, Susan K

    2015-11-01

    Increased levels of reactive oxygen species (ROS) such as superoxide anions and hydrogen peroxide have been reported in many cancer cells and they have been implicated in carcinogenesis and tumor progression. Antioxidant enzymes, such as Manganese Superoxide Dismutase (MnSOD or SOD2) and Glutathione Peroxidase-1 (GPx1), act coordinately to neutralize ROS. These enzymes are also thought to contribute to cancer cell resistance to conventional radio-chemo-therapies. Although some relationships have been reported between psychosocial factors and the regulation of antioxidant enzymes, little is known about these relationships in the context of cancer progression. The current study investigated the levels of MnSOD and GPx1in confirmed serous, high-grade tumor tissue from 60 ovarian cancer patients, and explored the relationship between the activity of these enzymes, the levels of tumor norepinephrine (NE), and patient mood as determined via pre-operative questionnaires. MnSOD activity was positively related to depressed mood (p=0.025) and tumor NE (p=0.023). In contrast, GPx1 activity was inversely related to fatigue (p=0.015) and tumor NE (p=0.009), and was positively associated with vigor (p=0.024). These findings suggest that psychological state and adrenergic signaling are linked with antioxidant enzyme activity in ovarian cancer and may have implications for patient treatments and outcomes.

  11. 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.

  12. Structure-based drug design: exploring the proper filling of apolar pockets at enzyme active sites.

    PubMed

    Zürcher, Martina; Diederich, François

    2008-06-20

    The proper filling of apolar pockets at enzyme active sites is central for increasing binding activity and selectivity of hits and leads in medicinal chemistry. In our structure-based design approach toward the generation of potent enzyme inhibitors, we encountered a variety of challenges in gaining suitable binding affinity from the occupation of such pockets. We summarize them here for the first time. A fluorine scan of tricyclic thrombin inhibitors led to the discovery of favorable orthogonal dipolar C-F...CO interactions. Efficient cation-pi interactions were established in the S4 pocket of factor Xa, another serine protease from the blood coagulation cascade. Changing from mono- to bisubstrate inhibitors of catechol O-methyltransferase, a target in the L-Dopa-based treatment of Parkinson's disease, enabled the full exploitation of a previously unexplored hydrophobic pocket. Conformational preorganization of a pocket at an enzyme active site is crucial for harvesting binding affinity. This is demonstrated for two enzymes from the nonmevalonate pathway of isoprenoid biosynthesis, IspE and IspF, which are pursued as antimalarial targets. Disrupting crystallographically defined water networks on the way into a pocket might cost all of the binding free enthalpy gained from its occupation, as revealed in studies with tRNA-guanine transglycosylase, a target against shigellosis. Investigations of the active site of plasmepsin II, another antimalarial target, showed that principles for proper apolar cavity filling, originally developed for synthetic host-guest systems, are also applicable to enzyme environments.

  13. Enzyme renaturation to higher activity driven by the sol-gel transition: Carbonic anhydrase

    PubMed Central

    Vinogradov, Vladimir V.; Avnir, David

    2015-01-01

    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. PMID:26394694

  14. 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.

  15. 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.

  16. 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.

  17. Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The purification and properties of an endogenous activator of the enzyme.

    PubMed

    Neuberger, A; Sandy, J D; Tait, G H

    1973-11-01

    1. A low-molecular-weight activator of 5-aminolaevulinate synthetase was detected in extracts of Rhodopseudomonas spheroides. The compound activates the enzyme extracted from oxygenated semi-anaerobically grown organisms by a factor of 6-8. 2. The activator was extensively purified, but owing to the exceedingly small amounts that could be extracted in the active form its structure was not determined. 3. The activator contains an acetylatable amino group; it is more stable at acid than at alkaline pH values; it is stable to treatment with I(2)-KI or potassium ferricyanide, but irreversibly inactivated by Na(2)S(2)O(4) or NaBH(4). 4. The chromatographic, electrophoretic, chemical and stability properties of the activator are similar to those of pteridines; purified activator preparations contain pteridines, as shown by their fluorescence spectrum. This does not, however, constitute an identification of the activator. 5. The activator enhances the activity of crude and partially purified enzyme and does not appear to require other endogenous factors or a supply of air to produce activation. Activation of the purified enzyme, however, requires the presence of either pyridoxal phosphate or sodium succinate. In the absence of both these factors the activator produces a time- and temperature-dependent decay of activity.

  18. NanoCluster Beacons as reporter probes in rolling circle enhanced enzyme activity detection.

    PubMed

    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

    2015-05-14

    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.

  19. Temperature affects the production, activity and stability of ligninolytic enzymes in Pleurotus ostreatus and Trametes versicolor.

    PubMed

    Snajdr, J; Baldrian, P

    2007-01-01

    Enzyme activity was determined in cultures of Pleurotus ostreatus and Trametes versicolor with cellulose as a sole C source and high C/N ratio. The fungi were able to grow and produce laccase and Mn-peroxidase (MnP) at 5-35 degrees C, the highest production being recorded at 25-30 degrees C in P. ostreatus and at 35 degrees C in T. versicolor. Production of both enzymes at 10 degrees C accounted only for 4-20% of the maximum value. Temperature optima for enzyme activity were 50 and 55 degrees C for P. ostreatus and T. versicolor laccases, respectively, and 60 degrees C for MnP. Temperatures causing 50% loss of activity after 24 h were 32 and 47 degrees C for laccases and 36 and 30 degrees C for MnP from P. ostreatus and T. versicolor, respectively.

  20. Effect of cell substrate on antioxidant enzyme activities in cultured renal glomerular epithelium.

    PubMed Central

    Yang, A. H.; Oberley, T. D.; Oberley, L. W.; Ramanathan, R.

    1988-01-01

    The activities of three antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, were monitored in isolated guinea pig glomeruli and primary or subcultured glomerular epithelial cells. Cell injury was assessed by morphologic studies and by measurement of cellular lipid peroxidation (levels of malondialdehyde). Antioxidant enzyme activities were very different in cultured cells than in parent glomeruli. The possible effect of culture substrates (tissue culture plastic, bovine corneal endothelial [BCE] cell basement membrane, and PF-HR-9 endodermal cell basement membrane) on antioxidant enzyme status, cell morphology, and lipid peroxidation was also assessed. Glomerular epithelial cells cultured on the BCE cell basement membrane substrate survived longer and showed less lipid peroxidation than cells cultured on plastic or the HR-9 substrate. Cells cultured on a plastic substrate had substantially less glutathione peroxidase activity than cells cultured on either BCE or HR-9 basement membranes. Images Figure 3 Figure 1 Figure 2 Figure 5 PMID:3348362

  1. [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.

  2. [Variation of microflora and enzyme activity in continuous cropping cucumber soil in solar greenhouse].

    PubMed

    Ma, Yunhua; Wei, Min; Wang, Xiufeng

    2004-06-01

    Variation of microflora and enzyme activity in solar greenhouse soil continuous cropping for 1, 3, 5, 7, 9 years was studied, in addition to the relationship between soil properities and microflora and enzyme activity. The results showed that number of bacteria, actinomyce as well as total microflora showed a trend with a saddle-shaped curve, while the number of fungi appeared an liner increase. Continuous cropping soil microflora shifted from bacteria type to fungi type significantly, of which Ammoniation bacterium and Fusarium oxysporum were main physiology groups. Path analysis results showed that microelements (Mn, Cu, Fe), organic matter, available N and bulk density are main restricted factors of soil microflora and enzyme activity in solar greenhouse.

  3. [The antioxidant enzyme activity in mouse liver mitochondria after nanosecond pulsed periodic X-ray exposure].

    PubMed

    Kniazeva, I R; Ivanov, V V; Bol'shakov, M A; Zharkova, L P; Kereia, A V; Kutenkov, O P; Rostov, V V

    2013-01-01

    The effect of repetitive pulsed X-ray (4 ns pulse duration, 300 kV accelerating voltage; 2.5 kA electron beam current) on the antioxidant enzyme activity in mouse liver mitochondria has been investigated. The mitochondrial suspension was exposed to single 4000 pulse X-ray radiation with repetition rates ranging between 10 and 22 pps (pulsed dose was 0.3-1.8 x 10(-6) Gy/pulse, the total absorbed dose following a single exposure was 7.2 x 10(-3) Gy). It was shown that a short-time exposure to X-ray radiation changes the antioxidant enzyme activity in mouse liver mitochondria. The greatest effect was observed in the changes of the activity of the metal-containing enzymes: superoxide dismutase and glutathione peroxidase. The effect depends on the pulse repetition frequency and radiation dose.

  4. Brownian dynamics simulation of substrate motion near active site of enzyme entrapped inside reverse micelle.

    PubMed

    Ermakova, Elena A; Zakhartchenko, Nataliya L; Zuev, Yuri F

    2010-08-01

    Brownian dynamics simulation has been applied to analyze the influence of the electrostatic field of a reverse micelle on the enzyme-substrate complex formation inside a micelle. The probability that the enzyme-substrate complex will form from serine protease (trypsin) and the specific hydrophilic cationic substrate Nalpha-benzoyl-L: -arginine ethyl ester has been studied within the framework of the encounter complex formation theory. It has been shown that surfactant charge, dipole moments created by charged surfactant molecules and counterions, and permittivity of the inner core of reverse micelles can all be used as regulatory parameters to alter the substrate orientation near the active site of the enzyme and to change the probability that the enzyme-substrate complex will form.

  5. [Responses of soil enzyme activities to re-vegetation in gully Loess Plateau of Northwest China].

