Sample records for acid cycle enzymes

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

    PubMed Central

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

    1971-01-01

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

  2. Diffusion of tricarboxylic acid cycle enzymes in the mitochondrial matrix in vivo. Evidence for restricted mobility of a multienzyme complex.

    PubMed

    Haggie, Peter M; Verkman, A S

    2002-10-25

    It has been proposed that enzymes in many metabolic pathways, including the tricarboxylic acid cycle in the mitochondrial matrix, are physically associated to facilitate substrate channeling and overcome diffusive barriers. We have used fluorescence recovery after photobleaching to measure the diffusional mobilities of chimeras consisting of green fluorescent protein (GFP) fused to the C terminus of four tricarboxylic acid cycle enzymes: malate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and succinyl-CoA synthetase. The GFP-enzyme chimeras were localized selectively in the mitochondrial matrix in transfected Chinese hamster ovary (CHO) and COS7 cells. Laser photobleaching using a 0.7-microm diameter spot demonstrated restricted diffusion of the GFP-enzyme chimeras. Interestingly, all four chimeras had similar diffusional characteristics, approximately 45% of each chimera was mobile and had a diffusion coefficient of 4 x 10(-8) cm(2)/s. In contrast, unconjugated GFP in the mitochondrial matrix (targeted using COX8 leader sequence) diffused freely (nearly 100% mobility) with a greater diffusion coefficient of 20 x 10(-8) cm(2)/s. The mobility of the GFP-enzyme chimeras was insensitive to substrate source, ATP depletion, or inhibition of the adenine nucleotide translocase. These results indicate similar mobility characteristics of unrelated tricarboxylic acid cycle enzymes having different sizes and physical properties, providing biophysical evidence for a diffusible multienzyme complex in the mitochondrial matrix.

  3. Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. A case study of endosymbiotic gene transfer.

    PubMed

    Schnarrenberger, Claus; Martin, William

    2002-02-01

    The citric acid or tricarboxylic acid cycle is a central element of higher-plant carbon metabolism which provides, among other things, electrons for oxidative phosphorylation in the inner mitochondrial membrane, intermediates for amino-acid biosynthesis, and oxaloacetate for gluconeogenesis from succinate derived from fatty acids via the glyoxylate cycle in glyoxysomes. The tricarboxylic acid cycle is a typical mitochondrial pathway and is widespread among alpha-proteobacteria, the group of eubacteria as defined under rRNA systematics from which mitochondria arose. Most of the enzymes of the tricarboxylic acid cycle are encoded in the nucleus in higher eukaryotes, and several have been previously shown to branch with their homologues from alpha-proteobacteria, indicating that the eukaryotic nuclear genes were acquired from the mitochondrial genome during the course of evolution. Here, we investigate the individual evolutionary histories of all of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle using protein maximum likelihood phylogenies, focusing on the evolutionary origin of the nuclear-encoded proteins in higher plants. The results indicate that about half of the proteins involved in this eukaryotic pathway are most similar to their alpha-proteobacterial homologues, whereas the remainder are most similar to eubacterial, but not specifically alpha-proteobacterial, homologues. A consideration of (a) the process of lateral gene transfer among free-living prokaryotes and (b) the mechanistics of endosymbiotic (symbiont-to-host) gene transfer reveals that it is unrealistic to expect all nuclear genes that were acquired from the alpha-proteobacterial ancestor of mitochondria to branch specifically with their homologues encoded in the genomes of contemporary alpha-proteobacteria. Rather, even if molecular phylogenetics were to work perfectly (which it does not), then some nuclear-encoded proteins that were acquired from the alpha

  4. Enzymes in Glycolysis and the Citric Acid Cycle in the Yeast and Mycelial Forms of Paracoccidioides brasiliensis

    PubMed Central

    Kanetsuna, Fuminori; Carbonell, Luis M.

    1966-01-01

    Kanetsuna, Fuminori (Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela), and Luis M. Carbonell. Enzymes in glycolysis and the citric acid cycle in the yeast and mycelial forms of Paracoccidioides brasiliensis. J. Bacteriol. 92:1315–1320. 1966.—Enzymatic activities in glycolysis, the hexose monophosphate shunt, and the citric acid cycle in cell-free extracts of the yeast and mycelial forms of Paracoccidioides brasiliensis were examined comparatively. Both forms have the enzymes of these pathways. Activities of glucose-6-phosphate dehydrogenase and malic dehydrogenase of the mycelial form were higher than those of the yeast form. Another 15 enzymatic activities of the mycelial form were lower than those of the yeast form. The activity of glyceraldehyde-3-phosphate dehydrogenase showed the most marked difference between the two forms, its activity in the mycelial form being about 20% of that in the yeast form. PMID:5924267

  5. Orthotopic Liver Transplantation for Urea Cycle Enzyme Deficiency

    PubMed Central

    Todo, Satoru; Starzl, Thomas E.; Tzakis, Andreas; Benkov, Keith J.; Kalousek, Frantisek; Saheki, Takeyori; Tanikawa, Kyuichi; Fenton, Wayne A.

    2010-01-01

    Hyperammonemia, abnormalities in plasma amino acids and abnormalities of standard liver functions were corrected by orthotopic liver transplantation in a 14-day-old boy with carbamyl phosphate synthetase-I deficiency and in a 35-yr-old man with argininosuccinic acid synthetase deficiency. The first patient had high plasma glutamine levels and no measureable citrulline, whereas citrulline values were markedly increased in Patient 2. Enzyme analysis of the original livers showed undetectable activity of carbamyl phosphate synthetase-I in Patient 1 and arginosuccinic acid synthetase in Patient 2. Both patients were comatose before surgery. Intellectual recovery of patient 1 has been slightly retarded because of a brain abscess caused by Aspergillus infection after surgery. Both patients are well at 34 and 40 mo, respectively, after surgery. Our experience has shown that orthotopic liver transplantation corrects the life-threatening metabolic abnormalities caused by deficiencies in the urea cycle enzymes carbamyl phosphate synthetase-I and arginosuccinic acid synthetase. Seven other patients–six with ornithine transcarbamylase deficiency and another with carbamyl phosphate synthetase-I deficiency–are known to have been treated elsewhere with liver transplantation 1½ yr or longer ago. Four of these seven recipients also are well, with follow-ups of 1½ to 5 yr. Thus liver transplantation corrects the metabolic abnormalities of three of the six urea cycle enzyme deficiencies, and presumably would correct all. PMID:1544622

  6. Microbial extracellular enzymes in biogeochemical cycling of ecosystems.

    PubMed

    Luo, Ling; Meng, Han; Gu, Ji-Dong

    2017-07-15

    Extracellular enzymes, primarily produced by microorganisms, affect ecosystem processes because of their essential roles in degradation, transformation and mineralization of organic matter. Extracellular enzymes involved in the cycling of carbon (C), nitrogen (N) and phosphorus (P) have been widely investigated in many different ecosystems, and several enzymes have been recognized as key components in regulating C storage and nutrient cycling. In this review, it was the first time to summarize the specific extracellular enzymes related to C storage and nutrient cycling for better understanding the important role of microbial extracellular enzymes in biogeochemical cycling of ecosystems. Subsequently, ecoenzymatic stoichiometry - the relative ratio of extracellular enzyme, has been reviewed and further provided a new perspective for understanding biogeochemical cycling of ecosystems. Finally, the new insights of using microbial extracellular enzyme in indicating biogeochemical cycling and then protecting ecosystems have been suggested. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Metabolic interaction between urea cycle and citric acid cycle shunt: A guided approach.

    PubMed

    Pesi, Rossana; Balestri, Francesco; Ipata, Piero L

    2018-03-01

    This article is a guided pedagogical approach, devoted to postgraduate students specializing in biochemistry, aimed at presenting all single reactions and overall equations leading to the metabolic interaction between ureagenesis and citric acid cycle to be incorporated into a two-three lecture series about the interaction of urea cycle with other metabolic pathways. We emphasize that citrate synthetase, aconitase, and isocitrate dehydrogenase, three enzymes of the citric acid cycle are not involved, thus creating a shunt in citric acid cycle. In contrast, the glutamic-oxaloacetate transaminase, which does not belong to citric acid cycle, has a paramount importance in the metabolic interaction of the two cycles, because it generates aspartate, one of the two fuel molecules of urea cycle, and a-ketoglutarate, an intermediate of the citric acid cycle. Finally, students should appreciate that balancing equations for all atoms and charges is not only a stoichiometric task, but strongly facilitates the discussion of the physiological roles of metabolic pathways. Indeed, this exercise has been used in the classroom, to encourage a deeper level of understanding of an important biochemical issue. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(2):182-185, 2018. © 2017 The International Union of Biochemistry and Molecular Biology.

  8. Enzymes of the γ-Glutamyl Cycle in the Ciliary Body and Lens

    PubMed Central

    Ross, Leonard L.; Barber, Lee; Tate, Suresh S.; Meister, Alton

    1973-01-01

    The enzymes of the γ-glutamyl cycle have been found in rabbit ciliary body and, except for 5-oxoprolinase, also in the ocular lens. Histochemical studies show that γ-glutamyl transpeptidase is localized mainly in the basal portions of the epithelial cells of the ciliary body; the findings are similar to those observed in the chloroid plexuses. The histochemical staining reaction in the ciliary epithelium is more intense than in the chloroid plexus, intestine, and kidney. γ-Glutamyl transpeptidase staining activity in the epithelium of the intestinal and renal proximal convoluted tubules is confined to the microvillus border. Moderate transpeptidase activity was found in the cytoplasm of nonpigmented epithelial cells of the iris at the posterior pupillary margin. The histochemical and enzyme activity studies are consistent with the thesis that the γ-glutamyl cycle functions in transport of amino acids across the blood-aqueous humor barrier. Images PMID:4152058

  9. The Krebs Uric Acid Cycle: A Forgotten Krebs Cycle.

    PubMed

    Salway, Jack G

    2018-05-25

    Hans Kornberg wrote a paper entitled 'Krebs and his trinity of cycles' commenting that every school biology student knows of the Krebs cycle, but few know that Krebs discovered two other cycles. These are (i) the ornithine cycle (urea cycle), (ii) the citric acid cycle (tricarboxylic acid or TCA cycle), and (iii) the glyoxylate cycle that was described by Krebs and Kornberg. Ironically, Kornberg, codiscoverer of the 'glyoxylate cycle', overlooked a fourth Krebs cycle - (iv) the uric acid cycle. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. 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

  11. Protein-protein interactions and metabolite channelling in the plant tricarboxylic acid cycle

    PubMed Central

    Zhang, Youjun; Beard, Katherine F. M.; Swart, Corné; Bergmann, Susan; Krahnert, Ina; Nikoloski, Zoran; Graf, Alexander; Ratcliffe, R. George; Sweetlove, Lee J.; Fernie, Alisdair R.; Obata, Toshihiro

    2017-01-01

    Protein complexes of sequential metabolic enzymes, often termed metabolons, may permit direct channelling of metabolites between the enzymes, providing increased control over metabolic pathway fluxes. Experimental evidence supporting their existence in vivo remains fragmentary. In the present study, we test binary interactions of the proteins constituting the plant tricarboxylic acid (TCA) cycle. We integrate (semi-)quantitative results from affinity purification-mass spectrometry, split-luciferase and yeast-two-hybrid assays to generate a single reliability score for assessing protein–protein interactions. By this approach, we identify 158 interactions including those between catalytic subunits of sequential enzymes and between subunits of enzymes mediating non-adjacent reactions. We reveal channelling of citrate and fumarate in isolated potato mitochondria by isotope dilution experiments. These results provide evidence for a functional TCA cycle metabolon in plants, which we discuss in the context of contemporary understanding of this pathway in other kingdoms. PMID:28508886

  12. Functional Study of the Vitamin K Cycle Enzymes in Live Cells

    PubMed Central

    Tie, J.-K.; Stafford, D.W.

    2018-01-01

    Vitamin K-dependent carboxylation, an essential posttranslational modification catalyzed by gamma-glutamyl carboxylase, is required for the biological functions of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylation, reduced vitamin K (KH2) is oxidized to vitamin K epoxide (KO). KO must be recycled back to KH2 by the enzymes vitamin K epoxide reductase and vitamin K reductase in a pathway known as the vitamin K cycle. Our current knowledge about the enzymes of the vitamin K cycle is mainly based on in vitro studies of each individual enzymes under artificial conditions, which are of limited usefulness in understanding how the complex carboxylation process is carried out in the physiological environment. In this chapter, we review the current in vitro activity assays for vitamin K cycle enzymes. We describe the rationale, establishment, and application of cell-based assays for the functional study of these enzymes in the native cellular milieu. In these cell-based assays, different vitamin K-dependent proteins were designed and stably expressed in mammalian cells as reporter proteins to accommodate the readily used enzyme-linked immunosorbent assay for carboxylation efficiency evaluation. Additionally, recently emerged genome-editing techniques TALENs and CRISPR-Cas9 were used to knock out the endogenous enzymes in the reporter cell lines to eliminate the background. These cell-based assays are easy to scale up for high-throughput screening of inhibitors of vitamin K cycle enzymes and have been successfully used to clarify the genotypes and their clinical phenotypes of enzymes of the vitamin K cycle. PMID:28065270

  13. The γ-Glutamyl Cycle in the Choroid Plexus: Its Possible Function in Amino Acid Transport

    PubMed Central

    Tate, Suresh S.; Ross, Leonard L.; Meister, Alton

    1973-01-01

    Various anatomic regions of rabbit brain have been examined for activities of the enzymes of the γ-glutamyl cycle. While these enzyme activities were widely distributed in the brain, they are present in much higher concentrations in the choroid plexus than in other parts of the brain. The activities observed are of about the same order of magnitude as found in the kidney. These observations and other considerations suggest that the γ-glutamyl cycle may play a significant role in the transport of amino acids between blood and cerebrospinal fluid. PMID:4145786

  14. NF-κB controls four genes encoding core enzymes of tricarboxylic acid cycle.

    PubMed

    Zhou, Fei; Xu, Xinhui; Wu, Jian; Wang, Danyang; Wang, Jinke

    2017-07-20

    NF-κB may promote tumor progression by altering cell metabolism. Hence, finding its target genes that are involved in cell metabolism is helpful for understanding its role in tumor growth. Here we discovered four metabolism-related target genes of this transcription factor. By analyzing a chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) data that characterizing the global binding sites (BSs) of NF-κB RelA in the TNFα-stimulated HeLa cells, we found that four genes that encode core enzymes of the tricarboxylic acid (TCA) cycle, including IDH1, IDH3A, ACO2, and SUCLA2, were multiply bound by this transcription factor. The subsequent bioinformatic analysis revealed that the NF-κB BSs contained many canonical κB sequences and the NF-κB-like DNA-binding motifs. Detection of ChIPed DNA with polymerase chain reaction (ChIP-PCR) also indicated that the NF-κB BSs were bound by NF-κB in both TNFα-treated HeLa and HepG2 cells. The reporter construct showed that the NF-κB BSs could activate the luciferase expression in cells in a NF-κB-specific manner. The quantitative PCR and Western blot detections demonstrated that NF-κB could regulate the expressions of IDH1, IDH3A, and ACO2 genes at both mRNA and protein levels and that of SUCLA2 gene at mRNA level in the TNFα-treated HeLa and HepG2 cells. Based on these investigations we identified the four genes as new target genes of NF-κB. The finding provides new insights into the role of NF-κB in cellular energetic metabolism, which may be beneficial for understanding the metabolic physiology of tumor growth. Copyright © 2017. Published by Elsevier B.V.

  15. Extending enzyme molecular recognition with an expanded amino acid alphabet

    PubMed Central

    Windle, Claire L.; Simmons, Katie J.; Ault, James R.; Trinh, Chi H.; Nelson, Adam

    2017-01-01

    Natural enzymes are constructed from the 20 proteogenic amino acids, which may then require posttranslational modification or the recruitment of coenzymes or metal ions to achieve catalytic function. Here, we demonstrate that expansion of the alphabet of amino acids can also enable the properties of enzymes to be extended. A chemical mutagenesis strategy allowed a wide range of noncanonical amino acids to be systematically incorporated throughout an active site to alter enzymic substrate specificity. Specifically, 13 different noncanonical side chains were incorporated at 12 different positions within the active site of N-acetylneuraminic acid lyase (NAL), and the resulting chemically modified enzymes were screened for activity with a range of aldehyde substrates. A modified enzyme containing a 2,3-dihydroxypropyl cysteine at position 190 was identified that had significantly increased activity for the aldol reaction of erythrose with pyruvate compared with the wild-type enzyme. Kinetic investigation of a saturation library of the canonical amino acids at the same position showed that this increased activity was not achievable with any of the 20 proteogenic amino acids. Structural and modeling studies revealed that the unique shape and functionality of the noncanonical side chain enabled the active site to be remodeled to enable more efficient stabilization of the transition state of the reaction. The ability to exploit an expanded amino acid alphabet can thus heighten the ambitions of protein engineers wishing to develop enzymes with new catalytic properties. PMID:28196894

  16. The role of CYP26 enzymes in retinoic acid clearance.

    PubMed

    Thatcher, Jayne E; Isoherranen, Nina

    2009-08-01

    Retinoic acid (RA) is a critical signaling molecule that regulates gene transcription and the cell cycle. Understanding of RA signaling has increased dramatically over the past decades, but the connection between whole body RA homeostasis and gene regulation in individual cells is still unclear. It has been proposed that cytochrome P450 family 26 (CYP26) enzymes have a role in determining the cellular exposure to RA by inactivating RA in cells that do not need RA. The CYP26 enzymes have been shown to metabolize RA efficiently and they are also inducible by RA in selected systems. However, their expression patterns in different cell types and a mechanistic understanding of their function is still lacking. Based on preliminary kinetic data and protein expression levels, one may predict that if CYP26A1 is expressed in the liver at even very low levels, it will be the major RA hydroxylase in this tissue. As such, it is an attractive pharmacological target for drug development when one aims to increase circulating or cellular RA concentrations. To further the understanding of how CYP26 enzymes contribute to the regulation of RA homeostasis, structural information of the CYP26s, commercially available recombinant enzymes and good specific and sensitive antibodies are needed.