    PubMed

    Li, Lin-Hai; Qiu, Li-Ping; Meng, Meng

    2012-12-01

    In combining field investigation with laboratory analysis, this paper studied the distribution characteristics of soil enzyme activities along the soil profiles and natural slopes with different re-vegetation treatments in gully Loess Plateau, aimed to assess the responses of the soil enzyme activities to re-vegetation. In the study area, the activities of soil urease, invertase and alkaline phosphatase along natural slopes were highly varied, but the activity of soil catalase was in adverse. The profile distribution of the soil enzyme activities varied significantly with vegetation type, and with increasing soil depth, the activities of soil urease, invertase and alkaline phosphatase decreased while the catalase activity increased. There existed significant positive correlation among the three hydrolases activities. The activities of the three hydrolases were all significantly negatively correlated with soil physical properties and positively correlated with soil chemical properties, while the soil catalase activity was positively correlated with soil moisture content and pH and negatively correlated with other soil physiochemical properties. It was suggested that the activities of soil urease, invertase and alkaline phosphatase in gully Loess Plateau could be used as the sensitive indicators for the soil responses to the re-vegetation in the Plateau, and re-vegetation could improve the biological properties in both surface and deeper soil layers.

  6. Enzyme activity electrophoresis and rocket immunoelectrophoresis for the qualitative and quantitative analysis of Geotrichum candidum lipase activity.

    PubMed

    Jacobsen, T; Poulsen, O M; Hau, J

    1989-01-01

    The development and application of a rocket immunoelectrophoretic and an enzyme activity electrophoretic assay for the qualitative analysis of Geotrichum candidum lipase activity is presented. The sensitivities of the four assays were (in arbitrary units): enzyme activity electrophoresis, 1-0.5; rocket immunoelectrophoresis, 0.5-0.2; radial diffusion, 1; titrimetry, 1. The electrophoretic methods made it possible to distinguish between high and low molecular weight forms of the G. candidum lipases. The enzyme activity electrophoretic methods can be combined with other electrophoretic techniques, as demonstrated here with isoelectric focusing, and produce useful information on physico-chemical differences between different molecular forms of the lipase, e.g. forms with different pI.

  7. Analysis of the DXD motifs in human xylosyltransferase I required for enzyme activity.

    PubMed

    Götting, Christian; Müller, Sandra; Schöttler, Manuela; Schön, Sylvia; Prante, Christian; Brinkmann, Thomas; Kuhn, Joachim; Kleesiek, Knut

    2004-10-08

    Human xylosyltransferase I (XT-I) is the initial enzyme involved in the biosynthesis of the glycosaminoglycan linker region in proteoglycans. Here, we tested the importance of the DXD motifs at positions 314-316 and 745-747 for enzyme activity and the nucleotide binding capacity of human XT-I. Mutations of the 314DED316 motif did not have any effect on enzyme activity, whereas alterations of the 745DWD747 motif resulted in reduced XT-I activity. Loss of function was observed after exchange of the highly conserved aspartic acid at position 745 with glycine. However, mutation of Asp745 to glutamic acid retained full enzyme activity, indicating the importance of an acidic amino acid at this position. Reduced substrate affinity was observed for mutants D747G (Km=6.9 microm) and D747E (Km=4.4 microm) in comparison with the wild-type enzyme (Km=0.9 microm). Changing the central tryptophan to a neutral, basic, or acidic amino acid resulted in a 6-fold lower Vmax, with Km values comparable with those of the wild-type enzyme. Despite the major effect of the DWD motif on XT-I activity, nucleotide binding was not abolished in the D745G and D747G mutants, as revealed by UDP-bead binding assays. Ki values for inhibition by UDP were determined to be 1.9-24.6 microm for the XT-I mutants. The properties of binding of XT-I to heparin-beads, the Ki constants for noncompetitive inhibition by heparin, and the activation by protamine were not altered by the generated mutations.

  8. 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

  9. Enzyme activity assays within microstructured optical fibers enabled by automated alignment

    PubMed Central

    Warren-Smith, Stephen C.; Nie, Guiying; Schartner, Erik P.; Salamonsen, Lois A.; Monro, Tanya M.

    2012-01-01

    A fluorescence-based enzyme activity assay has been demonstrated within a small-core microstructured optical fiber (MOF) for the first time. To achieve this, a reflection-based automated alignment system has been developed, which uses feedback and piezoelectric actuators to maintain optical alignment. The auto-alignment system provides optical stability for the time required to perform an activity assay. The chosen assay is based on the enzyme proprotein convertase 5/6 (PC6) and has important applications in women’s health. PMID:23243579

  10. Genistein inhibits activities of methylenetetrahydrofolate reductase and lactate dehydrogenase, enzymes which use NADH as a substrate.

    PubMed

    Grabowski, Michał; Banecki, Bogdan; Kadziński, Leszek; Jakóbkiewicz-Banecka, Joanna; Kaźmierkiewicz, Rajmund; Gabig-Cimińska, Magdalena; Węgrzyn, Grzegorz; Węgrzyn, Alicja; Banecka-Majkutewicz, Zyta

    2015-09-25

    Genistein (5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a natural isoflavone revealing many biological activities. Thus, it is considered as a therapeutic compound in as various disorders as cancer, infections and genetic diseases. Here, we demonstrate for the first time that genistein inhibits activities of bacterial methylenetetrahydrofolate reductase (MetF) and lactate dehydrogenase (LDH). Both enzymes use NADH as a substrate, and results of biochemical as well as molecular modeling studies with MetF suggest that genistein may interfere with binding of this dinucleotide to the enzyme. These results have implications for our understanding of biological functions of genistein and its effects on cellular metabolism.

  11. Effects of pyrite sludge pollution on soil enzyme activities: ecological dose-response model.

    PubMed

    Hinojosa, M Belén; Carreira, José A; Rodríguez-Maroto, José M; García-Ruíz, Roberto

    2008-06-25

    A laboratory study was conducted to evaluate the response of soil enzyme activities (acid and alkaline phosphatase, beta-glucosidase, arylsulfatase, urease and dehydrogenase) to different levels of trace elements pollution in soils representative of the area affected by the pyrite sludge mining spill of Aznalcóllar (Guadiamar basin, SW Spain). Three uncontaminated soils from the study area were mixed with different loads of pyrite sludge to resemble field conditions and criteria applied for reclamation practices following the pollution incident: 0% ("reference" or background level), 1.3% ("attention level", further monitoring required), 4% ("intervention level", further cleaning and liming required) and 13% (ten times the "attention level"). Enzyme activities were analysed 4, 7, 14, 21, 34 and 92 days after pollutant addition and those measured after 92 days were used to calculate the ecological dose value (ED50). Soil enzyme activities and pH decreased after the pyrite sludge addition with respect to the "reference level" (0% pyrite sludge), whereas soil bioavailable (DTPA-extractable) trace elements concentration increased. Arylsulfatase, beta-glucosidase and phosphatase activities were reduced by more than 50% at 1.3% pyrite sludge dose. Arylsulfasate was the most sensitive soil enzyme (in average, ED50=0.99), whereas urease activity showed the lowest inhibition (in average, ED50=7.87) after pyrite sludge addition. Our results showed that the ecological dose concept, applied to enzyme activities, was satisfactory to quantify the effect of a multi-metalic pollutant (pyrite sludge) on soil functionality, and would provide manageable data to establish permissible limits of trace elements in polluted soils. Additionally, we evaluate the recovery of enzyme activities after addition of sugar-beet lime (calcium carbonate) to each experimentally polluted soil. The amount of lime added to each soil was enough to raise the pH to the original value (equal to control soil

  12. Evaluation of fungicidal activity of extracellular filtrates of cyanobacteria--possible role of hydrolytic enzymes.

    PubMed

    Prasanna, Radha; Nain, Lata; Tripathi, Ravindramani; Gupta, Vishal; Chaudhary, Vidhi; Middha, Sheetal; Joshi, Monica; Ancha, Radhika; Kaushik, Brahma D

    2008-06-01

    A set of seventy axenised and unicyanobacterial isolates belonging to the genus Anabaena were evaluated for biocidal activity against a set of phytopathogenic fungi. Among them, 35 Anabaena strains showed zone of inhibition against one or more fungi. The extracellular filtrates from 4 and 8 weeks old cultures of these Anabaena strains were further evaluated in terms of hydrolytic enzymes, proteins and IAA employing standard methods. Significant differences were also observed among the strains in terms of their FPase, chitosanase and xylanase activity, while low and relatively similar values of CMCase, cellobiase and protease activity were recorded in the strains analyzed. IAA production was also observed in all the strains. Comparative evaluation of activity of hydrolytic enzymes and antifungal activity revealed that such enzymes may contribute to the fungicidal activity of the cyanobacterial strains, besides other bioactive compounds, including IAA, which are established promising traits for biocontrol agents. This study is a first time report on the production of hydrolytic enzymes by these oxygenic photosynthetic prokaryotes, which can be potential candidates for the development of biocontrol agent(s) against selected phytopathogenic fungi.

  13. Molecular and cellular effects of NEDD8-activating enzyme inhibition in myeloma.

    PubMed

    McMillin, Douglas W; Jacobs, Hannah M; Delmore, Jake E; Buon, Leutz; Hunter, Zachary R; Monrose, Val; Yu, Jie; Smith, Peter G; Richardson, Paul G; Anderson, Kenneth C; Treon, Steven P; Kung, Andrew L; Mitsiades, Constantine S

    2012-04-01

    The NEDD8-activating enzyme is upstream of the 20S proteasome in the ubiquitin/proteasome pathway and catalyzes the first step in the neddylation pathway. NEDD8 modification of cullins is required for ubiquitination of cullin-ring ligases that regulate degradation of a distinct subset of proteins. The more targeted impact of NEDD8-activating enzyme on protein degradation prompted us to study MLN4924, an investigational NEDD8-activating enzyme inhibitor, in preclinical multiple myeloma models. In vitro treatment with MLN4924 led to dose-dependent decrease of viability (EC(50) = 25-150 nmol/L) in a panel of human multiple myeloma cell lines. MLN4924 was similarly active against a bortezomib-resistant ANBL-6 subline and its bortezomib-sensitive parental cells. MLN4924 had submicromolar activity (EC(50) values <500 nmol/L) against primary CD138(+) multiple myeloma patient cells and exhibited at least additive effect when combined with dexamethasone, doxorubicin, and bortezomib against MM.1S cells. The bortezomib-induced compensatory upregulation of transcripts for ubiquitin/proteasome was not observed with MLN4924 treatment, suggesting distinct functional roles of NEDD8-activating enzyme versus 20S proteasome. MLN4924 was well tolerated at doses up to 60 mg/kg 2× daily and significantly reduced tumor burden in both a subcutaneous and an orthotopic mouse model of multiple myeloma. These studies provide the framework for the clinical investigation of MLN4924 in multiple myeloma.

  14. [Study on soil enzyme activities and microbial biomass carbon in greenland irrigated with reclaimed water].

    PubMed

    Pan, Neng; Hou, Zhen-An; Chen, Wei-Ping; Jiao, Wen-Tao; Peng, Chi; Liu, Wen

    2012-12-01

    The physicochemical properties of soils might be changed under the long-term reclaimed water irrigation. Its effects on soil biological activities have received great attentions. We collected surface soil samples from urban green spaces and suburban farmlands of Beijing. Soil microbial biomass carbon (SMBC), five types of soil enzyme activities (urease, alkaline phosphatase, invertase, dehydrogenase and catalase) and physicochemical indicators in soils were measured subsequently. SMBC and enzyme activities from green land soils irrigated with reclaimed water were higher than that of control treatments using drinking water, but the difference is not significant in farmland. The SMBC increased by 60.1% and 14.2% than those control treatments in 0-20 cm soil layer of green land and farmland, respectively. Compared with their respective controls, the activities of enzymes in 0-20 cm soil layer of green land and farmland were enhanced by an average of 36.7% and 7.4%, respectively. Investigation of SMBC and enzyme activities decreased with increasing of soil depth. Significantly difference was found between 0-10 cm and 10-20 cm soil layer in green land. Soil biological activities were improved with long-term reclaimed water irrigation in Beijing.

  15. Ethanol tolerance and membrane fatty acid adaptation in adh multiple and null mutants of Kluyveromyces lactis.

    PubMed

    Heipieper, H J; Isken, S; Saliola, M

    2000-11-01

    The effects of ethanol and 1-octanol on growth and fatty acid composition of different strains of Kluyveromyces lactis containing a mutation in the four different alcohol dehydrogenase (KlADH) genes were investigated. In the presence of ethanol and 1-octanol K. lactis reduced the fluidity of its lipids by decreasing the unsaturation index (UI) of its membrane fatty acids. In this way, a direct correlation between nonlethal ethanol concentrations and the decrease in the UI could be observed. At concentrations which totally inhibited cell growth no reaction occurred. These adaptive modifications of the fatty acid pattern of K. lactis to ethanol contrasted with those reported for Saccharomyces cerevisiae and Schizosaccharomyces pombe. Whereas these two yeasts increased the fluidity of their membrane lipids in the presence of ethanol, K. lactis reduced the fluidity (UI) of its lipids. Among the different isogenic adh negative strains tested, the strain containing no ADH (adh0) and that containing only KlADH1 were the most alcohol-sensitive. The strain with only KlADH2 showed nearly the same tolerance as reference strain CBS 2359/152 containing all four ADH genes. This suggests that the KlADH2 product could play an important role in the adaptation/detoxification reactions of K. lactis to high ethanol concentrations.

  16. Structure, Expression, Chromosomal Location and Product of the Gene Encoding Adh2 in Petunia

    PubMed Central

    Gregerson, R. G.; Cameron, L.; McLean, M.; Dennis, P.; Strommer, J.

    1993-01-01

    In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes. PMID:8096485

  17. Disconnect of microbial structure and function: enzyme activities and bacterial communities in nascent stream corridors

    PubMed Central

    Frossard, Aline; Gerull, Linda; Mutz, Michael; Gessner, Mark O

    2012-01-01

    A fundamental issue in microbial and general ecology is the question to what extent environmental conditions dictate the structure of communities and the linkages with functional properties of ecosystems (that is, ecosystem function). We approached this question by taking advantage of environmental gradients established in soil and sediments of small stream corridors in a recently created, early successional catchment. Specifically, we determined spatial and temporal patterns of bacterial community structure and their linkages with potential microbial enzyme activities along the hydrological flow paths of the catchment. Soil and sediments were sampled in a total of 15 sites on four occasions spread throughout a year. Denaturing gradient gel electrophoresis (DGGE) was used to characterize bacterial communities, and substrate analogs linked to fluorescent molecules served to track 10 different enzymes as specific measures of ecosystem function. Potential enzyme activities varied little among sites, despite contrasting environmental conditions, especially in terms of water availability. Temporal changes, in contrast, were pronounced and remarkably variable among the enzymes tested. This suggests much greater importance of temporal dynamics than spatial heterogeneity in affecting specific ecosystem functions. Most strikingly, bacterial community structure revealed neither temporal nor spatial patterns. The resulting disconnect between bacterial community structure and potential enzyme activities indicates high functional redundancy within microbial communities even in the physically and biologically simplified stream corridors of early successional landscapes. PMID:22030674

  18. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  19. Circadian pancreatic enzyme pattern and relationship between secretory and motor activity in fasting humans.

    PubMed

    Keller, Jutta; Layer, Peter

    2002-08-01

    It is unknown whether nonparallel pancreatic enzyme output occurs under basal conditions in humans. We aimed to determine whether the circadian or wake-sleep cycle influences the relationship among pancreatic enzymes or between pancreatic secretory and jejunal motor activity. Using orojejunal multilumen intubation, we measured enzyme outputs and proximal jejunal motility index during consecutive daytime and nighttime periods in each of seven fasting, healthy volunteers. Enzyme outputs were correlated tightly during daytime phases of wakefulness and nighttime phases of sleep (r > 0.72, P < 0.001). During nocturnal phases of wakefulness, output of proteases (r = 0.84, P < 0.001), but not of amylase and trypsin (r = 0.12), remained associated. Nocturnally, particularly during sleep, pancreatic secretory activity was directly correlated with jejunal motility index (r > 0.50, P < 0.001). In conclusion, parallel secretion of pancreatic enzymes dominates throughout the circadian cycle. Nonparallel secretion during nocturnal phases of wakefulness may be due to merely circadian effects or to the coupling of the wake-sleep and the circadian cycle. The association between fluctuations of secretory and motor activity appears to be particularly tight during the night.

  20. [Effects of stereoscopic cultivation on soil microorganism, enzyme activity and the agronomic characters of Panax notoginseng].

    PubMed

    Liao, Pei-ran; Cui, Xiu-ming; Lan, Lei; Chen, Wei-dong; Wang, Cheng-xiao; Yang, Xiao-yan; Liu, Da-hui; Yang, Ye

    2015-08-01

    Compartments of soil microorganism and enzymes between stereoscopic cultivation (three storeys) and field cultivation (CK) of Panax notoginseng were carried out, and the effects on P. notoginseng agronomic characters were also studied. Results show that concentration of soil microorganism of stereoscopic cultivation was lower than field cultivation; the activity of soil urea enzyme, saccharase and neutral phosphatase increased from lower storey to upper storey; the activity of soil urea enzyme and saccharase of lower and upper storeys were significantly lower than CK; agronomic characters of stereoscopic cultivated P. notoginsengin were inferior to field cultivation, the middle storey with the best agronomic characters among the three storeys. The correlation analysis showed that fungi, actinomycetes and neutral phosphatase were significantly correlated with P. notoginseng agronomic characters; concentration of soil fungi and bacteria were significantly correlated with the soil relative water content; actinomycete and neutral phosphatase were significantly correlated with soil pH and relative water content, respectively; the activities of soil urea enzyme and saccharase were significantly correlated with the soil daily maximum temperature difference. Inconclusion, The current research shows that the imbalance of soil microorganism and the acutely changing of soil enzyme activity were the main reasons that caused the agronomic characters of stereoscopic cultivated P. notoginseng were worse than field cultivation. Thus improves the concentration of soil microorganism and enzyme activity near to field soil by improving the structure of stereoscopic cultivation is very important. And it was the direction which we are endeavoring that built better soil ecological environment for P. notoginseng of stereoscopic cultivation.

  1. Molecular dynamics explorations of active site structure in designed and evolved enzymes.

    PubMed

    Osuna, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L; Houk, K N

    2015-04-21

    This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP

  2. [Degradation of phthalate esters in soil and the effects on soil enzyme activities].

    PubMed

    Zhang, Jian; Shi, Yi-Jing; Cui, Yin; Xie, Hui-Jun; Wang, Wen-Xing

    2010-12-01

    Phthalate esters (PAEs) are a kind of widespread toxic organic compounds in the environment. We discussed the different degradation rate of four kinds of PAEs in the soil and its impact on different soil enzyme activities. We used GC-MS methods to determine the concentration of PAEs in soil. The results showed that soil microorganisms play a major role in the degradation of PAEs. The biodegradation diagram of PAEs was accord with first-order kinetics equation. And the shorter carbon chain, the better degradation efficiency. With the high concentration of PAE30, DnOP, which has long carbon chain, the degradation efficiency is lower than that of PAE1 and PAE10, only 73% was degraded after 40 days. We use standard methods to determine the matrix enzyme activities, after adding the PAEs into soil, beta-glucosidase, phosphatase, urease, protease activity have changed. Phosphatase activity decreased at first and then increased, beta-glucosidase activity decreased slowly, protease activity increased at first and then decreased, the activity of urease increased gradually. After 20 days, except for beta-glucosidase activity continued decreasing, the activities of others enzyme recovered gradually, and higher than the control group.

  3. Steroidogenic enzyme activity and ovarian differentiation in the saltwater crocodile, Crocodylus porosus.

    PubMed

    Smith, C A; Joss, J M

    1994-02-01

    It has been hypothesized that steroid hormone production is involved in sexual differentiation of the gonads in reptiles with temperature-dependent sex determination. We have therefore examined steroidogenic enzymes and ovarian differentiation in Crocodylus porosus embryos incubated at 30 degrees, a temperature producing 100% female hatchlings. delta 5-3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) activity was detected only in the adrenal gland before, during, and after the period of ovary differentiation. The same pattern was observed during testis differentiation in embryos incubated at 32 degrees (predominantly male-producing). At no stage was 3 beta-HSD activity detected in the gonads. The tritiated water assay was used to measure aromatase enzyme activity in the gonad-adrenal-mesonephric complex (GAM) during development at 30 degrees C. Aromatase activity in the GAM increased during the period of ovary differentiation. In female C. porosus hatchlings, 85% of the aromatase activity within the GAM was derived from the ovary, 10% from the adrenal, and 5% from the regressing mesonephros. The aromatase inhibitors aminoglutethimide and 4-hydroxyandrostenedione significantly reduced aromatase activity in female hatchling GAMs. In some stage 25 embryos incubated at 32.5 degrees, aromatase activity was low in testes and high in ovaries. These observations show that urogenital tissues of C. porosus are steroidogenically active during gonadal differentiation and that increased aromatase enzyme activity accompanies ovary development at 30 degrees. Steroid synthesis in the GAM of C. porosus embryos may involve interaction between the adrenal glands and the gonads.