  17. The role of CYP26 enzymes in retinoic acid clearance

    PubMed Central

    Thatcher, Jayne E.; Isoherranen, Nina

    2009-01-01

    Retinoic acid (RA) is a critical signaling molecule that regulates gene transcription and the cell cycle. Understanding of RA signaling has increased dramatically over the past decades, but the connection between whole body RA homeostasis and gene regulation in individual cells is still unclear. It has been proposed that cytochrome P450 family 26 (CYP26) enzymes have a role in determining the cellular exposure to RA by inactivating RA in cells that do not need RA. The CYP26 enzymes have been shown to metabolize RA efficiently and they are also inducible by RA in selected systems. However, their expression patterns in different cell types and a mechanistic understanding of their function is still lacking. Based on preliminary kinetic data and protein expression levels, one may predict that if CYP26A1 is expressed in the liver at even very low levels, it will be the major RA hydroxylase in this tissue. As such, it is an attractive pharmacological target for drug development when one aims to increase circulating or cellular RA concentrations. To further the understanding of how CYP26 enzymes contribute to the regulation of RA homeostasis, structural information of the CYP26’s, commercially available recombinant enzymes and good specific and sensitive antibodies are needed. PMID:19519282

  18. Diacylglycerol, phosphatidic acid, and their metabolic enzymes in synaptic vesicle recycling.

    PubMed

    Tu-Sekine, Becky; Goldschmidt, Hana; Raben, Daniel M

    2015-01-01

    The synaptic vesicle (SV) cycle includes exocytosis of vesicles loaded with a neurotransmitter such as glutamate, coordinated recovery of SVs by endocytosis, refilling of vesicles, and subsequent release of the refilled vesicles from the presynaptic bouton. SV exocytosis is tightly linked with endocytosis, and variations in the number of vesicles, and/or defects in the refilling of SVs, will affect the amount of neurotransmitter available for release (Sudhof, 2004). There is increasing interest in the roles synaptic vesicle lipids and lipid metabolizing enzymes play in this recycling. Initial emphasis was placed on the role of polyphosphoinositides in SV cycling as outlined in a number of reviews (Lim and Wenk, 2009; Martin, 2012; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). Other lipids are now recognized to also play critical roles. For example, PLD1 (Humeau et al., 2001; Rohrbough and Broadie, 2005) and some DGKs (Miller et al., 1999; Nurrish et al., 1999) play roles in neurotransmission which is consistent with the critical roles for phosphatidic acid (PtdOH) and diacylglycerol (DAG) in the regulation of SV exo/endocytosis (Cremona et al., 1999; Exton, 1994; Huttner and Schmidt, 2000; Lim and Wenk, 2009; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). PLD generates phosphatidic acid by catalyzing the hydrolysis of phosphatidylcholine (PtdCho) and in some systems this PtdOH is de-phosphorylated to generate DAG. In contrast, DGK catalyzes the phosphorylation of DAG thereby converting it into PtdOH. While both enzymes are poised to regulate the levels of DAG and PtdOH, therefore, they both lead to the generation of PtdOH and could have opposite effects on DAG levels. This is particularly important for SV cycling as PtdOH and DAG are both needed for evoked exocytosis (Lim and Wenk, 2009; Puchkov and Haucke, 2013; Rohrbough and Broadie, 2005). Two lipids and their involved metabolic enzymes, two sphingolipids have also been implicated in

  19. D-lysergic acid-activating enzyme from the ergot fungus Claviceps purpurea.

    PubMed Central

    Keller, U; Zocher, R; Krengel, U; Kleinkauf, H

    1984-01-01

    A D-lysergic acid-activating enzyme from the ergot fungus Claviceps purpurea was purified about 145-fold. The enzyme was able to catalyse both the D-lysergic acid-dependent ATP-pyrophosphate exchange and the formation of ATP from D-lysergic acid adenylate and pyrophosphate. Both reactions were also catalysed to a decreased but significant extent with respect to dihydrolysergic acid. The molecular mass of the enzyme was estimated to lie between 135 and 140 kDa. The involvement of the enzyme in the biosynthesis of ergot peptide alkaloids is discussed. Images Fig. 4. PMID:6326747

  20. Ethosuximide: liver enzyme induction and D-glucaric acid excretion.

    PubMed

    Gilbert, J C; Scott, A K; Galloway, D B; Petrie, J C

    1974-06-01

    1 A study has been carried out to determine if ethosuximide induces liver enzymes. 2 Ethosuximide did not affect the urinary excretion of D-glucaric acid by healthy adult subjects nor was the mean daily D-glucaric acid excretion of three epileptic children on long term ethosuximide therapy different from that of three matched controls. 3 Ethosuximide (10 mg/kg or 50 mg/kg daily) did not influence D-glucaric acid excretion or liver microsomal protein and cytochrome P450 contents of guinea pigs but at a dose of 100 mg/kg daily in rats it increased liver microsomal protein and cytochrome P450 without altering D-glucaric acid excretion. 4 These results suggest that at anticonvulsant doses ethosuximide is unlikely to induce liver enzymes. The precise relationship between D-glucaric acid excretion and liver enzyme induction remains in doubt.

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

    PubMed

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

    2015-07-01

    The synthesis of novel amphiphilic hyaluronic acid (HYA) and poly(lactic acid) (PLA) block copolymers is reported as the key element of a strategy to detect the presence of pathogenic bacterial enzymes. In addition to the formation of defined HYA-block-PLA assemblies, the encapsulation of fluorescent reporter dyes and the selective enzymatic degradation of the capsules by hyaluronidase and proteinase K are studied. The synthesis of the dual enzyme-responsive HYA-b-PLA is carried out by copper-catalyzed Huisgen 1,3-dipolar cycloaddition. The resulting copolymers are assembled in water to form vesicular structures, which are characterized by scanning electron microscopy, transmission electron microscopy, dynamic light scattering (DLS), and fluorescence lifetime imaging microscopy (FLIM). DLS measurements show that both enzymes cause a rapid decrease in the hydrodynamic diameter of the nanocapsules. Fluorescence spectroscopy data confirm the liberation of encapsulated dye, which indicates the disintegration of the capsules and validates the concept of enzymatically triggered payload release. Finally, cytotoxicity assays confirm that the HYA-b-PLA nanocapsules are biocompatible with primary human dermal microvascular endothelial cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Inhibitors of the Glyoxylate Cycle Enzyme ICL1 in Candida albicans for Potential Use as Antifungal Agents

    PubMed Central

    Cheah, Hong-Leong; Lim, Vuanghao; Sandai, Doblin

    2014-01-01

    Candida albicans is an opportunistic pathogen that causes candidiasis in humans. In recent years, metabolic pathways in C. albicans have been explored as potential antifungal targets to treat candidiasis. The glyoxylate cycle, which enables C. albicans to survive in nutrient-limited host niches and its. Key enzymes (e.g., isocitrate lyase (ICL1), are particularly attractive antifungal targets for C. albicans. In this study, we used a new screening approach that better reflects the physiological environment that C. albicans cells experience during infection to identify potential inhibitors of ICL. Three compounds (caffeic acid (CAFF), rosmarinic acid (ROS), and apigenin (API)) were found to have antifungal activity against C. albicans when tested under glucose-depleted conditions. We further confirmed the inhibitory potential of these compounds against ICL using the ICL enzyme assay. Lastly, we assessed the bioavailability and toxicity of these compounds using Lipinski's rule-of-five and ADMET analysis. PMID:24781056

  3. Oxidation of indole-3-acetic acid to oxindole-3-acetic acid by an enzyme preparation from Zea mays

    NASA Technical Reports Server (NTRS)

    Reinecke, D. M.; Bandurski, R. S.

    1988-01-01

    Indole-3-acetic acid is oxidized to oxindole-3-acetic acid by Zea mays tissue extracts. Shoot, root, and endosperm tissues have enzyme activities of 1 to 10 picomoles per hour per milligram protein. The enzyme is heat labile, is soluble, and requires oxygen for activity. Cofactors of mixed function oxygenase, peroxidase, and intermolecular dioxygenase are not stimulatory to enzymic activity. A heat-stable, detergent-extractable component from corn enhances enzyme activity 6- to 10-fold. This is the first demonstration of the in vitro enzymic oxidation of indole-3-acetic acid to oxindole-3-acetic acid in higher plants.

  4. ENZYME ACTIVITIES DURING THE ASEXUAL CYCLE OF NEUROSPORA CRASSA

    PubMed Central

    Stine, G. J.

    1968-01-01

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

  5. Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides.

    PubMed

    Cravatt, B F; Giang, D K; Mayfield, S P; Boger, D L; Lerner, R A; Gilula, N B

    1996-11-07

    Endogenous neuromodulatory molecules are commonly coupled to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolysed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the structure and sleep-inducing properties of cis-9-octadecenamide, a lipid isolated from the cerebrospinal fluid of sleep-deprived cats. cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase, from rat liver plasma membranes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor, to arachidonic acid, indicating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling molecules, the fatty-acid amides. Therefore we will hereafter refer to oleamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates.

  6. Kinetics of leather dyeing pretreated with enzymes: role of acid protease.

    PubMed

    Kanth, Swarna Vinodh; Venba, Rajangam; Jayakumar, Gladstone Christopher; Chandrababu, Narasimhan Kannan

    2009-04-01

    In the present investigation, kinetics of dyeing involving pretreatment with acid protease has been presented. Application of acid protease in dyeing process resulted in increased absorption and diffusion of dye into the leather matrix. Enzyme treatment at 1% concentration, 60 min duration and 50 degrees C resulted in maximum of 98% dye exhaustion and increased absorption rate constants. The final exhaustion (C(infinity)) for the best fit of CI Acid Black 194 dye has been 98.5% with K and r2 values from the modified Cegarra-Puente isotherm as 0.1033 and 0.0631. CI Acid Black 194 being a 2:1 metal complex acid dye exhibited higher absorption rate than the acid dye CI Acid Black 210. A reduction in 50% activation energy calculated from Arrhenius equation has been observed in enzyme assisted dyeing process of both the dyes that substantiates enhanced dye absorption. The absorption rate constant calculated with modified Cegarra-Puente equation confirm higher rate constants and faster kinetics for enzyme assisted dyeing process. Enzyme treated leather exhibited richness of color and shade when compared with control. The present study substantiates the essential role of enzyme pretreatment as an eco-friendly leather dyeing process.

  7. Novel insights into the bioenergetics of mixed-acid fermentation: can hydrogen and proton cycles combine to help maintain a proton motive force?

    PubMed

    Trchounian, Armen; Gary Sawers, R

    2014-01-01

    Escherichia coli possesses four [NiFe]-hydrogenases that catalyze the reversible redox reaction of 2H(+) + 2e(-) ↔ H2. These enzymes together have the potential to form a hydrogen cycle across the membrane. Their activity, operational direction, and interaction with each other depend on the fermentation substrate and particularly pH. The enzymes producing H2 are likely able to translocate protons through the membrane. Moreover, the activity of some of these enzymes is dependent on the F0 F1 -ATPase, thus linking a proton cycle with the cycling of hydrogen. These two cycles are suggested to have a primary basic role in modulating the cell's energetics during mixed-acid fermentation, particularly in response to pH. Nevertheless, the mechanisms underlying the physical interactions between these enzyme complexes, as well as how this is controlled, are still not clearly understood. Here, we present a synopsis of the potential impact of proton-hydrogen cycling in fermentative bioenergetics. © 2013 International Union of Biochemistry and Molecular Biology.

  8. C-Myc Induced Compensated Cardiac Hypertrophy Increases Free Fatty Acid Utilization for the Citric Acid Cycle

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Olson, Aaron; Ledee, Dolena; Iwamoto, Kate

    The protooncogene C-Myc (Myc) regulates cardiac hypertrophy. Myc promotes compensated cardiac function, suggesting that the operative mechanisms differ from those leading to heart failure. Myc regulation of substrate metabolism is a reasonable target, as Myc alters metabolism in other tissues. We hypothesize that Myc-induced shifts in substrate utilization signal and promote compensated hypertrophy. We used cardiac specific Myc-inducible C57/BL6 male mice between 4-6 months old that develop hypertrophy with tamoxifen (tam). Isolated working hearts and 13Carbon (13C )-NMR were used to measure function and fractional contributions (Fc) to the citric acid cycle by using perfusate containing 13C-labeled free fatty acids,more » acetoacetate, lactate, unlabeled glucose and insulin. Studies were performed at pre-hypertrophy (3-days tam, 3dMyc), established hypertrophy (7-days tam, 7dMyc) or vehicle control (cont). Non-transgenic siblings (NTG) received 7-days tam or vehicle to assess drug effect. Hypertrophy was confirmed by echocardiograms and heart weights. Western blots were performed on key metabolic enzymes. Hypertrophy occurred in 7dMyc only. Cardiac function did not differ between groups. Tam alone did not affect substrate contribution in NTG. Substrate utilization was not significantly altered in 3dMyc versus cont. The free fatty acid FC was significantly greater in 7dMyc vs cont with decreased unlabeled Fc, which is predominately exogenous glucose. Free fatty acid flux to the citric acid cycle increased while lactate flux was diminished in 7dMyc compared to cont. Total protein levels of a panel of key metabolic enzymes were unchanged; however total protein O-GlcNAcylation was increased in 7dMyc. Substrate utilization changes did not precede hypertrophy; therefore they are not the primary signal for cardiac growth in this model. Free fatty acid utilization and oxidation increase at established hypertrophy. Understanding the mechanisms whereby this change

  9. Teaching about citric acid cycle using plant mitochondrial preparations: Some assays for use in laboratory courses*.

    PubMed

    Vicente, Joaquim A F; Gomes-Santos, Carina S S; Sousa, Ana Paula M; Madeira, Vítor M C

    2005-03-01

    Potato tubers and turnip roots were used to prepare purified mitochondria for laboratory practical work in the teaching of the citric acid cycle (TCA cycle). Plant mitochondria are particularly advantageous over the animal fractions to demonstrate the TCA cycle enzymatic steps, by using simple techniques to measure O(2) consumption and transmembrane potential (ΔΨ). The several TCA cycle intermediates induce specific enzyme activities, which can be identified by respiratory parameters. Such a strategy is also used to evidence properties of the TCA cycle enzymes: ADP stimulation of isocitrate dehydrogenase and α-ketoglutarate dehydrogenase; activation by citrate of downstream oxidation steps, e.g. succinate dehydrogenase; and regulation of the activity of isocitrate dehydrogenase by citrate action on the citrate/isocitrate carrier. Furthermore, it has been demonstrated that, in the absence of exogenous Mg(2+) , isocitrate-dependent respiration favors the alternative oxidase pathway, as judged by changes of the ADP/O elicited by the inhibitor n-propyl galate. These are some examples of assays related with TCA cycle intermediates we can use in laboratory courses. Copyright © 2005 International Union of Biochemistry and Molecular Biology, Inc.

  10. Sirt3 promotes the urea cycle and fatty acid oxidation during dietary restriction

    PubMed Central

    Hallows, William C.; Yu, Wei; Smith, Brian C.; Devries, Mark K.; Ellinger, James J.; Someya, Shinichi; Shortreed, Michael R.; Prolla, Tomas; Markley, John L.; Smith, Lloyd M.; Zhao, Shimin; Guan, Kun-Liang; Denu, John M.

    2011-01-01

    Summary Emerging evidence suggests that protein acetylation is a broad-ranging regulatory mechanism. Here we utilize acetyl-peptide arrays and metabolomic analyses to identify substrates of mitochondrial deacetylase Sirt3. We identified ornithine transcarbamoylase (OTC) from the urea cycle, and enzymes involved in β-oxidation. Metabolomic analyses of fasted mice lacking Sirt3 (sirt3−/−) revealed alterations in β-oxidation and the urea cycle. Biochemical analysis demonstrated that Sirt3 directly deacetylates OTC and stimulates its activity. Mice under caloric restriction (CR) increased Sirt3 protein levels, leading to deacetylation and stimulation of OTC activity. In contrast, sirt3−/− mice failed to deacetylate OTC in response to CR. Inability to stimulate OTC under CR led to a failure to reduce orotic acid levels, a known outcome of OTC deficiency. Thus, Sirt3 directly regulates OTC activity and promotes the urea cycle during CR, and the results suggest that under low energy input, Sirt3 modulates mitochondria by promoting amino-acid catabolism and β-oxidation. PMID:21255725

  11. Integrated structural biology and molecular ecology of N-cycling enzymes from ammonia-oxidizing archaea

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tolar, Bradley B.; Herrmann, Jonathan; Bargar, John R.

    In this paper, knowledge of the molecular ecology and environmental determinants of ammonia-oxidizing organisms is critical to understanding and predicting the global nitrogen (N) and carbon cycles, but an incomplete biochemical picture hinders in vitro studies of N-cycling enzymes. Although an integrative structural and dynamic characterization at the atomic scale would advance our understanding of function tremendously, structural knowlede of key N-cycling enzymes from ecologically-relevant ammonia oxidizers is unfortunately extremely limited. Here, we discuss the challenges and opportunities for examining the ecology of ammonia-oxidizing organisms, particularly uncultivated Thaumarchaeota, though (meta)genome-driven structural biology of the enzymes ammonia monooxygenase (AMO) andmore » nitrite reductase (NirK).« less

  12. Integrated structural biology and molecular ecology of N-cycling enzymes from ammonia-oxidizing archaea

    DOE PAGES

    Tolar, Bradley B.; Herrmann, Jonathan; Bargar, John R.; ...