  4. The inhibitory effect of the cerebellar fastigial stimulation on ADH secretion.

    PubMed

    Hata, N; Miura, M

    1974-11-01

    1. Both cardiovascular and antidiuretic hormone (ADH) responses to some neural inputs were examined in paralysed anaesthetized cats.2. Carotid occlusion elicited cardiovascular responses and increased ADH secretion. When the electrical stimulation of discrete loci of the cerebellar fastigial nucleus (fastigial pressor area) was superimposed on carotid occlusion, cardiovascular responses were further facilitated, while ADH secretion was inhibited.3. The fastigial stimulation alone elicited facilitory cardiovascular responses composed of hypertension and tachycardia, and the fastigial pressor response (FPR), but did not evoke any consistent ADH response.4. These facts indicate that cerebellar modulation of ADH secretion occurs not directly via the hypothalamo-hypophysial system but through the lower brain stem to which both carotid sinus nerves and outflows from the fastigial pressor area project.5. We conclude that the fastigial pressor area is specific for not only cardiovascular and other autonomic responses but pituitary hormonal response.

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

    PubMed Central

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

    2014-01-01

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

  6. Influence of microcystin-LR on the activity of membrane enzymes in rat intestinal mucosa.

    PubMed

    Moreno, I M; Mate, A; Repetto, G; Vázquez, C M; Cameán, A M

    2003-12-01

    The objective of the present study was to evaluate the effects of microcystin-LR (MCLR) on the activity of membrane enzymes from intestinal mucosa. In addition, serum chemistry and peroxidative status of both serum and intestinal homogenate were evaluated after treatment with MCLR. Wistar rats were treated with intraperitoneal injection of either 100 microg pure MCLR/Kg body weight or saline solution. A significant increase in liver weight and altered serum enzyme activities were found in MCLR-treated rats, indicating damage to the liver in these rats, as previously suggested. A higher specific activity of sucrase (1.5-fold) was observed after the administration of MCLR, whereas other intestinal apical membrane enzymes, such as lactase, maltase and alkaline phosphatase were not modified by the treatment. The specific activities of acid phosphatase and succinate dehydrogenase, markers for lysosomal and mitochondrial membranes, respectively, were also increased (32% and 60%, respectively) in treated rats. The analysis of lipid peroxidation showed that the peroxidative status was increased in both serum and intestinal mucosa from MCLR-treated rats, reflecting an excess production of oxygen free radicals induced by this cyanobacterial toxin. In conclusion, this study shows that acute exposure to MCLR affects the intestinal physiology by modifying the intestinal peroxidation status as well as the activity of membrane enzymes.

  7. Facet Energy versus Enzyme-like Activities: The Unexpected Protection of Palladium Nanocrystals against Oxidative Damage.

    PubMed

    Ge, Cuicui; Fang, Ge; Shen, Xiaomei; Chong, Yu; Wamer, Wayne G; Gao, Xingfa; Chai, Zhifang; Chen, Chunying; Yin, Jun-Jie

    2016-11-22

    To develop nanomaterials as artificial enzymes, it is necessary to better understand how their physicochemical properties affect their enzyme-like activities. Although prior research has demonstrated that nanomaterials exhibit tunable enzyme-like activities depending on their size, structure, and composition, few studies have examined the effect of surface facets, which determine surface energy or surface reactivity. Here, we use electron spin-resonance spectroscopy to report that lower surface energy {111}-faceted Pd octahedrons have greater intrinsic antioxidant enzyme-like activity than higher surface energy {100}-faceted Pd nanocubes. Our in vitro experiments found that those same Pd octahedrons are more effective than Pd nanocubes at scavenging reactive oxygen species (ROS). Those reductions in ROS preserve the homogeneity of mitochondrial membrane potential and attenuate damage to important biomolecules, thereby allowing a substantially higher number of cells to survive oxidative challenges. Our computations of molecular mechanisms for the antioxidant activities of {111}- and {100}-faceted Pd nanocrystals, as well as their activity order, agree well with experimental observations. These findings can guide the design of antioxidant-mimicking nanomaterials, which could have therapeutic or preventative potential against oxidative stress related diseases.

  8. Probiotic activity of lignocellulosic enzyme as bioactivator for rice husk degradation

    NASA Astrophysics Data System (ADS)

    Lamid, Mirni; Al-Arif, Anam; Warsito, Sunaryo Hadi

    2017-02-01

    The utilization of lignocellulosic enzyme will increase nutritional value of rice husk. Cellulase consists of C1 (β-1, 4-glucan cellobiohydrolase or exo-β-1,4glucanase), Cc (endo-β-1,4-glucanase) and component and cellobiose (β-glucocidase). Hemicellulase enzyme consists of endo-β-1,4-xilanase, β-xilosidase, α-L arabinofuranosidase, α-D-glukuronidaseand asetil xilan esterase. This research aimed to study the activity of lignocellulosic enzyme, produced by cows in their rumen, which can be used as a bioactivator in rice husk degradation. This research resulted G6 and G7 bacteria, producing xylanase and cellulase with the activity of 0.004 U mL-1 and 0.021 U mL-1; 0.003 ( U mL-1) and 0.026 (U mL-1) respectively.

  9. The universal epitope of influenza A viral neuraminidase fundamentally contributes to enzyme activity and viral replication.

    PubMed

    Doyle, Tracey M; Jaentschke, Bozena; Van Domselaar, Gary; Hashem, Anwar M; Farnsworth, Aaron; Forbes, Nicole E; Li, Changgui; Wang, Junzhi; He, Runtao; Brown, Earl G; Li, Xuguang

    2013-06-21

    The only universally conserved sequence among all influenza A viral neuraminidases is located between amino acids 222 and 230. However, the potential roles of these amino acids remain largely unknown. Through an array of experimental approaches including mutagenesis, reverse genetics, and growth kinetics, we found that this sequence could markedly affect viral replication. Additional experiments revealed that enzymes with mutations in this region demonstrated substantially decreased catalytic activity, substrate binding, and thermostability. Consistent with viral replication analyses and enzymatic studies, protein modeling suggests that these amino acids could either directly bind to the substrate or contribute to the formation of the active site in the enzyme. Collectively, these findings reveal the essential role of this unique region in enzyme function and viral growth, which provides the basis for evaluating the validity of this sequence as a potential target for antiviral intervention and vaccine development.

  10. Hydrolytic enzyme activities in shiitake mushroom (Lentinula edodes) strains cultivated on coffee pulp.

    PubMed

    Mata, Gerardo; Salmones, Dulce; Pérez-Merlo, Rosalía

    Hydrolytic enzyme production (cellulases, laminarinases and xylanases) was studied in cultures of Lentinula edodes on sterilized coffee pulp. Samples of substrate colonized by mycelia were taken after 7, 14, 21, 28 and 35 days of incubation at 25°C (W1 to W5) and during the fruiting period at different stages: formation of primordia (PF), first harvest (H) and one week after the first harvest (PH). The enzymatic activity was lower during the early mycelial growth and showed higher levels during the formation and development of fruiting bodies. During the reproductive stage of the fungus, the samples were subjected to a soaking treatment; however, it was not possible to relate this soaking treatment to the increase in enzyme production. The levels of enzymatic activity suggest that secretion of the studied enzymes does not influence the adaptability of the strains to the substrate.

  11. Soil extracellular enzyme activities, soil carbon and nitrogen storage under nitrogen fertilization: A meta-analysis

    SciTech Connect

    Jian, Siyang; Li, Jianwei; Chen, Ji; Wang, Gangsheng; Mayes, Melanie A.; Dzantor, Kudjo E.; Hui, Dafeng; Luo, Yiqi

    2016-07-08

    Nitrogen (N) fertilization affects the rate of soil organic carbon (SOC) decomposition by regulating extracellular enzyme activities (EEA). Extracellular enzymes have not been represented in global biogeochemical models. Understanding the relationships among EEA and SOC, soil N (TN), and soil microbial biomass carbon (MBC) under N fertilization would enable modeling of the influence of EEA on SOC decomposition. Based on 65 published studies, we synthesized the activities of α-1,4-glucosidase (AG), β-1,4-glucosidase (BG), β-d-cellobiosidase (CBH), β-1,4-xylosidase (BX), β-1,4-N-acetyl-glucosaminidase (NAG), leucine amino peptidase (LAP), urease (UREA), acid phosphatase (AP), phenol oxidase (PHO), and peroxidase (PEO) in response to N fertilization. Here, the proxy variables for hydrolytic C acquisition enzymes (C-acq), N acquisition (N-acq), and oxidative decomposition (OX) were calculated as the sum of AG, BG, CBH and BX; AG and LAP; PHO and PEO, respectively.

  12. Markers of oxidative stress and erythrocyte antioxidant enzyme activity in older men and women with differing physical activity.

    PubMed

    Rowiński, Rafał; Kozakiewicz, Mariusz; Kędziora-Kornatowska, Kornelia; Hübner-Woźniak, Elżbieta; Kędziora, Józef

    2013-11-01

    The aim of the present study was to examine the relationship between markers of oxidative stress and erythrocyte antioxidant enzyme activity and physical activity in older men and women. The present study included 481 participants (233 men and 248 women) in the age group 65-69 years (127 men and 125 women) and in the age group 90 years and over (106 men and 123 women). The classification of respondents by physical activity was based on answers to the question if, in the past 12 months, they engaged in any pastimes which require physical activity. The systemic oxidative stress status was assessed by measuring plasma iso-PGF2α and protein carbonyl concentration as well as erythrocyte antioxidant enzymes activity, i.e., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR). The concentration of plasma iso-PGF2α and protein carbonyls (CP) was lower in groups of younger men and women compared to the respective older groups. In all examined groups, physical activity resulted in decrease of these oxidative stress markers and simultaneously caused adaptive increase in the erythrocyte SOD activity. Additionally, in active younger men CAT, GPx, and GR activities were higher than in sedentary ones. In conclusion, oxidative stress increase is age-related, but physical activity can reduce oxidative stress markers and induce adaptive increase in the erythrocyte antioxidant enzyme activity, especially SOD, even in old and very old men and women.

  13. Apoferritin Nanoparticle: A Novel and Biocompatible Carrier for Enzyme Immobilization with Enhanced Activity and Stability

    SciTech Connect

    Zhang, Youyu; Tang, Zhiwen; Wang, Jun; Wu, Hong J.; Lin, Chiann Tso; Lin, Yuehe

    2011-11-01

    Apoferritin is a nanostructured material with a uniform size and spherical structure, and it has excellent bio-compatibility. In this work, we report the use of apoferritin as a novel and biocompatible carrier for stabilizing enzymes and their activities. We used glucose oxidase (GOx) as a model enzyme. GOx was immobilized on the surface of the apoferritin through a green synthetic approach taking advantage of bioaffinity binding between streptavidin and biotin. As a result, a glucose oxidase-biotin/streptavidin/biotin-apoferritin conjugate (Apo-GOx) was prepared using streptavidin as a bridge. The synthesized Apo-GOx was characterized with transmission electron microscopy, ultraviolet, and fluorescence spectroscopy. The activity and stability of GOx on the surface of the apoferritin were studied in different environments, such as temperature, chemicals, and pH, in comparison with the biotinylated GOx (B-GOx). The results showed that the activity of GOx on the apoferritin surface was significantly enhanced. The thermal and chemical stability of the GOx on the apoferritin was also greatly improved compared to free B-GOx in a solution. It was found that the activity of the GOx on the apoferritin only lost 30% in comparison to a 70% loss of free B-GOx after a 2 h incubation at 50oC. There was almost no decrease in activity for the GOx on the apoferritin as compared to an 80% activity decrease for free B-GOx after 30 min incubation in a 5 M urea solution. Glucose detection was used as a model application for the enzyme immobilization method developed in this work. The GOx immobilized apoferritin nanoparticles exhibited high sensitivity for glucose detection with a detection limit of 3 nM glucose. This work offers a novel approach for immobilizing enzymes with enhanced stability and activity, and this method may find a number of applications, such as in enzyme catalysis, DNA assays and immunoassays.

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

    PubMed Central

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

    2013-01-01

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

  15. Effects of long term irrigation with polluted water and sludge amendment on some soil enzyme activities

    SciTech Connect

    Topac, F.O.; Baskaya, H.S.; Alkan, U.; Katkat, A.V.

    2008-01-15

    The objective of this study was to determine the effects of wastewater sludge-fly ash mixtures on urease, dehydrogenase, alkaline phosphatase and beta-glucosidase activities in soils. In order to evaluate the probable effects of previous soil management practices (irrigation with polluted water) on soil enzymes, two different soil samples which were similar in physical properties, but different in irrigation practice were used. The application of wastewater sludges supplemented with varying doses of fly ash increased potential enzyme activities for a short period of time (3 months) in comparison to unamended soils. However, the activity levels generally showed a decreasing trend with increasing ash ratios indicating the inhibitory effect of fly ash. The urease and dehydrogenase activities were particularly lower in soils irrigated from a polluted stream, indicating the negative effects of the previous soil management on soil microbial activity.

  16. Influence of protoplast fusion between two Trichoderma spp. on extracellular enzymes production and antagonistic activity

    PubMed Central

    Hassan, Mohamed M.

    2014-01-01

    Biological control plays a crucial role in grapevine pathogens disease management. The cell-wall degrading enzymes chitinase, cellulase and β-glucanase have been suggested to be essential for the mycoparasitism activity of Trichoderma species against grapevine fungal pathogens. In order to develop a useful strain as a single source of these vital enzymes, it was intended to incorporate the characteristics of two parental fungicides tolerant mutants of Trichoderma belonging to the high chitinase producing species T. harzianum and the high cellulase producing species T. viride, by fusing their protoplasts. The phylogeny of the parental strains was carried out using a sequence of the 5.8S-ITS region. The BLAST of the obtained sequence identified these isolates as T. harzianum and T. viride. Protoplasts were isolated using lysing enzymes and were fused using polyethylene glycol. The fused protoplasts have been regenerated on protoplast regeneration minimal medium supplemented with two selective fungicides. Among the 40 fast growing fusants, 17 fusants were selected based on their enhanced growth on selective media for further studies. The fusant strains were growing 60%–70% faster than the parents up to third generation. All the 17 selected fusants exhibited morphological variations. Some fusant strains displayed threefold increased chitinase enzyme activity and twofold increase in β-glucanase enzyme activity compared to the parent strains. Most fusants showed powerful antagonistic activity against Macrophomin aphaseolina, Pythium ultimum and Sclerotium rolfsii pathogens. Fusant number 15 showed the highest inhibition percentage (92.8%) against M. phaseolina and P. ultimum, while fusant number 9 showed the highest inhibition percentage (98.2%) against the growth of S. rolfsii. A hyphal intertwining and degradation phenomenon was observed by scanning electron microscope. The Trichoderma antagonistic effect against pathogenic fungal mycelia was due to the

  17. Reconciling Apparent Variability in Effects of Biochar Amendment on Soil Enzyme Activities by Assay Optimization

    SciTech Connect

    Bailey, Vanessa L.; Fansler, Sarah J.; Smith, Jeffery L.; Bolton, Harvey

    2011-02-01

    Applying biochar to soils as an ameliorative substance and mechanism for C sequestration has received a great deal of interest in light of the sustained fertility observed in the Terra Preta soils of Brazil. The effects of synthetic biochars on biochemical processes needs to be better understood in order to determine if this is a reasonable practice in managed systems. The biochar studied was formed from the fast-pyrolysis of a switchgrass feedstock. Four soil enzymes were studied: β-glucosidase, β-N-acetylglucosaminidase, lipase, and leucine aminopeptidase. Both colorimetric and fluorescent assays were used for β-glucosidase and β-N-acetylglucosaminidase. Seven days after biochar was added to microcosms of a Palouse silt loam, the fluorescence-based assays indicated increased activities of the four enzymes, compared to non-amended soil. To clarify the mechanisms of the observed effects,in the absence of soil, purified enzymes or substrates were briefly exposed to biochar and then assayed. Except for β-N-acetylglucosaminidase, the exposure of substrate to biochar reduced the apparent activity of the remaining three enzymes in vitro, suggesting that sorption reactions between the substrate and biochar either removed the substrate from the assays or impeded the enzyme binding. The activity of purified β-N-acetylglucosaminidase increased significantly following biochar exposure, suggesting a chemical stimulation of enzyme functioning. We conclude that biochar added to soil acts as a substrate that can stimulate the soil microbial biomass and its activity. Our in vitro study suggests that biochar is not biochemically inert. Biochar amendments are likely to have effects that are currently difficult to predict, and that could impact overall soil function.

  18. Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

    PubMed Central

    Geiser, Elena; Reindl, Michèle; Blank, Lars M.; Feldbrügge, Michael

    2016-01-01

    ABSTRACT The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and β-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process. IMPORTANCE This study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis. As a plant pathogen, this fungus contains a

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

    SciTech Connect

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

    2008-11-10

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

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  1. Factors Influencing the Measurement of Lysosomal Enzymes Activity in Human Cerebrospinal Fluid

    PubMed Central

    Parnetti, Lucilla; Eusebi, Paolo; Paciotti, Silvia; De Carlo, Claudia; Codini, Michela; Tambasco, Nicola; Rossi, Aroldo; Agnaf, Omar M. El.; Calabresi, Paolo; Beccari, Tommaso

    2014-01-01

    Measurements of the activities of lysosomal enzymes in cerebrospinal fluid have recently been proposed as putative biomarkers for Parkinson's disease and other synucleinopathies. To define the operating procedures useful for ensuring the reliability of these measurements, we analyzed several pre-analytical factors that may influence the activity of β-glucocerebrosidase, α-mannosidase, β-mannosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, cathepsin D and cathepsin E in cerebrospinal fluid. Lysosomal enzyme activities were measured by well-established fluorimetric assays in a consecutive series of patients (n = 28) with different neurological conditions, including Parkinson's disease. The precision, pre-storage and storage conditions, and freeze/thaw cycles were evaluated. All of the assays showed within- and between-run variabilities below 10%. At −20°C, only cathepsin D was stable up to 40 weeks. At −80°C, the cathepsin D, cathepsin E, and β-mannosidase activities did not change significantly up to 40 weeks, while β-glucocerebrosidase activity was stable up to 32 weeks. The β-galactosidase and α-fucosidase activities significantly increased (+54.9±38.08% after 4 weeks and +88.94±36.19% after 16 weeks, respectively). Up to four freeze/thaw cycles did not significantly affect the activities of cathepsins D and E. The β-glucocerebrosidase activity showed a slight decrease (−14.6%) after two freeze/thaw cycles. The measurement of lysosomal enzyme activities in cerebrospinal fluid is reliable and reproducible if pre-analytical factors are accurately taken into consideration. Therefore, the analytical recommendations that ensue from this study may contribute to the establishment of actual values for the activities of cerebrospinal fluid lysosomal enzymes as putative biomarkers for Parkinson's disease and other neurodegenerative disorders. PMID:24983953