    2017-07-05

    In this paper, knowledge of the molecular ecology and environmental determinants of ammonia-oxidizing organisms is critical to understanding and predicting the global nitrogen (N) and carbon cycles, but an incomplete biochemical picture hinders in vitro studies of N-cycling enzymes. Although an integrative structural and dynamic characterization at the atomic scale would advance our understanding of function tremendously, structural knowlede of key N-cycling enzymes from ecologically-relevant ammonia oxidizers is unfortunately extremely limited. Here, we discuss the challenges and opportunities for examining the ecology of ammonia-oxidizing organisms, particularly uncultivated Thaumarchaeota, though (meta)genome-driven structural biology of the enzymes ammonia monooxygenase (AMO) andmore » nitrite reductase (NirK).« less

  13. Integrated structural biology and molecular ecology of N-cycling enzymes from ammonia-oxidizing archaea.

    PubMed

    Tolar, Bradley B; Herrmann, Jonathan; Bargar, John R; van den Bedem, Henry; Wakatsuki, Soichi; Francis, Christopher A

    2017-10-01

    Knowledge of the molecular ecology and environmental determinants of ammonia-oxidizing organisms is critical to understanding and predicting the global nitrogen (N) and carbon cycles, but an incomplete biochemical picture hinders in vitro studies of N-cycling enzymes. Although an integrative structural and dynamic characterization at the atomic scale would advance our understanding of function tremendously, structural knowledge of key N-cycling enzymes from ecologically relevant ammonia oxidizers is unfortunately extremely limited. Here, we discuss the challenges and opportunities for examining the ecology of ammonia-oxidizing organisms, particularly uncultivated Thaumarchaeota, through (meta)genome-driven structural biology of the enzymes ammonia monooxygenase (AMO) and nitrite reductase (NirK). © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  14. Exponential isothermal amplification of nucleic acids and amplified assays for proteins, cells, and enzyme activities.

    PubMed

    Reid, Michael S; Le, X Chris; Zhang, Hongquan

    2018-04-27

    Isothermal exponential amplification techniques, such as strand-displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA), have great potential for on-site, point-of-care, and in-situ assay applications. These amplification techniques eliminate the need for temperature cycling required for polymerase chain reaction (PCR) while achieving comparable amplification yield. We highlight here recent advances in exponential amplification reaction (EXPAR) for the detection of nucleic acids, proteins, enzyme activities, cells, and metal ions. We discuss design strategies, enzyme reactions, detection techniques, and key features. Incorporation of fluorescence, colorimetric, chemiluminescence, Raman, and electrochemical approaches enables highly sensitive detection of a variety of targets. Remaining issues, such as undesirable background amplification resulting from non-specific template interactions, must be addressed to further improve isothermal and exponential amplification techniques. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Combinatorial Effects of Fatty Acid Elongase Enzymes on Nervonic Acid Production in Camelina sativa

    PubMed Central

    Huai, Dongxin; Zhang, Yuanyuan; Zhang, Chunyu; Cahoon, Edgar B.; Zhou, Yongming

    2015-01-01

    Very long chain fatty acids (VLCFAs) with chain lengths of 20 carbons and longer provide feedstocks for various applications; therefore, improvement of VLCFA contents in seeds has become an important goal for oilseed enhancement. VLCFA biosynthesis is controlled by a multi-enzyme protein complex referred to as fatty acid elongase, which is composed of β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), β-hydroxyacyl-CoA dehydratase (HCD) and enoyl reductase (ECR). KCS has been identified as the rate-limiting enzyme, but little is known about the involvement of other three enzymes in VLCFA production. Here, the combinatorial effects of fatty acid elongase enzymes on VLCFA production were assessed by evaluating the changes in nervonic acid content. A KCS gene from Lunaria annua (LaKCS) and the other three elongase genes from Arabidopsis thaliana were used for the assessment. Five seed-specific expressing constructs, including LaKCS alone, LaKCS with AtKCR, LaKCS with AtHCD, LaKCS with AtECR, and LaKCS with AtKCR and AtHCD, were transformed into Camelina sativa. The nervonic acid content in seed oil increased from null in wild type camelina to 6-12% in LaKCS-expressing lines. However, compared with that from the LaKCS-expressing lines, nervonic acid content in mature seeds from the co-expressing lines with one or two extra elongase genes did not show further increases. Nervonic acid content from LaKCS, AtKCR and AtHCD co-expressing line was significantly higher than that in LaKCS-expressing line during early seed development stage, while the ultimate nervonic acid content was not significantly altered. The results from this study thus provide useful information for future engineering of oilseed crops for higher VLCFA production. PMID:26121034

  16. Improving a natural enzyme activity through incorporation of unnatural amino acids.

    PubMed

    Ugwumba, Isaac N; Ozawa, Kiyoshi; Xu, Zhi-Qiang; Ely, Fernanda; Foo, Jee-Loon; Herlt, Anthony J; Coppin, Chris; Brown, Sue; Taylor, Matthew C; Ollis, David L; Mander, Lewis N; Schenk, Gerhard; Dixon, Nicholas E; Otting, Gottfried; Oakeshott, John G; Jackson, Colin J

    2011-01-19

    The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.

  17. Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges.

    PubMed

    Qing, Taiping; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Wen, Li; Shangguan, Jingfang; Mao, Zhengui; Lei, Yanli

    2016-04-01

    Owing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as 'nano-tools' for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors.

  18. Krebs cycle metabolon formation: metabolite concentration gradient enhanced compartmentation of sequential enzymes.

    PubMed

    Wu, Fei; Pelster, Lindsey N; Minteer, Shelley D

    2015-01-25

    Dynamics of metabolon formation in mitochondria was probed by studying diffusional motion of two sequential Krebs cycle enzymes in a microfluidic channel. Enhanced directional co-diffusion of both enzymes against a substrate concentration gradient was observed in the presence of intermediate generation. This reveals a metabolite directed compartmentation of metabolic pathways.

  19. Jasmonic acid-amino acid conjugation enzyme assays.

    PubMed

    Rowe, Martha L; Staswick, Paul E

    2013-01-01

    Jasmonic acid (JA) is activated for signaling by its conjugation to isoleucine (Ile) through an amide linkage. The Arabidopsis thaliana JASMONIC ACID RESISTANT1 (JAR1) enzyme carries out this Mg-ATP-dependent reaction in two steps, adenylation of the free carboxyl of JA, followed by condensation of the activated group to Ile. This chapter details the protocols used to detect and quantify the enzymatic activity obtained from a glutathione-S-transferase:JAR1 fusion protein produced in Escherichia coli, including an isotope exchange assay for the adenylation step and assays for the complete reaction that involve the high-performance liquid chromatography quantitation of adenosine monophosphate, a stoichiometric by-product of the reaction, and detection of the conjugation product by thin-layer chromatography or gas -chromatography/mass spectrometry.

  20. Characterization of Enzymes Involved in Fatty Acid Elongation

    DTIC Science & Technology

    2007-04-11

    dihydroxyacetone reductase involved in phosphatidic acid biosynthesis [111]. Therefore, altered glycerophospholipid metabolism, along with reduced...2007 Title of Dissertation: "Characterization of Enzymes Involved in Fatty Acid Elongation" APPROVAL SHEET Ernest Maynard, P .D. Department of...Fatty Acid Elongation" is appropriately acknowledged and, beyond brief excerpts, is with the permission of the copyright owner. , /1:1 IJA"" 1< .IIVCf

  1. Ammonium Metabolism Enzymes Aid Helicobacter pylori Acid Resistance

    PubMed Central

    Miller, Erica F.

    2014-01-01

    The gastric pathogen Helicobacter pylori possesses a highly active urease to support acid tolerance. Urea hydrolysis occurs inside the cytoplasm, resulting in the production of NH3 that is immediately protonated to form NH4+. This ammonium must be metabolized or effluxed because its presence within the cell is counterproductive to the goal of raising pH while maintaining a viable proton motive force (PMF). Two compatible hypotheses for mitigating intracellular ammonium toxicity include (i) the exit of protonated ammonium outward via the UreI permease, which was shown to facilitate diffusion of both urea and ammonium, and/or (ii) the assimilation of this ammonium, which is supported by evidence that H. pylori assimilates urea nitrogen into its amino acid pools. We investigated the second hypothesis by constructing strains with altered expression of the ammonium-assimilating enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) and the ammonium-evolving periplasmic enzymes glutaminase (Ggt) and asparaginase (AsnB). H. pylori strains expressing elevated levels of either GS or GDH are more acid tolerant than the wild type, exhibit enhanced ammonium production, and are able to alkalize the medium faster than the wild type. Strains lacking the genes for either Ggt or AsnB are acid sensitive, have 8-fold-lower urea-dependent ammonium production, and are more acid sensitive than the parent. Additionally, we found that purified H. pylori GS produces glutamine in the presence of Mg2+ at a rate similar to that of unadenylated Escherichia coli GS. These data reveal that all four enzymes contribute to whole-cell acid resistance in H. pylori and are likely important for assimilation and/or efflux of urea-derived ammonium. PMID:24936052

  2. Correlations between FAS elongation cycle genes expression and fatty acid production for improvement of long-chain fatty acids in Escherichia coli.

    PubMed

    Lee, Sunhee; Jung, Yeontae; Lee, Seunghan; Lee, Jinwon

    2013-03-01

    Microorganisms have been used for biodiesel (fatty acid methyl ester) production due to their significant environmental and economic benefits. The aim of the present research was to develop new strains of Escherichia coli K-12 MG1655 and to increase the content of long-chain fatty acids by overexpressing essential enzymes that are involved in the fatty acid synthase elongation cycle. In addition, the relationship of β-ketoacyl-acyl carrier protein (ACP) synthase (fabH), β-ketoacyl-ACP reductase (fabG), β-hydroxyacyl-ACP dehydrase (fabZ), and β-enoyl-ACP reductase (fabI) with respect to fatty acid production was investigated. The four enzymes play a unique role in fatty acid biosynthesis and elongation processes. We report the generation of recombinant E. coli strains that produced long-chain fatty acids to amounts twofold over wild type. To verify the results, NAD(+)/NADH ratios and glucose analyses were performed. We also confirmed that FabZ plays an important role in producing unsaturated fatty acids (UFAs) as E. coli SGJS25 (overexpressing the fabZ gene) produced the highest percentage of UFAs (35 % of total long-chain fatty acids), over wild type and other recombinants. Indeed, cis-9-hexadecenoic acid, a major UFA in E. coli SGJS25, was produced at levels 20-fold higher than in wild type after 20 h in culture. The biochemically engineered E. coli presented in this study is expected to be more economical for producing long-chain fatty acids in quality biodiesel production processes.

  3. Eicosapentaenoic acid and docosahexaenoic acid increase the degradation of amyloid-β by affecting insulin-degrading enzyme.

    PubMed

    Grimm, Marcus O W; Mett, Janine; Stahlmann, Christoph P; Haupenthal, Viola J; Blümel, Tamara; Stötzel, Hannah; Grimm, Heike S; Hartmann, Tobias

    2016-12-01

    Omega-3 polyunsaturated fatty acids (PUFAs) have been proposed to be highly beneficial in Alzheimer's disease (AD). AD pathology is closely linked to an overproduction and accumulation of amyloid-β (Aβ) peptides as extracellular senile plaques in the brain. Total Aβ levels are not only dependent on its production by proteolytic processing of the amyloid precursor protein (APP), but also on Aβ-clearance mechanisms, including Aβ-degrading enzymes. Here we show that the omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) increase Aβ-degradation by affecting insulin-degrading enzyme (IDE), the major Aβ-degrading enzyme secreted into the extracellular space of neuronal and microglial cells. The identification of the molecular mechanisms revealed that EPA directly increases IDE enzyme activity and elevates gene expression of IDE. DHA also directly stimulates IDE enzyme activity and affects IDE sorting by increasing exosome release of IDE, resulting in enhanced Aβ-degradation in the extracellular milieu. Apart from the known positive effect of DHA in reducing Aβ production, EPA and DHA might ameliorate AD pathology by increasing Aβ turnover.

  4. Transcriptome mining and in silico structural and functional analysis of ascorbic acid and tartaric acid biosynthesis pathway enzymes in rose-scanted geranium.

    PubMed

    Narnoliya, Lokesh K; Sangwan, Rajender S; Singh, Sudhir P

    2018-06-01

    Rose-scented geranium (Pelargonium sp.) is widely known as aromatic and medicinal herb, accumulating specialized metabolites of high economic importance, such as essential oils, ascorbic acid, and tartaric acid. Ascorbic acid and tartaric acid are multifunctional metabolites of human value to be used as vital antioxidants and flavor enhancing agents in food products. No information is available related to the structural and functional properties of the enzymes involved in ascorbic acid and tartaric acid biosynthesis in rose-scented geranium. In the present study, transcriptome mining was done to identify full-length genes, followed by their bioinformatic and molecular modeling investigations and understanding of in silico structural and functional properties of these enzymes. Evolutionary conserved domains were identified in the pathway enzymes. In silico physicochemical characterization of the catalytic enzymes revealed isoelectric point (pI), instability index, aliphatic index, and grand average hydropathy (GRAVY) values of the enzymes. Secondary structural prediction revealed abundant proportion of alpha helix and random coil confirmations in the pathway enzymes. Three-dimensional homology models were developed for these enzymes. The predicted structures showed significant structural similarity with their respective templates in root mean square deviation analysis. Ramachandran plot analysis of the modeled enzymes revealed that more than 84% of the amino acid residues were within the favored regions. Further, functionally important residues were identified corresponding to catalytic sites located in the enzymes. To, our best knowledge, this is the first report which provides a foundation on functional annotation and structural determination of ascorbic acid and tartaric acid pathway enzymes in rose-scanted geranium.

  5. Development of a novel ultrasensitive enzyme immunoassay for human glutamic acid decarboxylase 65 antibody.

    PubMed

    Numata, Satoshi; Katakami, Hideki; Inoue, Shinobu; Sawada, Hirotake; Hashida, Seiichi

    2016-07-01

    We developed a novel, ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for determination of glutamic acid decarboxylase autoantibody concentrations in serum samples from patients with type 2 diabetes. We developed an immune complex transfer enzyme immunoassay for glutamic acid decarboxylase autoantibody and measured glutamic acid decarboxylase autoantibody from 22 patients with type 1 diabetes, 29 patients with type 2 diabetes, and 32 healthy controls. A conventional ELISA kit identified 10 patients with type 1 diabetes and one patient with type 2 diabetes as glutamic acid decarboxylase autoantibody positive, whereas 15 patients with type 1 diabetes and six patients with type 2 diabetes were identified as glutamic acid decarboxylase autoantibody positive using immune complex transfer enzyme immunoassay. Immune complex transfer enzyme immunoassay is a highly sensitive and specific assay for glutamic acid decarboxylase autoantibody and might be clinically useful for diabetic onset prediction and early diagnosis. © The Author(s) 2016.

  6. Beyond Vmax and Km: How details of enzyme function influence geochemical cycles

    NASA Astrophysics Data System (ADS)

    Steen, A. D.

    2015-12-01

    Enzymes catalyze the vast majority of chemical reactions relevant to geomicrobiology. Studies of the activities of enzymes in environmental systems often report Vmax (the maximum possible rate of reaction; often proportional to the concentration of enzymes in the system) and sometimes Km (a measure of the affinity between enzymes and their substrates). However, enzyme studies - particularly those related to enzymes involved in organic carbon oxidation - are often limited to only those parameters, and a relatively limited and mixed set of enzymes. Here I will discuss some novel methods to assay and characterize the specific sets of enzymes that may be important to the carbon cycle in aquatic environments. First, kinetic experiments revealed the collective properties of the complex mixtures of extracellular peptidases that occur where microbial communities are diverse. Crystal structures combined with biochemical characterization of specific enzymes can yield more detailed information about key steps in organic carbon transformations. These new techniques have the potential to provide mechanistic grounding to geomicrobiological models.

  7. Enzymes involved in branched-chain amino acid metabolism in humans.

    PubMed

    Adeva-Andany, María M; López-Maside, Laura; Donapetry-García, Cristóbal; Fernández-Fernández, Carlos; Sixto-Leal, Cristina

    2017-06-01

    Branched-chain amino acids (leucine, isoleucine and valine) are structurally related to branched-chain fatty acids. Leucine is 2-amino-4-methyl-pentanoic acid, isoleucine is 2-amino-3-methyl-pentanoic acid, and valine is 2-amino-3-methyl-butanoic acid. Similar to fatty acid oxidation, leucine and isoleucine produce acetyl-coA. Additionally, leucine generates acetoacetate and isoleucine yields propionyl-coA. Valine oxidation produces propionyl-coA, which is converted into methylmalonyl-coA and succinyl-coA. Branched-chain aminotransferase catalyzes the first reaction in the catabolic pathway of branched-chain amino acids, a reversible transamination that converts branched-chain amino acids into branched-chain ketoacids. Simultaneously, glutamate is converted in 2-ketoglutarate. The branched-chain ketoacid dehydrogenase complex catalyzes the irreversible oxidative decarboxylation of branched-chain ketoacids to produce branched-chain acyl-coA intermediates, which then follow separate catabolic pathways. Human tissue distribution and function of most of the enzymes involved in branched-chain amino acid catabolism is unknown. Congenital deficiencies of the enzymes involved in branched-chain amino acid metabolism are generally rare disorders. Some of them are associated with reduced pyruvate dehydrogenase complex activity and respiratory chain dysfunction that may contribute to their clinical phenotype. The biochemical phenotype is characterized by accumulation of the substrate to the deficient enzyme and its carnitine and/or glycine derivatives. It was established at the beginning of the twentieth century that the plasma level of the branched-chain amino acids is increased in conditions associated with insulin resistance such as obesity and diabetes mellitus. However, the potential clinical relevance of this elevation is uncertain.

  8. Succinyl-CoA:Mesaconate CoA-Transferase and Mesaconyl-CoA Hydratase, Enzymes of the Methylaspartate Cycle in Haloarcula hispanica.

    PubMed

    Borjian, Farshad; Johnsen, Ulrike; Schönheit, Peter; Berg, Ivan A

    2017-01-01

    Growth on acetate or other acetyl-CoA-generating substrates as a sole source of carbon requires an anaplerotic pathway for the conversion of acetyl-CoA into cellular building blocks. Haloarchaea (class Halobacteria ) possess two different anaplerotic pathways, the classical glyoxylate cycle and the novel methylaspartate cycle. The methylaspartate cycle was discovered in Haloarcula spp. and operates in ∼40% of sequenced haloarchaea. In this cycle, condensation of one molecule of acetyl-CoA with oxaloacetate gives rise to citrate, which is further converted to 2-oxoglutarate and then to glutamate. The following glutamate rearrangement and deamination lead to mesaconate (methylfumarate) that needs to be activated to mesaconyl-C1-CoA and hydrated to β-methylmalyl-CoA. The cleavage of β-methylmalyl-CoA results in the formation of propionyl-CoA and glyoxylate. The carboxylation of propionyl-CoA and the condensation of glyoxylate with another acetyl-CoA molecule give rise to two C 4 -dicarboxylic acids, thus regenerating the initial acetyl-CoA acceptor and forming malate, its final product. Here we studied two enzymes of the methylaspartate cycle from Haloarcula hispanica , succinyl-CoA:mesaconate CoA-transferase (mesaconate CoA-transferase, Hah_1336) and mesaconyl-CoA hydratase (Hah_1340). Their genes were heterologously expressed in Haloferax volcanii , and the corresponding enzymes were purified and characterized. Mesaconate CoA-transferase was specific for its physiological substrates, mesaconate and succinyl-CoA, and produced only mesaconyl-C1-CoA and no mesaconyl-C4-CoA. Mesaconyl-CoA hydratase had a 3.5-fold bias for the physiological substrate, mesaconyl-C1-CoA, compared to mesaconyl-C4-CoA, and virtually no activity with other tested enoyl-CoA/3-hydroxyacyl-CoA compounds. Our results further prove the functioning of the methylaspartate cycle in haloarchaea and suggest that mesaconate CoA-transferase and mesaconyl-CoA hydratase can be regarded as

  9. Design principles of autocatalytic cycles constrain enzyme kinetics and force low substrate saturation at flux branch points

    PubMed Central

    Barenholz, Uri; Davidi, Dan; Reznik, Ed; Bar-On, Yinon; Antonovsky, Niv; Noor, Elad; Milo, Ron

    2017-01-01

    A set of chemical reactions that require a metabolite to synthesize more of that metabolite is an autocatalytic cycle. Here, we show that most of the reactions in the core of central carbon metabolism are part of compact autocatalytic cycles. Such metabolic designs must meet specific conditions to support stable fluxes, hence avoiding depletion of intermediate metabolites. As such, they are subjected to constraints that may seem counter-intuitive: the enzymes of branch reactions out of the cycle must be overexpressed and the affinity of these enzymes to their substrates must be relatively weak. We use recent quantitative proteomics and fluxomics measurements to show that the above conditions hold for functioning cycles in central carbon metabolism of E. coli. This work demonstrates that the topology of a metabolic network can shape kinetic parameters of enzymes and lead to seemingly wasteful enzyme usage. DOI: http://dx.doi.org/10.7554/eLife.20667.001 PMID:28169831

  10. Alpha-lipoic acid supplementation protects enzymes from damage by nitrosative and oxidative stress.

    PubMed

    Hiller, Sylvia; DeKroon, Robert; Hamlett, Eric D; Xu, Longquan; Osorio, Cristina; Robinette, Jennifer; Winnik, Witold; Simington, Stephen; Maeda, Nobuyo; Alzate, Oscar; Yi, Xianwen

    2016-01-01

    S-nitrosylation of mitochondrial enzymes involved in energy transfer under nitrosative stress may result in ATP deficiency. We investigated whether α-lipoic acid, a powerful antioxidant, could alleviate nitrosative stress by regulating S-nitrosylation, which could result in retaining the mitochondrial enzyme activity. In this study, we have identified the S-nitrosylated forms of subunit 1 of dihydrolipoyllysine succinyltransferase (complex III), and subunit 2 of the α-ketoglutarate dehydrogenase complex by implementing a fluorescence-based differential quantitative proteomics method. We found that the activities of these two mitochondrial enzymes were partially but reversibly inhibited by S-nitrosylation in cultured endothelial cells, and that their activities were partially restored by supplementation of α-lipoic acid. We show that protein S-nitrosylation affects the activity of mitochondrial enzymes that are central to energy supply, and that α-lipoic acid protects mitochondrial enzymes by altering S-nitrosylation levels. Inhibiting protein S-nitrosylation with α-lipoic acid seems to be a protective mechanism against nitrosative stress. Identification and characterization of these new protein targets should contribute to expanding the therapeutic power of α-lipoic acid and to a better understanding of the underlying antioxidant mechanisms.

  11. Studies on the enzymes of Sarcocystis suicanis: purification and characterization of an acid phosphatase.

    PubMed

    Farooqui, A A; Adams, D D; Hanson, W L; Prestwood, A K

    1987-08-01

    Percoll density gradient centrifugation was used for isolating large quantities of bradyzoites of Sarcocystis suicanis, which were used for enzymatic analysis. Crude extracts of bradyzoites contained activities suggestive of several acid hydrolases. Levels of acid and alkaline phosphatase were higher than those of beta-N-acetylhexosaminidase and beta-galactosidase. Acid phosphatase was purified 156-fold with an overall recovery of 54% using DEAE-Sepharose 4B and Sephadex G-200 chromatography. The partially purified enzyme was not a glycoprotein and had a molecular weight of approximately 170,000. The enzyme was markedly inhibited by Cu++, Hg++, and iodoacetamide, suggesting the presence of a sulfhydryl group. Sodium tartrate caused strong inhibition of the enzyme. The acid phosphatase of S. suicanis appears to be a unique enzyme that cannot be classified under high or low molecular weight acid phosphatases of widely diverse origin.

  12. Cadmium Phytoavailability and Enzyme Activity under Humic Acid Treatment in Fluvo-aquic Soil

    NASA Astrophysics Data System (ADS)

    Liu, Borui; Huang, Qing; Su, Yuefeng

    2018-01-01

    A pot experiment was conducted to investigate the cadmium (Cd) availability to pakchois (Brassica chinensis L.) as well as the enzyme activities in fluvo-aquic soil under humic acid treatment. The results showed that the phytoavailability of Cd in soil decreased gradually as humic acid concentration rose (0 to 12 g·kg-1), while the activities of urease (UE), alkaline phosphatase (ALP) and catalase (CAT) kept increasing (P < 0.05). The correlation analysis indicated that humic acid was effective for reducing the devastation to soil enzymes due to the Cd pollution. In conclusion, humic acid is effective for the reduction of both Cd phytoavailability and the damage to enzyme activities due to Cd pollution in fluvo-aquic soil

  13. Variation in pH optima of hydrolytic enzyme activities in tropical rain forest soils.

    PubMed

    Turner, Benjamin L

    2010-10-01

    Extracellular enzymes synthesized by soil microbes play a central role in the biogeochemical cycling of nutrients in the environment. The pH optima of eight hydrolytic enzymes involved in the cycles of carbon, nitrogen, phosphorus, and sulfur, were assessed in a series of tropical forest soils of contrasting pH values from the Republic of Panama. Assays were conducted using 4-methylumbelliferone-linked fluorogenic substrates in modified universal buffer. Optimum pH values differed markedly among enzymes and soils. Enzymes were grouped into three classes based on their pH optima: (i) enzymes with acidic pH optima that were consistent among soils (cellobiohydrolase, β-xylanase, and arylsulfatase), (ii) enzymes with acidic pH optima that varied systematically with soil pH, with the most acidic pH optima in the most acidic soils (α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminidase), and (iii) enzymes with an optimum pH in either the acid range or the alkaline range depending on soil pH (phosphomonoesterase and phosphodiesterase). The optimum pH values of phosphomonoesterase were consistent among soils, being 4 to 5 for acid phosphomonoesterase and 10 to 11 for alkaline phosphomonoesterase. In contrast, the optimum pH for phosphodiesterase activity varied systematically with soil pH, with the most acidic pH optima (3.0) in the most acidic soils and the most alkaline pH optima (pH 10) in near-neutral soils. Arylsulfatase activity had a very acidic optimum pH in all soils (pH ≤3.0) irrespective of soil pH. The differences in pH optima may be linked to the origins of the enzymes and/or the degree of stabilization on solid surfaces. The results have important implications for the interpretation of hydrolytic enzyme assays using fluorogenic substrates.

  14. The Roles of Acids and Bases in Enzyme Catalysis

    ERIC Educational Resources Information Center

    Weiss, Hilton M.

    2007-01-01

    Many organic reactions are catalyzed by strong acids or bases that protonate or deprotonate neutral reactants leading to reactive cations or anions that proceed to products. In enzyme reactions, only weak acids and bases are available to hydrogen bond to reactants and to transfer protons in response to developing charges. Understanding this…

  15. Production of Glucaric Acid from Hemicellulose Substrate by Rosettasome Enzyme Assemblies.

    PubMed

    Lee, Charles C; Kibblewhite, Rena E; Paavola, Chad D; Orts, William J; Wagschal, Kurt

    2016-07-01

    Hemicellulose biomass is a complex polymer with many different chemical constituents that can be utilized as industrial feedstocks. These molecules can be released from the polymer and transformed into value-added chemicals through multistep enzymatic pathways. Some bacteria produce cellulosomes which are assemblies composed of lignocellulolytic enzymes tethered to a large protein scaffold. Rosettasomes are artificial engineered ring scaffolds designed to mimic the bacterial cellulosome. Both cellulosomes and rosettasomes have been shown to facilitate much higher rates of biomass hydrolysis compared to the same enzymes free in solution. We investigated whether tethering enzymes involved in both biomass hydrolysis and oxidative transformation to glucaric acid onto a rosettasome scaffold would result in an analogous production enhancement in a combined hydrolysis and bioconversion metabolic pathway. Three different enzymes were used to hydrolyze birchwood hemicellulose and convert the substituents to glucaric acid, a top-12 DOE value added chemical feedstock derived from biomass. It was demonstrated that colocalizing the three different enzymes to the synthetic scaffold resulted in up to 40 % higher levels of product compared to uncomplexed enzymes.

  16. Metabolism: Part II. The Tricarboxylic Acid (TCA), Citric Acid, or Krebs Cycle.

    ERIC Educational Resources Information Center

    Bodner, George M.

    1986-01-01

    Differentiates the tricarboxylic acid (TCA) cycle (or Krebs cycle) from glycolysis, and describes the bridge between the two as being the conversion of pyruvate into acetyl coenzyme A. Discusses the eight steps in the TCA cycle, the results of isotopic labeling experiments, and the net effects of the TCA cycle. (TW)

  17. Mutation for nonsyndromic mental retardation in the trans-2-enoyl-CoA reductase TER gene involved in fatty acid elongation impairs the enzyme activity and stability, leading to change in sphingolipid profile.

    PubMed

    Abe, Kensuke; Ohno, Yusuke; Sassa, Takayuki; Taguchi, Ryo; Çalışkan, Minal; Ober, Carole; Kihara, Akio

    2013-12-20

    Very long-chain fatty acids (VLCFAs, chain length >C20) exist in tissues throughout the body and are synthesized by repetition of the fatty acid (FA) elongation cycle composed of four successive enzymatic reactions. In mammals, the TER gene is the only gene encoding trans-2-enoyl-CoA reductase, which catalyzes the fourth reaction in the FA elongation cycle. The TER P182L mutation is the pathogenic mutation for nonsyndromic mental retardation. This mutation substitutes a leucine for a proline residue at amino acid 182 in the TER enzyme. Currently, the mechanism by which the TER P182L mutation causes nonsyndromic mental retardation is unknown. To understand the effect of this mutation on the TER enzyme and VLCFA synthesis, we have biochemically characterized the TER P182L mutant enzyme using yeast and mammalian cells transfected with the TER P182L mutant gene and analyzed the FA elongation cycle in the B-lymphoblastoid cell line with the homozygous TER P182L mutation (TER(P182L/P182L) B-lymphoblastoid cell line). We have found that TER P182L mutant enzyme exhibits reduced trans-2-enoyl-CoA reductase activity and protein stability, thereby impairing VLCFA synthesis and, in turn, altering the sphingolipid profile (i.e. decreased level of C24 sphingomyelin and C24 ceramide) in the TER(P182L/P182L) B-lymphoblastoid cell line. We have also found that in addition to the TER enzyme-catalyzed fourth reaction, the third reaction in the FA elongation cycle is affected by the TER P182L mutation. These findings provide new insight into the biochemical defects associated with this genetic mutation.

  18. Discrimination of acidic and alkaline enzyme using Chou's pseudo amino acid composition in conjunction with probabilistic neural network model.

    PubMed

    Khan, Zaheer Ullah; Hayat, Maqsood; Khan, Muazzam Ali

    2015-01-21

    Enzyme catalysis is one of the most essential and striking processes among of all the complex processes that have evolved in living organisms. Enzymes are biological catalysts, which play a significant role in industrial applications as well as in medical areas, due to profound specificity, selectivity and catalytic efficiency. Refining catalytic efficiency of enzymes has become the most challenging job of enzyme engineering, into acidic and alkaline. Discrimination of acidic and alkaline enzymes through experimental approaches is difficult, sometimes impossible due to lack of established structures. Therefore, it is highly desirable to develop a computational model for discriminating acidic and alkaline enzymes from primary sequences. In this study, we have developed a robust, accurate and high throughput computational model using two discrete sample representation methods Pseudo amino acid composition (PseAAC) and split amino acid composition. Various classification algorithms including probabilistic neural network (PNN), K-nearest neighbor, decision tree, multi-layer perceptron and support vector machine are applied to predict acidic and alkaline with high accuracy. 10-fold cross validation test and several statistical measures namely, accuracy, F-measure, and area under ROC are used to evaluate the performance of the proposed model. The performance of the model is examined using two benchmark datasets to demonstrate the effectiveness of the model. The empirical results show that the performance of PNN in conjunction with PseAAC is quite promising compared to existing approaches in the literature so for. It has achieved 96.3% accuracy on dataset1 and 99.2% on dataset2. It is ascertained that the proposed model might be useful for basic research and drug related application areas. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Determination Hypoiodous Acid (HIO) By Peroxidase System Using Peroxidase Enzyme

    NASA Astrophysics Data System (ADS)

    Al-Baarri, A. N.; Legowo, A. M.; Widayat; Abduh, S. B. M.; Hadipernata, M.; Wisnubroto; Ardianti, D. K.; Susanto, M. N.; Yusuf, M.; Demasta, E. K.

    2018-02-01

    It has been understood that peroxidase enzyme including peroxidase serves as catalyzer to enzymatic reaction among hydrogen peroxide and halides, therefore this research was done for generating hypoiodous acid (HIO) from peroxidase system using peroxidase enzyme. Hydrogen peroxide, potassium iodide, and peroxidase enzyme were used to produce HIO. Determination the amount of formed HIO was done using 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) or ABTS as substrate through the colorimetric measurement of hydrogen peroxide residue during reaction process using at 412 nm. The result indicated that residual hydrogen peroxide showed the minimum concentration after 60 minutes reaction time. Because the reaction started at the beginning time of mixing, hydrogen peroxide was unable to be eliminated totally to produce HIO. The reaction of peroxidase system was able to determine the beginning of mixing process but the reaction process could not eliminate the initial concentration of hydrogen peroxide indicating the maximum amount of production of HIO could be determined. In conclusion, the less of H2O2, higher HIO obtained and peroxidase enzymes can accelerate the formation of HIO.

  20. Determination of glutamine and glutamic acid in mammalian cell cultures using tetrathiafulvalene modified enzyme electrodes.

    PubMed

    Mulchandani, A; Bassi, A S

    1996-01-01

    Tetrathiafulvalene (TTF) mediated amperometric enzyme electrodes have been developed for the monitoring of L-glutamine and L-glutamic acid in growing mammalian cell cultures. The detection of glutamine was accomplished by a coupled enzyme system comprised of glutaminase plus glutamate oxidase, while the detection of glutamic acid was carried out by a single enzyme, glutamate oxidase. The appropriate enzyme(s) were immoblized on the Triton-X treated surface of tetrathiafulvalene modified carbon paste electrodes by adsorption, in conjunction with entrapment by an electrochemically deposited copolymer film of 1,3-phenylenediamine and resorcinol. Operating conditions for the glutamine enzyme electrode were optimized with respect to the amount of enzymes immoblized, pH, temperature and mobile phase flow rate for operation in a flow injection (FIA) system. When applied to glutamine and glutamic acid measurements in mammalian cell culture in FIA, the results obtained with enzyme electrodes were in excellent agreement with those determined by enzymatic analysis.

  1. Oxidation of d-Amino Acids by a Particulate Enzyme from Pseudomonas aeruginosa

    PubMed Central

    Marshall, Vincent P.; Sokatch, John R.