  2. Xyloglucan endotransglycosylase, a new wall-loosening enzyme activity from plants.

    PubMed

    Fry, S C; Smith, R C; Renwick, K F; Martin, D J; Hodge, S K; Matthews, K J

    1992-03-15

    1. Cell-free extracts of all plants tested contained a novel enzyme activity (xyloglucan endotransglycosylase, XET) able to transfer a high-Mr portion from a donor xyloglucan to a suitable acceptor such as a xyloglucan-derived nonasaccharide (Glc4Xyl3GalFuc; XG9). 2. A simple assay for the enzyme, using [3H]XG9 and based on the ability of the [3H]polysaccharide product to bind to filter paper, is described. 3. The enzyme was highly specific for xyloglucan as the glycosyl donor, and showed negligible transglycosylation of other polysaccharides, including CM-cellulose. 4. The Km for XG9 was 50 microM; certain other 3H-labelled xyloglucan oligosaccharides also acted as acceptors, and certain non-radioactive xyloglucan oligosaccharides competed with [3H]XG9 as acceptor; the minimum acceptor structure was deduced to be: [formula: see text] 5. The pH optimum was approx. 5.5 and the enzyme was less than half as active at pH 7.0. The enzyme was slightly activated by Ca2+, Mg2+, Mn2+, spermidine, ascorbate and 2-mercaptoethanol, and inhibited by Ag+, Hg2+, Zn2+ and La3+. 6. XET activity was essentially completely extracted by aqueous solutions of low ionic strength; Triton X-100, Ca2+, La3+, and Li+ did not enhance extraction. Negligible activity was left in the unextractable (cell-wall-rich) residue. 7. The enzyme differed from the major cellulases (EC 3.2.1.4) of pea in: (a) susceptibility to inhibition by cello-oligosaccharides, (b) polysaccharide substrate specificity, (c) inducibility by auxin, (d) requirement for salt in the extraction buffer and (e) activation by 2-mercaptoethanol. XET is therefore concluded to be a new enzyme activity (xyloglucan: xyloglucan xyloglucanotransferase; EC 2.4.1.-). 8. XET was detected in extracts of the growing portions of dicotyledons, monocotyledons (graminaceous and liliaceous) and bryophytes. 9. The activity was positively correlated with growth rate in different zones of the pea stem. 10. We propose that XET is responsible for

  3. Propene activation by the oxo-iron active species of taurine/alpha-ketoglutarate dioxygenase (TauD) enzyme. How does the catalysis compare to heme-enzymes?

    PubMed

    de Visser, Sam P

    2006-08-02

    Density functional calculations on the oxygenation reaction of propene by a model for taurine/alpha-ketoglutarate dioxygenase (TauD) enzyme are presented. The oxo-iron active species of TauD is shown to be a powerful and aggressive oxidant, which is able to hydroxylate C-H bonds and epoxidize C=C bonds with low barriers. In the case of propene oxygenation, the hydroxylation and epoxidation mechanisms are competitive on a dominant quintet spin state surface. We have compared the mechanism and thermodynamics of TauD with oxo-iron heme catalysts, such as the cytochromes P450, and found some critical differences. The TauD model is found to be much more reactive toward oxygenation of substrates than oxo-iron complexes in a heme environment with much lower reaction barriers. We have analyzed this and assigned this to the strength of the O-H bond formed after hydrogen abstraction from a substrate, which is at least 10 kcal mol(-)(1) stronger in five-coordinated oxo-iron nonheme complexes than in six-coordinated oxo-iron heme complexes. Since, the metal in TauD enzymes is five-coordinated, whereas in heme-enzymes it is six-coordinated there are some critical differences in the valence molecular orbitals. Thus, in oxo-iron heme catalysts one of the antibonding pi orbitals is replaced by a low-lying nonbonding delta orbital resulting in a lower overall spin state. Moreover, heme-enzymes have an extra oxidation equivalent located on the heme, which is missing in non-heme oxo-iron catalysts. As a result, the oxo-iron species of TauD reacts via single-state reactivity on a dominant quintet spin state surface, whereas oxo-iron heme catalysts react via two-state reactivity on competing doublet and quartet spin states.

  4. Modulation of digestive enzyme activities during ontogeny of Labeo rohita larvae fed ascorbic acid enriched zooplankton.

    PubMed

    Mitra, Gopa; Mukhopadhyay, P K; Ayyappan, S

    2008-04-01

    The effect of supplementation of ascorbic acid through enriched zooplankton [10%, 20% and 30% ascorbyl palmitate (AP) inclusion in diet of zooplankton] on different digestive enzyme activities during ontogeny of Labeo rohita larvae was studied from 4 day to 15 day post hatch. Ascorbic acid (AA) content in different groups of unenriched (8.6+/-0.71) and enriched zooplankton were, 750+/-29.3, 1409.1+/-45.5, 2009.21+/-199.2 mug/g respectively on dry matter basis with differences (P<0.05) between the treatments. A difference (P<0.05) was found in tissue AA level in different dietary groups. Low amylase, protease, lipase and alkaline phosphatase activities were present in rohu larvae from the mouth opening stage which showed increasing trend with the age of larvae and increasing dietary AA content. A clear dose-dependent modulation of digestive enzyme activities in response to 10%, 20% and 30% AP enriched zooplankton feeding was evidenced from positive correlations between dietary AA content with magnitude of elevation of enzyme activity in different groups. There were 57, 55, 29.2 and 2 fold increases in amylase activity; 7.35, 7.02, 4.43 and 2.73 fold increases in protease activity; 45.636, 41.50, 19.83 and 13.69 fold increases in lipase activity and 6, 5, 3, and 2 fold increases in alkaline phosphatase activity observed in the 15th day post hatch larvae fed 20%, 30%, 10%AP enriched and normal zooplankton respectively, than 4-day post hatch larvae of the respective groups. Enzyme activities were also positively correlated with specific growth rates of wet weight of rohu larvae at the 15th day post hatch. Increased AA might have played an important role in advancing morphological transformation of the digestive tract, protecting gastric mucosa and accelerating growth by the process of tissue formation, which necessitated the requirement of more nutrient thereby, increasing digestive enzyme activity. The regulatory role of AA in the modulation of different digestive

  5. Crystal Structure of Escherichia coli Diaminopropionate Ammonia-lyase Reveals Mechanism of Enzyme Activation and Catalysis*

    PubMed Central

    Bisht, Shveta; Rajaram, Venkatesan; Bharath, Sakshibeedu R.; Kalyani, Josyula Nitya; Khan, Farida; Rao, Appaji N.; Savithri, Handanahal S.; Murthy, Mathur R. N.

    2012-01-01

    Pyridoxal 5′-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp120 and Lys77 is suggested. PMID:22505717

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  7. Potential enzyme activities altered by increased nutrient availability in Arctic tundra soils

    NASA Astrophysics Data System (ADS)

    Koyama, A.; Wallenstein, M. D.; Moore, J. C.; Simpson, R. T.

    2012-12-01

    The Arctic tundra is a biome affected most by global warming predicted in the future. Such warming is expected to increase nutrient availability to soil microbes which, in turn, may accelerate soil organic matter decomposition. We investigated how extra-cellular enzyme activities in soils were affected by increasing nutrient availability in an Arctic tundra ecosystem. Specifically, we measured potential activities of seven enzymes at three profiles (organic, organic/mineral interface, and mineral) of soils which had been fertilized in long- (23 years) and short-terms (six years), assayed at four temperatures. The long-term site had a high fertilization treatment (10g N m-2 year-1 and 5g P m-2 year-1) and control, and the short-term site had a low fertilization treatment (5g N m-2 year-1 and 2.5g P m-2 year-1) in addition to the high fertilization treatment and control. The fertilization treatments significantly altered most of the enzyme activities in both sites. The fertilization treatments increased activities of enzymes hydrolyzing products for C and nitrogen N sources, but decreased phosphatase activities. Such alterations were most pronounced in the organic soils. The fertilization treatments also increased ratios of total enzyme activities involved in hydrolysis for C products to those for N products. This result is consistent with an observation that long-term N and P fertilization decreased soil organic C in the same tundra ecosystem. Altered enzymatic stoichiometry with increased nutrient availability should be considered when modeling biogeochemical cycles in Arctic tundra ecosystems in response to warming predicted in the future.

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

    PubMed Central

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

    2013-01-01

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

  9. Variation in the Activity of Some Enzymes of Photorespiratory Metabolism in C4 Grasses

    PubMed Central

    UENO, OSAMU; YOSHIMURA, YASUYUKI; SENTOKU, NAOKI

    2005-01-01

    • Background and Aims Photorespiration occurs in C4 plants, although rates are small compared with C3 plants. The amount of glycine decarboxylase in the bundle sheath (BS) varies among C4 grasses and is positively correlated with the granal index (ratio of the length of appressed thylakoid membranes to the total length of all thylakoid membranes) of the BS chloroplasts: C4 grasses with high granal index contained more glycine decarboxylase per unit leaf area than those with low granal index, probably reflecting the differences in O2 production from photosystem II and the potential photorespiratory capacity. Thus, it is hypothesized that the activities of peroxisomal enzymes involved in photorespiration are also correlated with the granal development. • Methods The granal development in BS chloroplasts was investigated and activities of the photorespiratory enzymes assayed in 28 C4 grasses and seven C3 grasses. • Key Results The NADP–malic enzyme grasses were divided into two groups: one with low granal index and the other with relatively high granal index in the BS chloroplasts. Both the NAD–malic enzyme and phosphoenolpyruvate carboxykinase grasses had high granal index in the BS chloroplasts. No statistically significant differences were found in activity of hydroxypyruvate reductase between the C3 and C4 grasses, or between the C4 subtypes. The activity of glycolate oxidase and catalase were smaller in the C4 grasses than in the C3 grasses. Among the C4 subtypes, glycolate oxidase activities were significantly smaller in the NADP–malic enzyme grasses with low granal index in the BS chloroplasts, compared with in the C4 grasses with substantial grana in the BS chloroplasts. • Conclusions There is interspecies variation in glycolate oxidase activity associated with the granal development in the BS chloroplasts and the O2 production from photosystem II, which suggests different potential photorespiration capacities among C4 grasses. PMID:16100226

  10. Crystal structure of Escherichia coli diaminopropionate ammonia-lyase reveals mechanism of enzyme activation and catalysis.