    1968-01-01

    A particulate d-amino acid dehydrogenase has been partially purified from cell free extracts of Pseudomonas aeruginosa grown on dl-valine as the source of carbon and energy. A standard assay was developed which utilized 2,6-dichlorophenol-indophenol as the electron acceptor. The pH optimum for enzyme activity ranged from 6.0 to 8.0, depending on the amino acid assayed. The enzyme was most active with monoamino-monocarboxylic amino acids and histidine. The Michaelis constant for d-phenylalanine was found to be 1.3 × 10-3m d-phenylalanine. Constants could not be calculated for the other amino acids oxidized because anomalous plots of V as a function of V/S were obtained. Spectra of enzyme preparations reduced with d-valine or sodium hydrosulfite exhibited adsorption bands typical of the α, β, and γ bands of cytochromes as well as bleaching in the flavin region of the spectrum. When dl-valine was added to a medium with glycerol as the energy source, d-amino acid dehydrogenase was detected after the addition of valine and was produced at a rate directly proportional to the synthesis of total protein. The enzyme was formed when d-valine, l-valine, or dl-alanine was the source of carbon and energy, but not when glucose, glycerol, or succinate was the energy source. PMID:4384679

  2. Genetic investigation of tricarboxylic acid metabolism during the Plasmodium falciparum life cycle.

    PubMed

    Ke, Hangjun; Lewis, Ian A; Morrisey, Joanne M; McLean, Kyle J; Ganesan, Suresh M; Painter, Heather J; Mather, Michael W; Jacobs-Lorena, Marcelo; Llinás, Manuel; Vaidya, Akhil B

    2015-04-07

    New antimalarial drugs are urgently needed to control drug-resistant forms of the malaria parasite Plasmodium falciparum. Mitochondrial electron transport is the target of both existing and new antimalarials. Herein, we describe 11 genetic knockout (KO) lines that delete six of the eight mitochondrial tricarboxylic acid (TCA) cycle enzymes. Although all TCA KOs grew normally in asexual blood stages, these metabolic deficiencies halted life-cycle progression in later stages. Specifically, aconitase KO parasites arrested as late gametocytes, whereas α-ketoglutarate-dehydrogenase-deficient parasites failed to develop oocysts in the mosquitoes. Mass spectrometry analysis of (13)C-isotope-labeled TCA mutant parasites showed that P. falciparum has significant flexibility in TCA metabolism. This flexibility manifested itself through changes in pathway fluxes and through altered exchange of substrates between cytosolic and mitochondrial pools. Our findings suggest that mitochondrial metabolic plasticity is essential for parasite development. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  3. The Viability of a Nonenzymatic Reductive Citric Acid Cycle Kinetics and Thermochemistry

    NASA Astrophysics Data System (ADS)

    Ross, David S.

    2007-02-01

    The likelihood of a functioning nonenzymatic reductive citric acid cycle, recently proposed as the precursor to biosynthesis on early Earth, is examined on the basis of the kinetics and thermochemistry of the acetate → pyruvate → oxaloacetate → malate sequence. Using data derived from studies of the Pd-catalyzed phosphinate reduction of carbonyl functions it is shown that the rate of conversion of pyruvate to malate with that system would have been much too slow to have played a role in the early chemistry of life, while naturally occurring reduction systems such as the fayalite magnetite quartz and pyrrhotite pyrite magnetite mineral assemblages would have provided even slower conversions. It is also shown that the production of pyruvate from acetate is too highly endoergic to be driven by a naturally occurring energy source such as pyrophosphate. It is thus highly doubtful that the cycle can operate at suitable rates without enzymes, and most unlikely that it could have participated in the chemistry leading to life.

  4. Acetaminophen Toxicity and 5-Oxoproline (Pyroglutamic Acid): A Tale of Two Cycles, One an ATP-Depleting Futile Cycle and the Other a Useful Cycle

    PubMed Central

    2014-01-01

    Summary The acquired form of 5-oxoproline (pyroglutamic acid) metabolic acidosis was first described in 1989 and its relationship to chronic acetaminophen ingestion was proposed the next year. Since then, this cause of chronic anion gap metabolic acidosis has been increasingly recognized. Many cases go unrecognized because an assay for 5-oxoproline is not widely available. Most cases occur in malnourished, chronically ill women with a history of chronic acetaminophen ingestion. Acetaminophen levels are very rarely in the toxic range; rather, they are usually therapeutic or low. The disorder generally resolves with cessation of acetaminophen and administration of intravenous fluids. Methionine or N-acetyl cysteine may accelerate resolution and methionine is protective in a rodent model. The disorder has been attributed to glutathione depletion and activation of a key enzyme in the γ-glutamyl cycle. However, the specific metabolic derangements that cause the 5-oxoproline accumulation remain unclear. An ATP-depleting futile 5-oxoproline cycle can explain the accumulation of 5-oxoproline after chronic acetaminophen ingestion. This cycle is activated by the depletion of both glutathione and cysteine. This explanation contributes to our understanding of acetaminophen-induced 5-oxoproline metabolic acidosis and the beneficial role of N-acetyl cysteine therapy. The ATP-depleting futile 5-oxoproline cycle may also play a role in the energy depletions that occur in other acetaminophen-related toxic syndromes. PMID:24235282

  5. Acetaminophen toxicity and 5-oxoproline (pyroglutamic acid): a tale of two cycles, one an ATP-depleting futile cycle and the other a useful cycle.

    PubMed

    Emmett, Michael

    2014-01-01

    The acquired form of 5-oxoproline (pyroglutamic acid) metabolic acidosis was first described in 1989 and its relationship to chronic acetaminophen ingestion was proposed the next year. Since then, this cause of chronic anion gap metabolic acidosis has been increasingly recognized. Many cases go unrecognized because an assay for 5-oxoproline is not widely available. Most cases occur in malnourished, chronically ill women with a history of chronic acetaminophen ingestion. Acetaminophen levels are very rarely in the toxic range; rather, they are usually therapeutic or low. The disorder generally resolves with cessation of acetaminophen and administration of intravenous fluids. Methionine or N-acetyl cysteine may accelerate resolution and methionine is protective in a rodent model. The disorder has been attributed to glutathione depletion and activation of a key enzyme in the γ-glutamyl cycle. However, the specific metabolic derangements that cause the 5-oxoproline accumulation remain unclear. An ATP-depleting futile 5-oxoproline cycle can explain the accumulation of 5-oxoproline after chronic acetaminophen ingestion. This cycle is activated by the depletion of both glutathione and cysteine. This explanation contributes to our understanding of acetaminophen-induced 5-oxoproline metabolic acidosis and the beneficial role of N-acetyl cysteine therapy. The ATP-depleting futile 5-oxoproline cycle may also play a role in the energy depletions that occur in other acetaminophen-related toxic syndromes.

  6. Affinity labelling enzymes with esters of aromatic sulfonic acids

    DOEpatents

    Wong, Show-Chu; Shaw, Elliott

    1977-01-01

    Novel esters of aromatic sulfonic acids are disclosed. The specific esters are nitrophenyl p- and m-amidinophenylmethanesulfonate. Also disclosed is a method for specific inactivation of the enzyme, thrombin, employing nitrophenyl p-amidinophenylmethanesulfonate.

  7. Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism.

    PubMed

    Marelja, Zvonimir; Leimkühler, Silke; Missirlis, Fanis

    2018-01-01

    may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle.

  8. Iron Sulfur and Molybdenum Cofactor Enzymes Regulate the Drosophila Life Cycle by Controlling Cell Metabolism

    PubMed Central

    Marelja, Zvonimir; Leimkühler, Silke; Missirlis, Fanis

    2018-01-01

    may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle. PMID:29491838

  9. Characterization of Carboxylic Acid Reductases as Enzymes in the Toolbox for Synthetic Chemistry.

    PubMed

    Finnigan, William; Thomas, Adam; Cromar, Holly; Gough, Ben; Snajdrova, Radka; Adams, Joseph P; Littlechild, Jennifer A; Harmer, Nicholas J

    2017-03-20

    Carboxylic acid reductase enzymes (CARs) meet the demand in synthetic chemistry for a green and regiospecific route to aldehydes from their respective carboxylic acids. However, relatively few of these enzymes have been characterized. A sequence alignment with members of the ANL (Acyl-CoA synthetase/ NRPS adenylation domain/Luciferase) superfamily of enzymes shed light on CAR functional dynamics. Four unstudied enzymes were selected by using a phylogenetic analysis of known and hypothetical CARs, and for the first time, a thorough biochemical characterization was performed. Kinetic analysis of these enzymes with various substrates shows that they have a broad but similar substrate specificity. Electron-rich acids are favored, which suggests that the first step in the proposed reaction mechanism, attack by the carboxylate on the α-phosphate of adenosine triphosphate (ATP), is the step that determines the substrate specificity and reaction kinetics. The effects of pH and temperature provide a clear operational window for the use of these CARs, whereas an investigation of product inhibition by NADP + , adenosine monophosphate, and pyrophosphate indicates that the binding of substrates at the adenylation domain is ordered with ATP binding first. This study consolidates CARs as important and exciting enzymes in the toolbox for sustainable chemistry and provides specifications for their use as a biocatalyst.

  10. The krebs cycle enzyme α-ketoglutarate decarboxylase is an essential glycosomal protein in bloodstream African trypanosomes.

    PubMed

    Sykes, Steven; Szempruch, Anthony; Hajduk, Stephen

    2015-03-01

    α-Ketoglutarate decarboxylase (α-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei. The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, α-KDE1 was expressed in BF T. brucei and RNA interference knockdown of α-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagellar and plasma membranes into the pocket. BF T. brucei expressing an epitope-tagged copy of α-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of α-KDE1 fused to green fluorescent protein to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of α-KDE1 contain overlapping mitochondrion- and peroxisome-targeting sequences and are sufficient to direct localization to the glycosome in BF T. brucei. These results suggest that α-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Immunolocalization of steroidogenic enzymes in the vaginal mucous of Galea spixii during the estrous cycle.

    PubMed

    Dos Santos, Amilton Cesar; Conley, Alan James; de Oliveira, Moacir Franco; Oliveira, Gleidson Benevides; Viana, Diego Carvalho; Assis Neto, Antônio Chaves de

    2017-04-24

    The synthesis of sex steroids is controlled by several enzymes such as17α-hydroxylase cytochrome P450 (P450c17) catalyzing androgen synthesis and aromatase cytochrome P450 (P450arom) catalyzing estrogen synthesis, both of which must complex with the redox partner NADPH-cytochrome P450 oxidoreductase (CPR) for activity. Previous studies have identified expression of steroidogenic enzymes in vaginal tissue, suggesting local sex steroid synthesis. The current studies investigate P450c17, P450aromatase and CPR expression in vaginal mucosa of Galea spixii (Spix cavy) by immuno-histochemical and western immunoblot analyses. Stages of estrous cyclicity were monitored by vaginal exfoliative cytology. After euthanasia, vaginal tissues were retrieved, fixed and frozen at diestrus, proestrus, estrus and metestrus. The ovaries and testis were used as positive control tissues for immunohistochemistry. Data from cytological study allowed identification of different estrous cycle phases. Immunohistochemical analysis showed different sites of expression of steroidogenic enzymes along with tissue response throughout different phases of the estrous cycle. However, further studies are needed to characterize the derived hormones synthesized by, and the enzymes activities associated with, vaginal tissues. Current results not only support the expression of enzymes involved in sex steroid synthesis in the wall of the vagina, they also indicate that expression changes with the stage of the cycle, both the levels and types of cells exhibiting expression. Thus, changes in proliferation of vaginal epithelial cells and the differentiation of the mucosa may be influenced by local steroid synthesis as well as circulating androgens and estrogens.

  12. [Modification of L-asparaginase with colominic acid and the new characteristics of the modified enzyme].

    PubMed

    Wang, Y D; Guo, L; Qian, S J; Meng, G Z; Zhang, S Z

    2000-07-01

    The colominic acid was covalently coupled to L-asparaginase molecule by reductive amination. Depending on the molar ratios of colominic acid-asparaginase (30:1, 50:1 and 100:1), a modified enzyme molecule contained 4.7, 7.2 and 12 colominic acid molecule, they retained 58%, 56% and 33.2% of the initial asparaginase activity, respectively. In comparison with the native enzyme, modified enzyme had lower immunogenicity and antigenicity, longer half-life time (in vitro), more resistance ability to trypsin proteolysis, and similar Km value for L-asparagine.

  13. α-Lactalbumin-oleic acid complex kills tumor cells by inducing excess energy metabolism but inhibiting mRNA expression of the related enzymes.

    PubMed

    Fang, B; Zhang, M; Ge, K S; Xing, H Z; Ren, F Z

    2018-06-01

    Previous studies have demonstrated that the anti-tumor α-lactalbumin-oleic acid complex (α-LA-OA) may target the glycolysis of tumor cells. However, few data are available regarding the effects of α-LA-OA on energy metabolism. In this study, we measured glycolysis and mitochondrial functions in HeLa cells in response to α-LA-OA using the XF flux analyzer (Seahorse Bioscience, North Billerica, MA). The gene expression of enzymes involved in glycolysis, tricarboxylic acid cycle, electron transfer chain, and ATP synthesis were also evaluated. Our results show that α-LA-OA significantly enhanced the basal glycolysis and glycolytic capacity. Mitochondrial oxidative phosphorylation, including the basal respiration, maximal respiration, spare respiratory capacity and ATP production were also improved in response to α-LA-OA. The enhanced mitochondrial functions maybe partly due to the increased capacity of utilizing fatty acids and glutamine as the substrate. However, the gene expressions of pyruvate kinase M2, lactate dehydrogenase A, aconitate hydratase, and isocitrate dehydrogenase 1 were inhibited, suggesting an insufficient ability for the glycolysis process and the tricarboxylic acid cycle. The increased expression of acetyl-coenzyme A acyltransferase 2, a central enzyme involved in the β-oxidation of fatty acids, would enhance the unbalance due to the decreased expression of electron transfer flavoprotein β subunit, which acts as the electron acceptor. These results indicated that α-LA-OA may induce oxidative stress due to conditions in which the ATP production is exceeding the energy demand. Our results may help clarify the mechanism of apoptosis induced by reactive oxygen species and mitochondrial destruction. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  14. Production of L-lactic Acid from Biomass Wastes Using Scallop Crude Enzymes and Novel Lactic Acid Bacterium

    NASA Astrophysics Data System (ADS)

    Yanagisawa, Mitsunori; Nakamura, Kanami; Nakasaki, Kiyohiko

    In the present study, biomass waste raw materials including paper mill sludge, bamboo, sea lettuce, and shochu residue (from a distiller) and crude enzymes derived from inedible and discarded scallop parts were used to produce L-lactic acid for the raw material of biodegradable plastic poly-lactic acid. The activities of cellulase and amylase in the crude enzymes were 22 and 170units/L, respectively, and L-lactic acid was produced from every of the above mentioned biomass wastes, by the method of liquid-state simultaneous saccharification and fermentation (SSF) . The L-lactic acid concentrations produced from sea lettuce and shochu residue, which contain high concentration of starch were 3.6 and 9.3g/L, respectively, and corresponded to greater than 25% of the conversion of glucans contained in these biomass wastes. Furthermore, using the solid state SSF method, concentrations as high as 13g/L of L-lactic acid were obtained from sea lettuce and 26g/L were obtained from shochu residue.

  15. Engineering acidic Streptomyces rubiginosus D-xylose isomerase by rational enzyme design.

    PubMed

    Waltman, Mary Jo; Yang, Zamin Koo; Langan, Paul; Graham, David E; Kovalevsky, Andrey

    2014-02-01

    To maximize bioethanol production from lignocellulosic biomass, all sugars must be utilized. Yeast fermentation can be improved by introducing the d-xylose isomerase enzyme to convert the pentose sugar d-xylose, which cannot be fermented by Saccharomyces cerevisiae, into the fermentable ketose d-xylulose. The low activity of d-xylose isomerase, especially at the low pH required for optimal fermentation, limits its use. A rational enzyme engineering approach was undertaken, and seven amino acid positions were replaced to improve the activity of Streptomyces rubiginosus d-xylose isomerase towards its physiological substrate at pH values below 6. The active-site design was guided by mechanistic insights and the knowledge of amino acid protonation states at low pH obtained from previous joint X-ray/neutron crystallographic experiments. Tagging the enzyme with 6 or 12 histidine residues at the N-terminus resulted in a significant increase in the active-site affinity towards substrate at pH 5.8. Substituting an asparagine at position 215, which hydrogen bonded to the metal-bound Glu181 and Asp245, with an aspartate gave a variant with almost an order of magnitude lower KM than measured for the native enzyme, with a 4-fold increase in activity. Other studied variants showed similar (Asp57Asn, Glu186Gln/Asn215Asp), lower (Asp57His, Asn247Asp, Lys289His, Lys289Glu) or no (Gln256Asp, Asp287Asn, ΔAsp287) activity in acidic conditions relative to the native enzyme.

  16. Metabolomics and transcriptomics profiles reveal the dysregulation of the tricarboxylic acid cycle and related mechanisms in prostate cancer.

    PubMed

    Shao, Yaping; Ye, Guozhu; Ren, Shancheng; Piao, Hai-Long; Zhao, Xinjie; Lu, Xin; Wang, Fubo; Ma, Wang; Li, Jia; Yin, Peiyuan; Xia, Tian; Xu, Chuanliang; Yu, Jane J; Sun, Yinghao; Xu, Guowang

    2018-07-15

    Genetic alterations drive metabolic reprograming to meet increased biosynthetic precursor and energy demands for cancer cell proliferation and survival in unfavorable environments. A systematic study of gene-metabolite regulatory networks and metabolic dysregulation should reveal the molecular mechanisms underlying prostate cancer (PCa) pathogenesis. Herein, we performed gas chromatography-mass spectrometry (GC-MS)-based metabolomics and RNA-seq analyses in prostate tumors and matched adjacent normal tissues (ANTs) to elucidate the molecular alterations and potential underlying regulatory mechanisms in PCa. Significant accumulation of metabolic intermediates and enrichment of genes in the tricarboxylic acid (TCA) cycle were observed in tumor tissues, indicating TCA cycle hyperactivation in PCa tissues. In addition, the levels of fumarate and malate were highly correlated with the Gleason score, tumor stage and expression of genes encoding related enzymes and were significantly related to the expression of genes involved in branched chain amino acid degradation. Using an integrated omics approach, we further revealed the potential anaplerotic routes from pyruvate, glutamine catabolism and branched chain amino acid (BCAA) degradation contributing to replenishing metabolites for TCA cycle. Integrated omics techniques enable the performance of network-based analyses to gain a comprehensive and in-depth understanding of PCa pathophysiology and may facilitate the development of new and effective therapeutic strategies. © 2018 UICC.

  17. Enzymes involved in plastid-targeted phosphatidic acid synthesis are essential for Plasmodium yoelii liver-stage development.

    PubMed

    Lindner, Scott E; Sartain, Mark J; Hayes, Kiera; Harupa, Anke; Moritz, Robert L; Kappe, Stefan H I; Vaughan, Ashley M

    2014-02-01

    Malaria parasites scavenge nutrients from their host but also harbour enzymatic pathways for de novo macromolecule synthesis. One such pathway is apicoplast-targeted type II fatty acid synthesis, which is essential for late liver-stage development in rodent malaria. It is likely that fatty acids synthesized in the apicoplast are ultimately incorporated into membrane phospholipids necessary for exoerythrocytic merozoite formation. We hypothesized that these synthesized fatty acids are being utilized for apicoplast-targeted phosphatidic acid synthesis, the phospholipid precursor. Phosphatidic acid is typically synthesized in a three-step reaction utilizing three enzymes: glycerol 3-phosphate dehydrogenase, glycerol 3-phosphate acyltransferase and lysophosphatidic acid acyltransferase. The Plasmodium genome is predicted to harbour genes for both apicoplast- and cytosol/endoplasmic reticulum-targeted phosphatidic acid synthesis. Our research shows that apicoplast-targeted Plasmodium yoelii glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate acyltransferase are expressed only during liver-stage development and deletion of the encoding genes resulted in late liver-stage growth arrest and lack of merozoite differentiation. However, the predicted apicoplast-targeted lysophosphatidic acid acyltransferase gene was refractory to deletion and was expressed solely in the endoplasmic reticulum throughout the parasite life cycle. Our results suggest that P. yoelii has an incomplete apicoplast-targeted phosphatidic acid synthesis pathway that is essential for liver-stage maturation. © 2013 John Wiley & Sons Ltd.

  18. Production of 5-aminolevulinic acid by cell free multi-enzyme catalysis.

    PubMed

    Meng, Qinglong; Zhang, Yanfei; Ju, Xiaozhi; Ma, Chunling; Ma, Hongwu; Chen, Jiuzhou; Zheng, Ping; Sun, Jibin; Zhu, Jun; Ma, Yanhe; Zhao, Xueming; Chen, Tao

    2016-05-20

    5-Aminolevulinic acid (ALA) is the precursor for the biosynthesis of tetrapyrroles and has broad agricultural and medical applications. Currently ALA is mainly produced by chemical synthesis and microbial fermentation. Cell free multi-enzyme catalysis is a promising method for producing high value chemicals. Here we reported our work on developing a cell free process for ALA production using thermostable enzymes. Cheap substrates (succinate and glycine) were used for ALA synthesis by two enzymes: 5-aminolevulinic acid synthase (ALAS) from Laceyella sacchari (LS-ALAS) and succinyl-CoA synthase (Suc) from Escherichia coli. ATP was regenerated by polyphosphate kinase (Ppk) using polyphosphate as the substrate. Succinate was added into the reaction system in a fed-batch mode to avoid its inhibition effect on Suc. After reaction for 160min, ALA concentration was increased to 5.4mM. This is the first reported work on developing the cell free process for ALA production. Through further process and enzyme optimization the cell free process could be an effective and economic way for ALA production. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Two enzyme immunoassays to screen for 2,4-dichlorophenoxyacetic acid in water.

    PubMed

    Fleeker, J

    1987-01-01

    Two solid-phase enzyme immunoassays were developed to measure 2,4-dichlorophenoxyacetic acid (2,4-D), using 2 sets of structurally distinct immunogens and enzyme ligands. The 2,4-D analog, 2-methyl-4-chlorophenoxyacetic acid (MCPA), gave a similar response with both methods, whereas other phenoxy herbicides cross-reacted differently. In method A, the aromatic moiety of 2,4-D was distal from the carrier protein and labeled enzyme, whereas in method B, the acetic acid portion of the herbicide was distal. The use of both methods to screen for this herbicide in ground water and municipal and river water reduced the number of false-positive responses. Water sources having a low background response could be monitored with either method alone. When a concentration step, with disposable C18 extraction columns, was used, the limit of sensitivity was 5 micrograms/L. Method A was the more sensitive of the 2 methods with a limit of detection of 10 micrograms/L without the concentration step.

  20. Formation of conjugated delta8,delta10-double bonds by delta12-oleic-acid desaturase-related enzymes: biosynthetic origin of calendic acid.

    PubMed

    Cahoon, E B; Ripp, K G; Hall, S E; Kinney, A J

    2001-01-26

    Divergent forms of the plant Delta(12)-oleic-acid desaturase (FAD2) have previously been shown to catalyze the formation of acetylenic bonds, epoxy groups, and conjugated Delta(11),Delta(13)-double bonds by modification of an existing Delta(12)-double bond in C(18) fatty acids. Here, we report a class of FAD2-related enzymes that modifies a Delta(9)-double bond to produce the conjugated trans-Delta(8),trans-Delta(10)-double bonds found in calendic acid (18:3Delta(8trans,10trans,12cis)), the major component of the seed oil of Calendula officinalis. Using an expressed sequence tag approach, cDNAs for two closely related FAD2-like enzymes, designated CoFADX-1 and CoFADX-2, were identified from a C. officinalis developing seed cDNA library. The deduced amino acid sequences of these polypeptides share 40-50% identity with those of other FAD2 and FAD2-related enzymes. Expression of either CoFADX-1 or CoFADX-2 in somatic soybean embryos resulted in the production of calendic acid. In embryos expressing CoFADX-2, calendic acid accumulated to as high as 22% (w/w) of the total fatty acids. In addition, expression of CoFADX-1 and CoFADX-2 in Saccharomyces cerevisiae was accompanied by calendic acid accumulation when induced cells were supplied exogenous linoleic acid (18:2Delta(9cis,12cis)). These results are thus consistent with a route of calendic acid synthesis involving modification of the Delta(9)-double bond of linoleic acid. Regiospecificity for Delta(9)-double bonds is unprecedented among FAD2-related enzymes and further expands the functional diversity found in this family of enzymes.

  1. Blood metabolomics analysis identifies abnormalities in the citric acid cycle, urea cycle, and amino acid metabolism in bipolar disorder.

    PubMed

    Yoshimi, Noriko; Futamura, Takashi; Kakumoto, Keiji; Salehi, Alireza M; Sellgren, Carl M; Holmén-Larsson, Jessica; Jakobsson, Joel; Pålsson, Erik; Landén, Mikael; Hashimoto, Kenji

    2016-06-01

    Bipolar disorder (BD) is a severe and debilitating psychiatric disorder. However, the precise biological basis remains unknown, hampering the search for novel biomarkers. We performed a metabolomics analysis to discover novel peripheral biomarkers for BD. We quantified serum levels of 116 metabolites in mood-stabilized male BD patients (n = 54) and age-matched male healthy controls (n = 39). After multivariate logistic regression, serum levels of pyruvate, N-acetylglutamic acid, α-ketoglutarate, and arginine were significantly higher in BD patients than in healthy controls. Conversely, serum levels of β-alanine, and serine were significantly lower in BD patients than in healthy controls. Chronic (4-weeks) administration of lithium or valproic acid to adult male rats did not alter serum levels of pyruvate, N-acetylglutamic acid, β-alanine, serine, or arginine, but lithium administration significantly increased serum levels of α-ketoglutarate. The metabolomics analysis demonstrated altered serum levels of pyruvate, N-acetylglutamic acid, β-alanine, serine, and arginine in BD patients. The present findings suggest that abnormalities in the citric acid cycle, urea cycle, and amino acid metabolism play a role in the pathogenesis of BD.

  2. Cell cycle nucleic acids, polypeptides and uses thereof

    DOEpatents

    Gordon-Kamm, William J [Urbandale, IA; Lowe, Keith S [Johnston, IA; Larkins, Brian A [Tucson, AZ; Dilkes, Brian R [Tucson, AZ; Sun, Yuejin [Westfield, IN

    2007-08-14

    The invention provides isolated nucleic acids and their encoded proteins that are involved in cell cycle regulation. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The present invention provides methods and compositions relating to altering cell cycle protein content, cell cycle progression, cell number and/or composition of plants.

  3. Glutamic acid is an active site residue of angiotensin I-converting enzyme. Use of the Lossen rearrangement for identification of dicarboxylic acid residues.

    PubMed

    Harris, R B; Wilson, I B

    1983-01-25

    A set of chemical reactions was used to show that one glutamic acid residue at the active site of bovine lung angiotensin I-converting enzyme is esterified with the alkylating agent p-[N,N-bis(chloroethyl)amino] phenylbutyryl-L-Pro (chlorambucyl-L-Pro), an affinity label for this enzyme (Harris, R. B., and Wilson, I. B. (1982) J. Biol. Chem. 257, 811-815). The same procedure was used to confirm that a glutamic acid residue of carboxypeptidase A alpha is esterified by reaction with bromoacetyl-N-methyl-L-phenylalanine (Haas, G. M., and Neurath, H. (1971) Biochemistry 10, 3535-3546). In the procedure described in this paper, the esterified residue at the active site is converted to the hydroxamic acid by reaction with hydroxylamine and the hydroxamic acid is subject to the Lossen rearrangement. If a glutamic acid residue was esterified, 1 eq of 2,4-diaminobutyric acid will be formed. Aspartyl esters will give 2,3-diaminopropionic acid. The diamino acids can be quantitatively measured using the short column of an amino acid analyzer if the amount of lysine and histidine is largely decreased by modification with suitable side chain protecting groups. With carboxypeptidase A, the reactions were done on the whole undigested enzyme. With the converting enzyme, we first cleaved the esterified enzyme with cyanogen bromide. Twenty-nine cleavage peptides were separated on high performance liquid chromatography and one of these contained all of the bound radioactive inhibitor. This active site peptide was then subjected to the derivatization and Lossen procedures, and 1 eq of 2,4-diaminobutyric acid was obtained.

  4. Enzymes of Glyoxylate in Conifers 12

    PubMed Central

    Firenzuoli, A. M.; Vanni, P.; Mastronuzzi, E.; Zanobini, A.; Baccari, V.

    1968-01-01

    The high level of lipids in seeds of some species of conifers suggested that the glyoxylate cycle might have a role in conifer seed metabolism. Six species (Pinus pinea, Pinus pinaster, Pinus canariensis, Pinus strobus, Abies alba, and Cupressus sempervirens) were investigated for their lipid content and malate synthase and isocitrate lyase level. The fatty acid composition of the triglyceride fraction was also investigated. The correlation between lipid content of germinating seed with the presence of the cycle was confirmed. The enzymes of the glyoxylate cycle were not detected in Cupressus sempervirens where the lipid content is very low. PMID:16656892

  5. Serum uric acid in relation to endogenous reproductive hormones during the menstrual cycle: findings from the BioCycle study

    PubMed Central

    Mumford, Sunni L.; Dasharathy, Sonya S.; Pollack, Anna Z.; Perkins, Neil J.; Mattison, Donald R.; Cole, Stephen R.; Wactawski-Wende, Jean; Schisterman, Enrique F.

    2013-01-01

    STUDY QUESTION Do uric acid levels across the menstrual cycle show associations with endogenous estradiol (E2) and reproductive hormone concentrations in regularly menstruating women? SUMMARY ANSWER Mean uric acid concentrations were highest during the follicular phase, and were inversely associated with E2 and progesterone, and positively associated with FSH. WHAT IS KNOWN ALREADY E2 may decrease serum levels of uric acid in post-menopausal women; however, the interplay between endogenous reproductive hormones and uric acid levels among regularly menstruating women has not been elucidated. STUDY DESIGN, SIZE, DURATION The BioCycle study was a prospective cohort study conducted at the University at Buffalo research centre from 2005 to 2007, which followed healthy women for one (n = 9) or 2 (n = 250) menstrual cycle(s). PARTICIPANTS/MATERIALS, SETTING, METHODS Participants were healthy women aged 18–44 years. Hormones and uric acid were measured in serum eight times each cycle for up to two cycles. Marginal structural models with inverse probability of exposure weights were used to evaluate the associations between endogenous hormones and uric acid concentrations. MAIN RESULTS AND THE ROLE OF CHANCE Uric acid levels were observed to vary across the menstrual cycle, with the lowest levels observed during the luteal phase. Every log-unit increase in E2 was associated with a decrease in uric acid of 1.1% (β = −0.011; 95% confidence interval (CI): −0.019, −0.004; persistent-effects model), and for every log-unit increase in progesterone, uric acid decreased by ∼0.8% (β = −0.008; 95% CI: −0.012, −0.004; persistent-effects model). FSH was positively associated with uric acid concentrations, such that each log-unit increase was associated with a 1.6% increase in uric acid (β = 0.016; 95% CI: 0.005, 0.026; persistent-effects model). Progesterone and FSH were also associated with uric acid levels in acute-effects models. Of 509 cycles, 42 were anovulatory

  6. The viability of a nonenzymatic reductive citric acid cycle - Kinetics and thermochemistry

    USGS Publications Warehouse

    Ross, D.S.

    2007-01-01

    The likelihood of a functioning nonenzymatic reductive citric acid cycle, recently proposed as the precursor to biosynthesis on early Earth, is examined on the basis of the kinetics and thermochemistry of the acetate ??? pyruvate ??? oxaloacetate ??? malate sequence. Using data derived from studies of the Pd-catalyzed phosphinate reduction of carbonyl functions it is shown that the rate of conversion of pyruvate to malate with that system would have been much too slow to have played a role in the early chemistry of life, while naturally occurring reduction systems such as the fayalite-magnetite-quartz and pyrrhotite-pyrite-magnetite mineral assemblages would have provided even slower conversions. It is also shown that the production of pyruvate from acetate is too highly endoergic to be driven by a naturally occurring energy source such as pyrophosphate. It is thus highly doubtful that the cycle can operate at suitable rates without enzymes, and most unlikely that it could have participated in the chemistry leading to life. ?? 2006 Springer Science + Business Media B.V.

  7. Origin of the Reductive Tricarboxylic Acid (rTCA) Cycle-Type CO2 Fixation: A Perspective

    PubMed Central

    Fujishima, Kosuke

    2017-01-01

    The reductive tricarboxylic acid (rTCA) cycle is among the most plausible candidates for the first autotrophic metabolism in the earliest life. Extant enzymes fixing CO2 in this cycle contain cofactors at the catalytic centers, but it is unlikely that the protein/cofactor system emerged at once in a prebiotic process. Here, we discuss the feasibility of non-enzymatic cofactor-assisted drive of the rTCA reactions in the primitive Earth environments, particularly focusing on the acetyl-CoA conversion to pyruvate. Based on the energetic and mechanistic aspects of this reaction, we propose that the deep-sea hydrothermal vent environments with active electricity generation in the presence of various sulfide catalysts are a promising setting for it to progress. Our view supports the theory of an autotrophic origin of life from primordial carbon assimilation within a sulfide-rich hydrothermal vent.

  8. Evolutionary History of the Enzymes Involved in the Calvin-Benson Cycle in Euglenids.

    PubMed

    Markunas, Chelsea M; Triemer, Richard E

    2016-05-01

    Euglenids are an ancient lineage that may have existed as early as 2 billion years ago. A mere 65 years ago, Melvin Calvin and Andrew A. Benson performed experiments on Euglena gracilis and elucidated the series of reactions by which carbon was fixed and reduced during photosynthesis. However, the evolutionary history of this pathway (Calvin-Benson cycle) in euglenids was more complex than Calvin and Benson could have imagined. The chloroplast present today in euglenophytes arose from a secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga. A long period of evolutionary time existed before this secondary endosymbiotic event took place, which allowed for other endosymbiotic events or gene transfers to occur prior to the establishment of the green chloroplast. This research revealed the evolutionary history of the major enzymes of the Calvin-Benson cycle throughout the euglenid lineage and showed that the majority of genes for Calvin-Benson cycle enzymes shared an ancestry with red algae and/or chromophytes suggesting they may have been transferred to the nucleus prior to the acquisition of the green chloroplast. © 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists.

  9. Linked cycles of oxidative decarboxylation of glyoxylate as protometabolic analogs of the citric acid cycle.

    PubMed

    Springsteen, Greg; Yerabolu, Jayasudhan Reddy; Nelson, Julia; Rhea, Chandler Joel; Krishnamurthy, Ramanarayanan

    2018-01-08

    The development of metabolic approaches towards understanding the origins of life, which have focused mainly on the citric acid (TCA) cycle, have languished-primarily due to a lack of experimentally demonstrable and sustainable cycle(s) of reactions. We show here the existence of a protometabolic analog of the TCA involving two linked cycles, which convert glyoxylate into CO 2 and produce aspartic acid in the presence of ammonia. The reactions proceed from either pyruvate, oxaloacetate or malonate in the presence of glyoxylate as the carbon source and hydrogen peroxide as the oxidant under neutral aqueous conditions and at mild temperatures. The reaction pathway demonstrates turnover under controlled conditions. These results indicate that simpler versions of metabolic cycles could have emerged under potential prebiotic conditions, laying the foundation for the appearance of more sophisticated metabolic pathways once control by (polymeric) catalysts became available.