    PubMed

    Bisht, Shveta; Rajaram, Venkatesan; Bharath, Sakshibeedu R; Kalyani, Josyula Nitya; Khan, Farida; Rao, Appaji N; Savithri, Handanahal S; Murthy, Mathur R N

    2012-06-08

    Pyridoxal 5'-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of d- and l-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 Å resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with dl-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp(120) and Lys(77) is suggested.

  11. [Effects of tillage and straw returning on microorganism quantity, enzyme activities in soils and grain yield].

    PubMed

    Zhao, Ya-li; Guo, Hai-bin; Xue, Zhi-wei; Mu, Xin-yuan; Li, Chao-hai

    2015-06-01

    A two-year field study with split plot design was conducted to investigate the effects of different soil tillage (conventional tillage, CT; deep tillage, DT; subsoil tillage, ST) and straw returning (all straw retention, AS; no straw returning, NS) on microorganism quantity, enzyme activities in soil and grain yield. The results showed that, deep or subsoil tillage and straw returning not only reduced the soil bulk density and promoted the content of organic carbon in soil, but increased the soil microbial quantity, soil enzyme activities and grain yield. Furthermore, such influences in maize season were greater than that in wheat season. Compared with CT+NS, DT+AS and ST+AS decreased the soil bulk density at 20-30 cm depth by 8.5% and 6.6%, increased the content of soil organic carbon by 14.8% and 12.4%, increased the microorganism quantity by 45.9% and 33.9%, increased the soil enzyme activities by 34.1% and 25.5%, increased the grain yield by 18.0% and 19.3%, respectively. No significant difference was observed between DT+AS and ST+AS. We concluded that retaining crop residue and deep or subsoil tillage improved soil microorganism quantity, enzyme activities and crop yield.

  12. Microbial Enzyme Activity, Nutrient Uptake, and Nutrient Limitation in Forested Streams

    EPA Science Inventory

    We measured NH4 + and PO4 -3 uptake length (Sw), uptake velocity (Vf), uptake rate (U), biofilm enzyme activity (BEA), and channel geomorphology in streams draining forested catchments in the Northwestern (Northern California Coast Range and Cascade Mountains) and Southeastern (A...

  13. VARIATIONS IN SOIL AGGREGATE STABILITY AND ENZYME ACTIVITIES IN A TEMPERATE AGROFORESTRY PRACTICE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Agroforestry and grass buffers have been shown to improve soil properties and overall environmental quality. The objective of this study was to examine management and landscape effects on water stable soil aggregates (WSA), soil carbon, soil nitrogen, enzyme activity, and microbial community DNA co...

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

    PubMed

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

    2013-03-01

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

  15. Detection of NAD(P)H-dependent enzyme activity with dynamic luminescence quenching of terbium complexes.

    PubMed

    Ito, Hiroki; Terai, Takuya; Hanaoka, Kenjiro; Ueno, Tasuku; Komatsu, Toru; Nagano, Tetsuo; Urano, Yasuteru

    2015-05-14

    We discovered that positively charged terbium complexes bearing 1,4,7,10-tetraazacyclododecane functionalized with amide ligands are highly sensitive to dynamic luminescence quenching by NAD(P)H. We exploited this phenomenon to establish a general time-resolved luminescence-based assay platform for sensitive detection of NAD(P)H-dependent enzyme activities.

  16. Steps in reductive activation of the disulfide-generating enzyme Ero1p

    PubMed Central

    Heldman, Nimrod; Vonshak, Ohad; Sevier, Carolyn S; Vitu, Elvira; Mehlman, Tevie; Fass, Deborah

    2010-01-01

    Ero1p is the primary catalyst of disulfide bond formation in the yeast endoplasmic reticulum (ER). Ero1p contains a pair of essential disulfide bonds that participate directly in the electron transfer pathway from substrate thiol groups to oxygen. Remarkably, elimination of certain other Ero1p disulfides by mutation enhances enzyme activity. In particular, the C150A/C295A Ero1p mutant exhibits increased thiol oxidation in vitro and in vivo and interferes with redox homeostasis in yeast cells by hyperoxidizing the ER. Inhibitory disulfides of Ero1p are thus important for enzyme regulation. To visualize the differences between de-regulated and wild-type Ero1p, we determined the crystal structure of Ero1p C150A/C295A. The structure revealed local changes compared to the wild-type enzyme around the sites of mutation, but no conformational transitions within 25 Å of the active site were observed. To determine how the C150—C295 disulfide nonetheless participates in redox regulation of Ero1p, we analyzed using mass spectrometry the changes in Ero1p disulfide connectivity as a function of time after encounter with reducing substrates. We found that the C150—C295 disulfide sets a physiologically appropriate threshold for enzyme activation by guarding a key neighboring disulfide from reduction. This study illustrates the diverse and interconnected roles that disulfides can play in redox regulation of protein activity. PMID:20669236

  17. Microbial respiration and extracellular enzyme activity in sediments from the Gulf of Mexico hypoxic zone

    EPA Science Inventory

    This study explores the relationship between sediment chemistry (TC, TN, TP) and microbial respiration (DHA) and extracellular enzyme activity (EEA) across the Gulf of Mexico (GOM) hypoxic zone. TC, TN, and TP were all positively correlated with each other (r=0.19-0.68). DHA was ...

  18. Affinity electrophoresis as a method for determining substrate-binding specificity of carbohydrate-active enzymes for soluble polysaccharides.

    PubMed

    Moraïs, Sarah; Lamed, Raphael; Bayer, Edward A

    2012-01-01

    Affinity electrophoresis is a simple and rapid tool for the analysis of protein-binding affinities to soluble polysaccharides. This approach is particularly suitable for the characterization of the carbohydrate-active enzymes that contain a carbohydrate-binding module and for their mutants and chimeras. Knowledge of the binding characteristics of these enzymes can be the first step to elucidate the enzymatic activity of a putative enzyme; moreover in some cases, enzymes are able to bind polysaccharides targets other than their specified substrate, and this knowledge can be essential to understand the basics of the intrinsic mechanism of these enzymes in their natural environment.

  19. Effect of sound wave stress on antioxidant enzyme activities and lipid peroxidation of Dendrobium candidum.

    PubMed

    Li, Biao; Wei, Jinmin; Wei, Xiaolan; Tang, Kun; Liang, Yilong; Shu, Kunxian; Wang, Bochu

    2008-06-01

    The effect of sound wave stress on important medicinal plant, Dendrobium candidum Wall. ex Lindl, was investigated, including the responses on malondialdehyde (MDA) content, the activities change of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX). Results were found that the activities of SOD, CAT, POD and APX enhanced totally in different organs of D. candidum, as leaves, stems and roots, in response to the stress. Furthermore there happened similar shift of antioxidant enzymes activities, which increased in the initial stimulation and decreased afterwards. Data showed SOD, CAT, POD and APX activities ascended to max at day 9, 6, 9 and 12 in leaves, at day 9, 6, 12 and 9 in stems, and at day 12, 6, 9 and 9 in roots, respectively. As a lipid peroxidation parameter, MDA content in different organs increased in the beginning, dropped afterward, and increased again in the late. Anyway the total trend was the rise of MDA level compared to the control. It was interesting that the MDA content appeared the lowest levels almost when the antioxidant enzymes activities were up to the highest. Our results demonstrated the different organs of D. candidum might produce accumulation of active oxygen species (AOS) under initial treatment of sound wave stress. Later AOS might start to reduce due to the enhancement of antioxidant enzymes activities treated by the stress. The data revealed that the antioxidant metabolism was to be important in determining the ability of plants to survive in sound stress, and the up regulation of these enzymes activities would help to reduce the build up of AOS, which could protect plant cells from oxidative damage. Moreover, different cell compartments might activate different defensive system to reduce excessive amount of AOS. Finally the mechanism of this action was also discussed simply.

  20. Relationship of lipogenic enzyme activities to the rate of rat liver fatty acid synthesis

    SciTech Connect

    Nelson, G.; Kelley, D.; Schmidt, P.; Virk, S.; Serrato, C.

    1986-05-01

    The mechanism by which diet regulates liver lipogenesis is unclear. Here the authors report how dietary alterations effect the activities of key enzymes of fatty acid (FA) synthesis. Male Sprague-Dawley rats, 400-500 g, were fasted for 48h and then refed a fat-free, high carbohydrate (HC) diet (75% cal. from sucrose) for 0,3,9,24 and 48h, or refed a HC diet for 48h, then fed a high-fat (HF) diet (44% cal. from corn oil) for 3,9,24 and 48h. The FA synthesis rate and the activities of acetyl CoA carboxylase (AC), fatty acid synthase (FAS), ATP citrate lyase (CL), and glucose 6-phosphate dehydrogenase (G6PDH) were determined in the livers. FA synthesis was assayed with /sup 3/H/sub 2/O, enzyme activities were measured spectrophotometrically except for AC which was assayed with /sup 14/C-bicarbonate. There was no change in the activity of AC during fasting or on the HC diet. Fasting decreased the rate of FA synthesis by 25% and the activities of FAS and CL by 50%; refeeding the HC diet induced parallel changes in FA synthesis and the activities of FAS, CL, and G6PDH. After 9h on the HF diet, FA synthesis had decreased sharply, AC activity increased significantly while no changes were detected in the other activities. Subsequently FA synthesis did not change while the activities of the enzymes decreased slowly. These enzymes did not appear to regulate FA synthesis during inhibition of lipogenesis, but FAS, CL or G6PDH may be rate limiting in the induction phase. Other key factors may regulate FA synthesis during dietary alterations.

  1. Activities of Extracellular Enzymes in Soils Following Woody Plant Invasion of Grassland

    NASA Astrophysics Data System (ADS)

    Filley, T. R.; Stott, D. E.; Dooling, V.; Sorg, L.; Boutton, T.