  10. Metabolic Interaction between Urea Cycle and Citric Acid Cycle Shunt: A Guided Approach

    ERIC Educational Resources Information Center

    Pesi, Rossana; Balestri, Francesco; Ipata, Piero L.

    2018-01-01

    This article is a guided pedagogical approach, devoted to postgraduate students specializing in biochemistry, aimed at presenting all single reactions and overall equations leading to the metabolic interaction between ureagenesis and citric acid cycle to be incorporated into a two-three lecture series about the interaction of urea cycle with other…

  11. Acidic amino acids: A new-type of enzyme mimics with application to biosensing and evaluating of antioxidant behaviour.

    PubMed

    Shi, Ying; Liu, Li; Yu, Yuanyuan; Long, Yijuan; Zheng, Huzhi

    2018-08-05

    Nanomaterials have triggered tremendous interest to mimick peroxidase but rarely attention has been paid to small molecules. Herein we first found that acidic amino acids including l-glutamic acid (L-Glu) and l-aspartic acid (L-Asp) exhibited an intrinsic peroxidase-like activity, endowing acidic amino acids with the capability of catalysing the oxidation of the peroxidase substrates 3,3',5,5'-tetramethylbenzidine (TMB) to produce color reaction in the presence of H 2 O 2 . Reaction mechanism was further investigated by means of electron spin resonance spectroscopy (ESR), enzyme kinetics assay and quantum theoretical calculations, to verify and provide a good deal of insight into the catalytic process. Based on the above discovery, a colorimetric platform was successfully developed for sensing glucose in the range of 0.10 μM to 10 μM with a detection limit of 40 nM, as well as evaluating the inhibitory effect of antioxidants on reactive oxygen species. This extraordinary finding not only extends the new biological function of acidic amino acids, but also opens new opportunities to deepen the knowledge of the new class of small molecule enzymes. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Golgi enzymes do not cycle through the endoplasmic reticulum during protein secretion or mitosis

    PubMed Central

    Villeneuve, Julien; Duran, Juan; Scarpa, Margherita; Bassaganyas, Laia; Van Galen, Josse; Malhotra, Vivek

    2017-01-01

    Golgi-specific sialyltransferase (ST) expressed as a chimera with the rapamycin-binding domain of mTOR, FRB, relocates to the endoplasmic reticulum (ER) in cells exposed to rapamycin that also express invariant chain (Ii)-FKBP in the ER. This result has been taken to indicate that Golgi-resident enzymes cycle to the ER constitutively. We show that ST-FRB is trapped in the ER even without Ii-FKBP upon rapamycin addition. This is because ER-Golgi–cycling FKBP proteins contain a C-terminal KDEL-like sequence, bind ST-FRB in the Golgi, and are transported together back to the ER by KDEL receptor–mediated retrograde transport. Moreover, depletion of KDEL receptor prevents trapping of ST-FRB in the ER by rapamycin. Thus ST-FRB cycles artificially by binding to FKBP domain–containing proteins. In addition, Golgi-specific O-linked glycosylation of a resident ER protein occurs only upon artificial fusion of Golgi membranes with ER. Together these findings support the consensus view that there is no appreciable mixing of Golgi-resident enzymes with ER under normal conditions. PMID:27807044

  13. Kinetic characteristics of polygalacturonase enzymes hydrolyzing galacturonic acid oligomers using isothermal titration calorimetry

    USDA-ARS?s Scientific Manuscript database

    Polygalacturonase enzymes hydrolyze the polygalacturonic acid chains found in pectin. Interest in polygalacturonase enzymes continues as they are useful in a number of industrial processes and conversely, detrimental, as they are involved in maceration of economically important crops. While a good...

  14. Key enzymes of the retinoid (visual) cycle in vertebrate retina

    PubMed Central

    Kiser, Philip D.; Golczak, Marcin; Maeda, Akiko; Palczewski, Krzysztof

    2011-01-01

    A major goal in vision research over the past few decades has been to understand the molecular details of retinoid processing within the retinoid (visual) cycle. This includes the consequences of side reactions that result from delayed all-trans-retinal clearance and condensation with phospholipids that characterize a variety of serious retinal diseases. Knowledge of the basic retinoid biochemistry involved in these diseases is essential for development of effective therapeutics. Photoisomerization of the 11-cis-retinal chromophore of rhodopsin triggers a complex set of metabolic transformations collectively termed phototransduction that ultimately lead to light perception. Continuity of vision depends on continuous conversion of all-trans-retinal back to the 11-cis-retinal isomer. This process takes place in a series of reactions known as the retinoid cycle, which occur in photoreceptor and RPE cells. All-trans-retinal, the initial substrate of this cycle, is a chemically reactive aldehyde that can form toxic conjugates with proteins and lipids. Therefore, much experimental effort has been devoted to elucidate molecular mechanisms of the retinoid cycle and all-trans-retinal-mediated retinal degeneration, resulting in delineation of many key steps involved in regenerating 11-cis-retinal. Three particularly important reactions are catalyzed by enzymes broadly classified as acyltransferases, short-chain dehydrogenases/reductases and carotenoid/retinoid isomerases/oxygenases. PMID:21447403

  15. In vitro enzymic hydrolysis of chlorogenic acids in coffee.

    PubMed

    da Encarnação, Joana Amarante; Farrell, Tracy L; Ryder, Alexandra; Kraut, Nicolai U; Williamson, Gary

    2015-02-01

    Coffee is rich in quinic acid esters of phenolic acids (chlorogenic acids) but also contains some free phenolic acids. A proportion of phenolic acids appear in the blood rapidly after coffee consumption due to absorption in the small intestine. We investigated in vitro whether this appearance could potentially be derived from free phenolic acids in instant coffee or from hydrolysis of chlorogenic acids by pancreatic or brush border enzymes. We quantified six free phenolic acids in instant coffees using HPLC-DAD-mass spectrometry. The highest was caffeic acid, but all were present at low levels compared to the chlorogenic acids. Roasting and decaffeination significantly reduced free phenolic acid content. We estimated, using pharmacokinetic modelling with previously published data, that the contribution of these compounds to small intestinal absorption is minimal. Hydrolysis of certain chlorogenic acids was observed with human-differentiated Caco-2 cell monolayers and with porcine pancreatin, which showed maximal rates on 3- and 5-O-caffeoylquinic acids, respectively. The amounts of certain free phenolic acids in coffee could only minimally account for small intestinal absorption based on modelling. The hydrolysis of caffeoylquinic, but not feruloylquinic acids, by enterocyte and pancreatic esterases is potentially a contributing mechanism to small intestinal absorption. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Aberrant Expression and Distribution of Enzymes of the Urea Cycle and Other Ammonia Metabolizing Pathways in Dogs with Congenital Portosystemic Shunts

    PubMed Central

    van Straten, Giora; van Steenbeek, Frank G.; Grinwis, Guy C. M.; Favier, Robert P.; Kummeling, Anne; van Gils, Ingrid H.; Fieten, Hille; Groot Koerkamp, Marian J. A.; Holstege, Frank C. P.; Rothuizen, Jan; Spee, Bart

    2014-01-01

    The detoxification of ammonia occurs mainly through conversion of ammonia to urea in the liver via the urea cycle and glutamine synthesis. Congenital portosystemic shunts (CPSS) in dogs cause hyperammonemia eventually leading to hepatic encephalopathy. In this study, the gene expression of urea cycle enzymes (carbamoylphosphate synthetase (CPS1), ornithine carbamoyltransferase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase (ARG1)), N-acetylglutamate synthase (NAGS), Glutamate dehydrogenase (GLUD1), and glutamate-ammonia ligase (GLUL) was evaluated in dogs with CPSS before and after surgical closure of the shunt. Additionally, immunohistochemistry was performed on urea cycle enzymes and GLUL on liver samples of healthy dogs and dogs with CPSS to investigate a possible zonal distribution of these enzymes within the liver lobule and to investigate possible differences in distribution in dogs with CPSS compared to healthy dogs. Furthermore, the effect of increasing ammonia concentrations on the expression of the urea cycle enzymes was investigated in primary hepatocytes in vitro. Gene-expression of CPS1, OTC, ASL, GLUD1 and NAGS was down regulated in dogs with CPSS and did not normalize after surgical closure of the shunt. In all dogs GLUL distribution was localized pericentrally. CPS1, OTC and ASS1 were localized periportally in healthy dogs, whereas in CPSS dogs, these enzymes lacked a clear zonal distribution. In primary hepatocytes higher ammonia concentrations induced mRNA levels of CPS1. We hypothesize that the reduction in expression of urea cycle enzymes, NAGS and GLUD1 as well as the alterations in zonal distribution in dogs with CPSS may be caused by a developmental arrest of these enzymes during the embryonic or early postnatal phase. PMID:24945279

  17. Aberrant expression and distribution of enzymes of the urea cycle and other ammonia metabolizing pathways in dogs with congenital portosystemic shunts.

    PubMed

    van Straten, Giora; van Steenbeek, Frank G; Grinwis, Guy C M; Favier, Robert P; Kummeling, Anne; van Gils, Ingrid H; Fieten, Hille; Groot Koerkamp, Marian J A; Holstege, Frank C P; Rothuizen, Jan; Spee, Bart

    2014-01-01

    The detoxification of ammonia occurs mainly through conversion of ammonia to urea in the liver via the urea cycle and glutamine synthesis. Congenital portosystemic shunts (CPSS) in dogs cause hyperammonemia eventually leading to hepatic encephalopathy. In this study, the gene expression of urea cycle enzymes (carbamoylphosphate synthetase (CPS1), ornithine carbamoyltransferase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase (ARG1)), N-acetylglutamate synthase (NAGS), Glutamate dehydrogenase (GLUD1), and glutamate-ammonia ligase (GLUL) was evaluated in dogs with CPSS before and after surgical closure of the shunt. Additionally, immunohistochemistry was performed on urea cycle enzymes and GLUL on liver samples of healthy dogs and dogs with CPSS to investigate a possible zonal distribution of these enzymes within the liver lobule and to investigate possible differences in distribution in dogs with CPSS compared to healthy dogs. Furthermore, the effect of increasing ammonia concentrations on the expression of the urea cycle enzymes was investigated in primary hepatocytes in vitro. Gene-expression of CPS1, OTC, ASL, GLUD1 and NAGS was down regulated in dogs with CPSS and did not normalize after surgical closure of the shunt. In all dogs GLUL distribution was localized pericentrally. CPS1, OTC and ASS1 were localized periportally in healthy dogs, whereas in CPSS dogs, these enzymes lacked a clear zonal distribution. In primary hepatocytes higher ammonia concentrations induced mRNA levels of CPS1. We hypothesize that the reduction in expression of urea cycle enzymes, NAGS and GLUD1 as well as the alterations in zonal distribution in dogs with CPSS may be caused by a developmental arrest of these enzymes during the embryonic or early postnatal phase.

  18. Metabolism of organic acids, nitrogen and amino acids in chlorotic leaves of 'Honeycrisp' apple (Malus domestica Borkh) with excessive accumulation of carbohydrates.

    PubMed

    Wang, Huicong; Ma, Fangfang; Cheng, Lailiang

    2010-07-01

    Metabolite profiles and activities of key enzymes in the metabolism of organic acids, nitrogen and amino acids were compared between chlorotic leaves and normal leaves of 'Honeycrisp' apple to understand how accumulation of non-structural carbohydrates affects the metabolism of organic acids, nitrogen and amino acids. Excessive accumulation of non-structural carbohydrates and much lower CO(2) assimilation were found in chlorotic leaves than in normal leaves, confirming feedback inhibition of photosynthesis in chlorotic leaves. Dark respiration and activities of several key enzymes in glycolysis and tricarboxylic acid (TCA) cycle, ATP-phosphofructokinase, pyruvate kinase, citrate synthase, aconitase and isocitrate dehydrogenase were significantly higher in chlorotic leaves than in normal leaves. However, concentrations of most organic acids including phosphoenolpyruvate (PEP), pyruvate, oxaloacetate, 2-oxoglutarate, malate and fumarate, and activities of key enzymes involved in the anapleurotic pathway including PEP carboxylase, NAD-malate dehydrogenase and NAD-malic enzyme were significantly lower in chlorotic leaves than in normal leaves. Concentrations of soluble proteins and most free amino acids were significantly lower in chlorotic leaves than in normal leaves. Activities of key enzymes in nitrogen assimilation and amino acid synthesis, including nitrate reductase, glutamine synthetase, ferredoxin and NADH-dependent glutamate synthase, and glutamate pyruvate transaminase were significantly lower in chlorotic leaves than in normal leaves. It was concluded that, in response to excessive accumulation of non-structural carbohydrates, glycolysis and TCA cycle were up-regulated to "consume" the excess carbon available, whereas the anapleurotic pathway, nitrogen assimilation and amino acid synthesis were down-regulated to reduce the overall rate of amino acid and protein synthesis.

  19. Increased resiliency and activity of microbial mediated carbon cycling enzymes in diversified bioenergy cropping systems

    NASA Astrophysics Data System (ADS)

    Upton, R.; Bach, E.; Hofmockel, K. S.

    2017-12-01

    Microbes are mediators of soil carbon (C) and are influenced in membership and activity by nitrogen (N) fertilization and inter-annual abiotic factors. Microbial communities and their extracellular enzyme activities (EEA) are important parameters that influence ecosystem C cycling properties and are often included in microbial explicit C cycling models. In an effort to generate model relevant, empirical findings, we investigated how both microbial community structure and C degrading enzyme activity are influenced by inter-annual variability and N inputs in bioenergy crops. Our study was performed at the Comparison of Biofuel Systems field-site from 2011 to 2014, in three bioenergy cropping systems, continuous corn (CC) and two restored prairies, both fertilized (FP) and unfertilized (P). We hypothesized microbial community structure would diverge during the prairie restoration, leading to changes in C cycling enzymes over time. Using a sequencing approach (16S and ITS) we determined the bacterial and fungal community structure response to the cropping system, fertilization, and inter-annual variability. Additionally, we used EEA of β-glucosidase, cellobiohydrolase, and β-xylosidase to determine inter-annual and ecosystem impacts on microbial activity. Our results show cropping system was a main effect for microbial community structure, with corn diverging from both prairies to be less diverse. Inter-annual changes showed that a drought occurring in 2012 significantly impacted microbial community structure in both the P and CC, decreasing microbial richness. However, FP increased in microbial richness, suggesting the application of N increased resiliency to drought. Similarly, the only year in which C cycling enzymes were impacted by ecosystem was 2012, with FP supporting higher potential enzymatic activity then CC and P. The highest EEA across all ecosystems occurred in 2014, suggesting the continued root biomass and litter build-up in this no till system

  20. Antagonistic Enzymes in a Biocatalytic pH Feedback System Program Autonomous DNA Hydrogel Life Cycles.

    PubMed

    Heinen, Laura; Heuser, Thomas; Steinschulte, Alexander; Walther, Andreas

    2017-08-09

    Enzymes regulate complex functions and active behavior in natural systems and have shown increasing prospect for developing self-regulating soft matter systems. Striving for advanced autonomous hydrogel materials with fully programmable, self-regulated life cycles, we combine two enzymes with an antagonistic pH-modulating effect in a feedback-controlled biocatalytic reaction network (BRN) and couple it to pH-responsive DNA hydrogels to realize hydrogel systems with distinct preprogrammable lag times and lifetimes in closed systems. The BRN enables precise and orthogonal internal temporal control of the "ON" and "OFF" switching times of the temporary gel state by modulation of programmable, nonlinear pH changes. The time scales are tunable by variation of the enzyme concentrations and additional buffer substances. The resulting material system operates in full autonomy after injection of the chemical fuels driving the BRN. The concept may open new applications inherent to DNA hydrogels, for instance, autonomous shape memory behavior for soft robotics. We further foresee general applicability to achieve autonomous life cycles in other pH switchable systems.

  1. Carboxylic acid reductase enzymes (CARs).

    PubMed

    Winkler, Margit

    2018-04-01

    Carboxylate reductases (CARs) are emerging as valuable catalysts for the selective one-step reduction of carboxylic acids to their corresponding aldehydes. The substrate scope of CARs is exceptionally broad and offers potential for their application in diverse synthetic processes. Two major fields of application are the preparation of aldehydes as end products for the flavor and fragrance sector and the integration of CARs in cascade reactions with aldehydes as the key intermediates. The latest applications of CARs are dominated by in vivo cascades and chemo-enzymatic reaction sequences. The challenge to fully exploit product selectivity is discussed. Recent developments in the characterization of CARs are summarized, with a focus on aspects related to the domain architecture and protein sequences of CAR enzymes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Production of Delta(1)-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris.

    PubMed

    Taura, Futoshi; Dono, Emi; Sirikantaramas, Supaart; Yoshimura, Kohji; Shoyama, Yukihiro; Morimoto, Satoshi

    2007-09-28

    Delta(1)-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of Delta(1)-tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted approximately 1.32nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a approximately 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure-function studies because it displayed much more activity than the previously reported native enzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.

  3. Influence of time, storage temperature and freeze/thaw cycles on the activity of digestive enzymes from gilthead sea bream (Sparus aurata).