    2008-12-01

    Extracellular enzymes produced by microbes and immobilize in the soil environment are the principle means by which complex plant and microbial compounds are degraded. The concentration of these enzymes and their ability to interact with litter and soil organic matter contributes both to the stabilization and destabilization of soil carbon. We quantified the activities of three extracellular enzymes, B-glucosidase, B- glucosaminidase, polyphenol oxidase (PPO), and a general marker for hydrolytic activity through fluorescein diacetate (FDA) hydrolysis activity, in a subtropical savanna parkland in southern Texas where woody plants have invaded a once open grassland. Previous research has demonstrated that areas which have shifted to woody vegetation are accruing soil carbon, undergoing a dramatic shift in the chemistry of plant input, and increasing in hyphal biomass. Soils were obtained along a successional chronosequence from grassland dominated by C4 grasses to woody patches dominated by C3 trees/shrubs in Oct 2006 and stored immediately frozen until thawing for enzyme assay. Most enzymes, with the exception of PPO, show distinct behavior when comparing grassland and clusters in that grasslands exhibit far lower mass normalized activity than clusters and no activity trend with respect to age of the adjacent cluster. Both FDA and B- glucosaminidase activities are positively correlated with the age of the woody clusters and increase their activity by as much as 10-fold across the age gradient from 14 yr to 86 yr old clusters. The cellulose degrading enzyme, B-glucosidase, always exhibited greater activity (1.5 -4 fold) in woody clusters than in grasslands, but did not exhibit a trend with increasing cluster age. The PPO activity is anomalous in that there is no quantitative difference in mass normalized activity between grassland and cluster and no trend with cluster age. The results for the FDA and B-glucosaminidase assays are consistent with concurrent studies

  2. Influence of electromagnetic fields on the enzyme activity of rheumatoid synovial fluid cells in vitro.

    PubMed

    Mohamed-Ali, H; Kolkenbrock, H; Ulbrich, N; Sörensen, H; Kramer, K D; Merker, H J

    1994-04-01

    Since positive clinical effects have been observed in the treatment of rheumatoid arthritis with electromagnetic fields of weak strength and low frequency range (magnetic field strength: 70 microT; frequency: 1.36-14.44 Hz), an attempt was made to analyse the effects of these electromagnetic fields on enzyme activity in monolayer cultures of rheumatoid synovial fluid cells after single irradiation of the cultures for 24 hours. We only investigated the matrix metalloproteinases (collagenase, gelatinase, proteinase 24.11 and aminopeptidases). It was found that electromagnetic fields of such a weak strength and low frequency range do not generally have a uniform effect on the activity of the different proteinases in vitro. While aminopeptidases do not show any great changes in activity, the peptidases hydrolysing N(2,4)-dinitrophenyl-peptide exhibit a distinct increase in activity in the late phase in culture medium without fetal calf serum. In the presence of fetal calf serum this effect is not observed and enzyme activity is diminished. Our experiments do not show whether such a phase-bound increase in the activity of proteinases in vitro is only one finding in a much broader range of effects of electromagnetic fields, or whether it is a specific effect of weak pulsed magnetic fields of 285 +/- 33 nT on enzyme activity after single irradiation. This question requires further elucidation.

  3. Costus afer Possesses Carbohydrate Hydrolyzing Enzymes Inhibitory Activity and Antioxidant Capacity In Vitro

    PubMed Central

    Tchamgoue, Armelle D.; Tchokouaha, Lauve R. Y.; Tarkang, Protus A.; Kuiate, Jules-Roger; Agbor, Gabriel A.

    2015-01-01

    Diabetes mellitus is a metabolic disorder of glucose metabolism which correlates with postprandial hyperglycemia and oxidative stress. Control of blood glucose level is imperative in the management of diabetes. The present study tested the hypothesis that Costus afer, an antihyperglycemic medicinal plant, possesses inhibitory activity against carbohydrate hydrolyzing enzymes. Hexane, ethyl acetate, methanol, and water extracts were prepared from the leaf, stem, and rhizome of C. afer and subjected to phytochemical screening, assayed for α-amylase and α-glucosidase inhibitory activities and antioxidant capacity (determined by total phenolic and total flavonoids contents, ferric reducing antioxidant power (FRAP), and DPPH radical scavenging activity). All extracts inhibited α-amylase and α-glucosidase activities. Ethyl acetate rhizome and methanol leaf extracts exhibited the best inhibitory activity against α-amylase and α-glucosidase (IC50: 0.10 and 5.99 mg/mL), respectively. Kinetic analysis revealed two modes of enzyme inhibition (competitive and mixed). All extracts showed antioxidant capacity, with hexane extracts exhibiting the best activity. DPPH assay revealed that methanol leaf, rhizome, and ethyl acetate stem extracts (IC50 < 5 mg/mL) were the best antioxidants. The presence of bioactive compounds such as flavonoids, alkaloids, phenols, and tannins may account for the antioxidant capacity and carbohydrate hydrolyzing enzyme inhibitory activity of C. afer. PMID:26246844

  4. Effect of antimony on the microbial growth and the activities of soil enzymes.

    PubMed

    An, Youn-Joo; Kim, Minjin

    2009-02-01

    The effects of antimony (Sb) on microbial growth inhibition and activities of soil enzymes were investigated in the present study. Test bacterial species were Escherichia coli, Bacillus subtilis and Streptococcus aureus. Among the microorganisms tested, S. aureus was the most sensitive. The 50% effects on the inhibition of specific growth rate of E. coli, B. subtilis, and, S. aureus were 555, 18.4, and 15.8 mg Sb L(-1), respectively. A silt loam soil was amended with antimony and incubated in a controlled condition. Microbial activities of dehydrogenase, acid phosphatase (P cycle), arylsulfatase (S cycle), beta-glucosidase (C cycle), urease (N cycle), and fluorescein diacetate hydrolase in soil were measured. Activities of urease and dehydrogenase were related with antimony and can be an early indication of antimony contamination. The maximum increase in soil urease activity by antimony was up to 168% after 3d compared with the control. The activities of other four enzymes (acid phosphatase, fluorescein diacetate hydrolase, arylsulfatase and ss-glucosidase) were less affected by antimony. This study suggested that antimony affects nitrogen cycle in soil by changing urease activity under the neutral pH, however, soil enzyme activities may not be a good protocol due to their complex response patterns to antimony pollution.

  5. Quantitative assessment on soil enzyme activities of heavy metal contaminated soils with various soil properties.

    PubMed

    Xian, Yu; Wang, Meie; Chen, Weiping

    2015-11-01

    Soil enzyme activities are greatly influenced by soil properties and could be significant indicators of heavy metal toxicity in soil for bioavailability assessment. Two groups of experiments were conducted to determine the joint effects of heavy metals and soil properties on soil enzyme activities. Results showed that arylsulfatase was the most sensitive soil enzyme and could be used as an indicator to study the enzymatic toxicity of heavy metals under various soil properties. Soil organic matter (SOM) was the dominant factor affecting the activity of arylsulfatase in soil. A quantitative model was derived to predict the changes of arylsulfatase activity with SOM content. When the soil organic matter content was less than the critical point A (1.05% in our study), the arylsulfatase activity dropped rapidly. When the soil organic matter content was greater than the critical point A, the arylsulfatase activity gradually rose to higher levels showing that instead of harm the soil microbial activities were enhanced. The SOM content needs to be over the critical point B (2.42% in our study) to protect its microbial community from harm due to the severe Pb pollution (500mgkg(-1) in our study). The quantitative model revealed the pattern of variation of enzymatic toxicity due to heavy metals under various SOM contents. The applicability of the model under wider soil properties need to be tested. The model however may provide a methodological basis for ecological risk assessment of heavy metals in soil.

  6. Vanadate and selenium inhibit the triiodothyronine induced enzyme activity and mRNA level for both fatty acid synthase and malic enzyme

    SciTech Connect

    Zhu, Y.; Mirmiran, R.; Goodridge, A.G.; Stapleton, S.R. Western Michigan Univ., Kalamazoo )

    1991-03-15

    In chick-embryo hepatocytes in culture, triiodothyronine stimulates enzyme activity, mRNA level and transcription rate for both fatty acid synthase (FAS) and malic enzyme (ME). Insulin alone has no effect but amplifies the induction by T3. Recent evidence has demonstrated the insulin-mimicking action of vanadate and selenium on various physiological processes. Little information, however, is available on the affects of vanadate and selenium on the expression of genes that are regulated by insulin. These studies were initiated to test the potential of vanadate and selenium to mimic the amplification affect of insulin on the T3 induction of FAS and ME. In chick-embryo hepatocytes incubated in a chemically defined medium, addition of T3 for 48h causes an increase in the enzyme activity and mRNA level for both FAS and ME. Addition of sodium vanadate or sodium selenate (20 {mu}M) coincident with the T3 almost completely inhibited the stimulation of FAS and ME activity and accumulation of their respective mRNA's. Fifty percent maximal inhibition occurred at about 3-40{mu}M vanadate or 5-10{mu}M selenium. Vanadate and selenium similarity inhibited FAS and ME enzyme activity and mRNA level when the cells were incubated in the presence of insulin and T3. The effect of these metals was selective; isocitrate dehydrogenase activity as well as the level of glyceraldehyde 3-phosphate mRNA were not affected by any of the additions made to the cells in culture. This effect by vanadate and selenium also does not appear to be a generalized effect of metals on lipogenic enzymes as molydate under similar experimental conditions has no effect on either the enzyme activity or mRNA level of FAS or ME. Studies are continuing to determine the mechanism of action of these agents on the regulation of lipogenic enzymes.

  7. Tracking a defined route for O₂ migration in a dioxygen-activating diiron enzyme.

    PubMed

    Song, Woon Ju; Gucinski, Grant; Sazinsky, Matthew H; Lippard, Stephen J

    2011-09-06

    For numerous enzymes reactive toward small gaseous compounds, growing evidence indicates that these substrates diffuse into active site pockets through defined pathways in the protein matrix. Toluene/o-xylene monooxygenase hydroxylase is a dioxygen-activating enzyme. Structural analysis suggests two possible pathways for dioxygen access through the α-subunit to the diiron center: a channel or a series of hydrophobic cavities. To distinguish which is utilized as the O(2) migration pathway, the dimensions of the cavities and the channel were independently varied by site-directed mutagenesis and confirmed by X-ray crystallography. The rate constants for dioxygen access to the diiron center were derived from the formation rates of a peroxodiiron(III) intermediate, generated upon treatment of the diiron(II) enzyme with O(2). This reaction depends on the concentration of dioxygen to the first order. Altering the dimensions of the cavities, but