    PubMed

    Solovyev, Mikhail; Gisbert, Enric

    2016-10-01

    In this study, we tested the effects of long-term storage (2 years) at -20 °C and short-term storage (several hours) in ice and freeze/thaw cycles on the activities of pancreatic, gastric and intestinal (brush border and cytosolic) digestive enzymes in a teleost fish species. The results revealed a significant lose in activity of pancreatic (trypsin, chymotrypsin, total alkaline proteases and α-amylase) and intestinal cytosolic (leucine-alanine peptidase) enzymes between 140 and 270 days of storage at -20 °C, whereas in contrast, the activity of all the assayed brush border enzymes remained constant during the first 2 years of storage at -20 °C. During short-term storage conditions, the most stable enzymes assayed were those of the enterocytes of the brush border, which did not show any change in activity after being held for 5 h in ice. Five freezing and thawing cycles did not affect the activity of the intestinal brush border enzymes and the cytosolic ones, whereas the activity of trypsin, α-amylase and bile-salt-activated lipase was significantly affected by the number of freezing and thawing cycles. No changes in pepsin activity were found in samples exposed to 1 and 2 freezing and thawing cycles.

  4. Engineering the intracellular metabolism of Escherichia coli to produce gamma-aminobutyric acid by co-localization of GABA shunt enzymes.

    PubMed

    Pham, Van Dung; Somasundaram, Sivachandiran; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

    2016-02-01

    To direct the carbon flux from Krebs cycle into the gamma-aminobutyric acid (GABA) shunt pathway for the production of GABA by protein scaffold introduction in Escherichia coli. Escherichia coli was engineered to produce GABA from glucose by the co-localization of enzymes succinate semialdehyde dehydrogenase (GadD), GABA aminotransferase (PuuE) and GABA transporter (GadC) by protein scaffold. 0.7 g GABA l(-1) was produced from 10 g glucose l(-1) while no GABA was produced in wild type E. coli. pH 6 and 30 °C were optimum for GABA production, and GABA concentration increased to 1.12 g GABA l(-1) when 20 g glucose l(-1) was used. When competing metabolic networks were inactivated, GABA increased by 24 % (0.87 g GABA l(-1)). The novel GABA production system was constructed by co-localization of GABA shunt enzymes.

  5. Closed cycle ion exchange method for regenerating acids, bases and salts

    DOEpatents

    Dreyfuss, Robert M.

    1976-01-01

    A method for conducting a chemical reaction in acidic, basic, or neutral solution as required and then regenerating the acid, base, or salt by means of ion exchange in a closed cycle reaction sequence which comprises contacting the spent acid, base, or salt with an ion exchanger, preferably a synthetic organic ion-exchange resin, so selected that the counter ions thereof are ions also produced as a by-product in the closed reaction cycle, and then regenerating the spent ion exchanger by contact with the by-product counter ions. The method is particularly applicable to closed cycle processes for the thermochemical production of hydrogen.

  6. Association of canalicular membrane enzymes with bile acid micelles and lipid aggregates in human and rat bile.

    PubMed

    Accatino, L; Pizarro, M; Solís, N; Koenig, C S

    1995-01-18

    This study was undertaken to gain insights into the characteristics of the polymolecular association between canalicular membrane enzymes, bile acids, cholesterol and phospholipids in bile and into the celular mechanisms whereby the enzymes are secreted into bile. With this purpose, we studied the distribution of bile acids, cholesterol, phospholipids, proteins and representative canalicular membrane enzymes (alkaline phosphatase, 5'-nucleotidase and gamma-glutamyl transpeptidase), which can be considered specific marker constituents, in bile fractions enriched in phospholipid-cholesterol lamellar structures (multilamellar and unilamellar vesicles) and bile acid-mixed micelles. These fractions were isolated by ultracentrifugation from human hepatic bile, normal rat bile and bile of rats treated with diosgenin, a steroid that induces a marked increase in biliary cholesterol secretion, and were characterized by density, lipid composition and transmission electron microscopy. These studies demonstrate that alkaline phosphatase, 5'-nucleotidase and gamma-glutamyl transpeptidase are secreted into both human and rat bile where they are preferentially associated with bile acid-mixed micelles, suggesting a role for bile acids in both release of these enzymes and lipids from the canalicular membrane and solubilization in bile. In addition, heterogeneous association of these enzymes with nonmicellar, lamellar structures in human and rat bile is consistent with the hypothesis that processes independent of the detergent effects of bile acids might also result in the release of specific intrinsic membrane proteins into bile.

  7. Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans-4-Hydroxylation of l-Pipecolic Acid

    PubMed Central

    Hibi, Makoto; Mori, Ryosuke; Miyake, Ryoma; Kawabata, Hiroshi; Kozono, Shoko; Takahashi, Satomi

    2016-01-01

    Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward l-pipecolic acid (l-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme l-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of l-Pip, and produces optically pure trans-4-hydroxy-l-pipecolic acid (trans-4-l-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had l-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the l and d forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-l-HyPip was successfully developed. Thus, we report a fungal family of l-Pip hydroxylases and the enzymatic preparation of trans-4-l-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities. PMID:26801577

  8. Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes.

    PubMed

    Greenough, Lucia; Schermerhorn, Kelly M; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E; Gardner, Andrew F

    2016-01-29

    Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3'-5' exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. N-Carbamoyl-β-alanine amidohydrolase from Agrobacterium tumefaciens C58: a promiscuous enzyme for the production of amino acids.

    PubMed

    Martínez-Gómez, A I; Andújar-Sánchez, M; Clemente-Jiménez, J M; Neira, J L; Rodríguez-Vico, F; Martínez-Rodríguez, S; Las Heras-Vázquez, F J

    2011-11-01

    The availability of enzymes with a high promiscuity/specificity relationship permits the hydrolysis of several substrates with a view to obtaining a certain product or using one enzyme for several productive lines. N-Carbamoyl-β-alanine amidohydrolase from Agrobacterium tumefaciens (Atβcar) has shown high versatility to hydrolyze different N-carbamoyl-, N-acetyl- and N-formyl-amino acids to produce different α, β, γ and δ amino acids. We have calculated the promiscuity index for the enzyme, obtaining a value of 0.54, which indicates that it is a modestly promiscuous enzyme. Atβcar presented the highest probability of hydrolysis for N-carbamoyl-amino acids, being the enzyme more efficient for the production of α-amino acids. We have also demonstrated by mutagenesis, modelling, kinetic and binding experiments that W218 and A359 indirectly influence the plasticity of the enzyme due to interaction with the environment of R291, the key residue for catalytic activity. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Uronic Acid Products Release from Enzymically Active Cell Wall from Tomato Fruit and Its Dependency on Enzyme Quantity and Distribution 1

    PubMed Central

    Huber, Donald J.; Lee, James H.

    1988-01-01

    Isolated cell wall from tomato (Lycopersicon esculentum Mill. cv Rutgers) fruit released polymeric (degree of polymerization [DP] > 8), oligomeric, and monomeric uronic acids in a reaction mediated by bound polygalacturonase (PG) (EC 3.2.1.15). Wall autolytic capacity increased with ripening, reflecting increased levels of bound PG; however, characteristic oligomeric and monomeric products were recovered from all wall isolates exhibiting net pectin release. The capacity of wall from fruit at early ripening (breaker, turning) to generate oligomeric and monomeric uronic acids was attributed to the nonuniform ripening pattern of the tomato fruit and, consequently, a locally dense distribution of enzyme in wall originating from those fruit portions at more temporally advanced stages of ripening. Artificial autolytically active wall, prepared by permitting solubilized PG to bind to enzymically inactive wall from maturegreen fruit, released products which were similar in size characteristics to those recovered from active wall isolates. Extraction of wall-bound PG using high concentrations of NaCl (1.2 molar) did not attenuate subsequent autolytic activity but greatly suppressed the production of oligomeric and monomeric products. An examination of water-soluble uronic acids recovered from ripe pericarp tissue disclosed the presence of polymeric and monomeric uronic acids but only trace quantities of oligomers. The significance in autolytic reactions of enzyme quantity and distribution and their possible relevance to in vivo pectin degradation will be discussed. PMID:16666191

  11. Recombinant microorganisms for increased production of organic acids

    DOEpatents

    Yi, Jian [East Lansing, MI; Kleff, Susanne [East Lansing, MI; Guettler, Michael V [Holt, MI

    2012-02-21

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  12. Recombinant microorganisms for increased production of organic acids

    DOEpatents

    Yi, Jian; Kleff, Susanne; Guettler, Michael V

    2013-04-30

    Disclosed are recombinant microorganisms for producing organic acids. The recombinant microorganisms express a polypeptide that has the enzymatic activity of an enzyme that is utilized in the pentose phosphate cycle. The recombinant microorganism may include recombinant Actinobacillus succinogenes that has been transformed to express a Zwischenferment (Zwf) gene. The recombinant microorganisms may be useful in fermentation processes for producing organic acids such as succinic acid and lactic acid. Also disclosed are novel plasmids that are useful for transforming microorganisms to produce recombinant microorganisms that express enzymes such as Zwf.

  13. Decreased glycolytic and tricarboxylic acid cycle intermediates coincide with peripheral nervous system oxidative stress in a murine model of type 2 diabetes.

    PubMed

    Hinder, Lucy M; Vivekanandan-Giri, Anuradha; McLean, Lisa L; Pennathur, Subramaniam; Feldman, Eva L

    2013-01-01

    Diabetic neuropathy (DN) is the most common complication of diabetes and is characterized by distal-to-proximal loss of peripheral nerve axons. The idea of tissue-specific pathological alterations in energy metabolism in diabetic complications-prone tissues is emerging. Altered nerve metabolism in type 1 diabetes models is observed; however, therapeutic strategies based on these models offer limited efficacy to type 2 diabetic patients with DN. Therefore, understanding how peripheral nerves metabolically adapt to the unique type 2 diabetic environment is critical to develop disease-modifying treatments. In the current study, we utilized targeted liquid chromatography-tandem mass spectrometry (LC/MS/MS) to characterize the glycolytic and tricarboxylic acid (TCA) cycle metabolomes in sural nerve, sciatic nerve, and dorsal root ganglia (DRG) from male type 2 diabetic mice (BKS.Cg-m+/+Lepr(db); db/db) and controls (db/+). We report depletion of glycolytic intermediates in diabetic sural nerve and sciatic nerve (glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate (sural nerve only), 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and lactate), with no significant changes in DRG. Citrate and isocitrate TCA cycle intermediates were decreased in sural nerve, sciatic nerve, and DRG from diabetic mice. Utilizing LC/electrospray ionization/MS/MS and HPLC methods, we also observed increased protein and lipid oxidation (nitrotyrosine; hydroxyoctadecadienoic acids) in db/db tissue, with a proximal-to-distal increase in oxidative stress, with associated decreased aconitase enzyme activity. We propose a preliminary model, whereby the greater change in metabolomic profile, increase in oxidative stress, and decrease in TCA cycle enzyme activity may cause distal peripheral nerves to rely on truncated TCA cycle metabolism in the type 2 diabetes environment.

  14. Quantitative RT-PCR Comparison of the Urea and Nitric Oxide Cycle Gene Transcripts in Adult Human Tissues

    PubMed Central

    Neill, Meaghan Anne; Aschner, Judy; Barr, Frederick; Summar, Marshall L.

    2009-01-01

    The urea cycle and nitric oxide cycle play significant roles in complex biochemical and physiologic reactions. These cycles have distinct biochemical goals including the clearance of waste nitrogen; the production of the intermediates ornithine, citrulline, and arginine for the urea cycle; and the production of nitric oxide for the nitric oxide pathway. Despite their disparate functions, the two pathways share two enzymes, argininosuccinic acid synthase and argininosuccinic acid lyase, and a transporter, citrin. Studying the gene expression of these enzymes is paramount in understanding these complex biochemical pathways. Here, we examine the expression of genes involved in the urea cycle and the nitric oxide cycle in a panel of eleven different tissue samples obtained from individual adults without known inborn errors of metabolism. In this study, the pattern of co-expressed enzymes provides a global view of the metabolic activity of the urea and nitric oxide cycles in human tissues. Our results show that these transcripts are differentially expressed in different tissues. The pattern of co-expressed enzymes provides a global view of the metabolic activity of the urea and nitric oxide cycles in human tissues. Using the co-expression profiles, we discovered that the combination of expression of enzyme transcripts as detected in our study, might serve to fulfill specific physiologic function(s) in tissue including urea production/nitrogen removal, arginine/citrulline production, nitric oxide production, and ornithine production. Our study reveals the importance of studying not only the expression profile of an enzyme of interest, but also studying the expression profiles of the other enzymes involved in a particular pathway so as to better understand the context of expression. The tissue patterns we observed highlight the variety of important functions they conduct and provide insight into many of the clinical observations from their disruption. PMID:19345634

  15. Analysis of hyaluronic acid concentration in rat vocal folds during estral and gravidic puerperal cycles.

    PubMed

    Pedroso, José Eduardo de Sá; Brasil, Osíris Camponês do; Martins, João Roberto Maciel; Nader, Helena Bociane; Simões, Manuel de Jesus

    2009-01-01

    Hormone plays an important role in the larynx. Among other substances, vocal folds contain hyaluronic acid, which tissue concentration may vary according to hormone action. the objective of this study is to analyze hyaluronic acid concentration in the vocal folds during estral and gravidic-puerperal cycles. Experimental study. 40 adult rats were divided into two groups. In the first group we used 20 rats to establish the concentration of hyaluronic acid during the estral cycle and in the second group, 20 animals were submitted to the same procedure but during the gravidic-puerperal cycle. Variations in hyaluronic acid concentration was not observed during the estral cycle. In the gravidic puerperal cycle group, an increase in hyaluronic acid concentration was observed in the puerperal subgroup. Comparing the two groups of estral and gravidic-puerperal cycles, no difference was observed. In comparing all subgroups of estral and gravidic-puerperal cycles, an increase in hyaluronic acid concentration was noticed only in the puerperal phase.

  16. Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme.

    PubMed

    Gallage, Nethaji J; Hansen, Esben H; Kannangara, Rubini; Olsen, Carl Erik; Motawia, Mohammed Saddik; Jørgensen, Kirsten; Holme, Inger; Hebelstrup, Kim; Grisoni, Michel; Møller, Birger Lindberg

    2014-06-19

    Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco.

  17. Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme

    PubMed Central

    Gallage, Nethaji J.; Hansen, Esben H.; Kannangara, Rubini; Olsen, Carl Erik; Motawia, Mohammed Saddik; Jørgensen, Kirsten; Holme, Inger; Hebelstrup, Kim; Grisoni, Michel; Møller, Birger Lindberg

    2014-01-01

    Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco. PMID:24941968

  18. Secretion of acid phosphatase by axenic Entamoeba histolytica NIH-200 and properties of the extracellular enzyme.

    PubMed

    Agrawal, A; Pandey, V C; Kumar, S; Sagar, P

    1989-01-01

    Entamoeba histolytica (NIH-200) secreted large amounts of acid phosphatase in its external environment when grown axenically in modified TPS-II medium. Fractionation by DEAE-cellulose chromatography of the precipitate obtained from the cell-free medium at 60% ammonium sulfate saturation yielded 3 distinct peaks of enzyme activity. The enzyme in all the peaks showed resistance to tartrate but was inhibited by fluoride, cupric chloride, ethylene diamine-tetra acetic acid, ammonium molybdate and cysteine; however, enzyme associated with different peaks differed in its polyacrylamide gel electrophoretic profiles and behavior towards concanavalin A.

  19. Single-Cell Measurements of Enzyme Levels as a Predictive Tool for Cellular Fates during Organic Acid Production

    PubMed Central

    Zdraljevic, Stefan; Wagner, Drew; Cheng, Kevin; Ruohonen, Laura; Jäntti, Jussi; Penttilä, Merja; Resnekov, Orna

    2013-01-01

    Organic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity. Cytosolic acidification affects cells asynchronously, suggesting that there is an underlying cell-to-cell heterogeneity in acid productivity and/or in resistance to toxicity. We used fluorescence microscopy to investigate the relationship between enzyme concentration, cytosolic pH, and viability at the single-cell level in Saccharomyces cerevisiae engineered to synthesize xylonic acid. We found that cultures producing xylonic acid accumulate cells with cytosolic pH below 5 (referred to here as “acidified”). Using live-cell time courses, we found that the probability of acidification was related to the initial levels of xylose dehydrogenase and sharply increased from 0.2 to 0.8 with just a 60% increase in enzyme abundance (Hill coefficient, >6). This “switch-like” relationship likely results from an enzyme level threshold above which the produced acid overwhelms the cell's pH buffering capacity. Consistent with this hypothesis, we showed that expression of xylose dehydrogenase from a chromosomal locus yields ∼20 times fewer acidified cells and ∼2-fold more xylonic acid relative to expression of the enzyme from a plasmid with variable copy number. These results suggest that strategies that further reduce cell-to-cell heterogeneity in enzyme levels could result in additional gains in xylonic acid productivity. Our results demonstrate a generalizable approach that takes advantage of the cell-to-cell variation of a clonal population to uncover causal relationships in the toxicity of engineered pathways. PMID:24038690

  20. Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes

    PubMed Central

    Greenough, Lucia; Schermerhorn, Kelly M.; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E.; Gardner, Andrew F.

    2016-01-01

    Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3′-5′ exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. PMID:26365239

  1. Metabolic engineering in the biotechnological production of organic acids in the tricarboxylic acid cycle of microorganisms: Advances and prospects.

    PubMed

    Yin, Xian; Li, Jianghua; Shin, Hyun-Dong; Du, Guocheng; Liu, Long; Chen, Jian

    2015-11-01

    Organic acids, which are chemically synthesized, are also natural intermediates in the metabolic pathways of microorganisms, among which the tricarboxylic acid (TCA) cycle is the most crucial route existing in almost all living organisms. Organic acids in the TCA cycle include citric acid, α-ketoglutaric acid, succinic acid, fumaric acid, l-malic acid, and oxaloacetate, which are building-block chemicals with wide applications and huge markets. In this review, we summarize the synthesis pathways of these organic acids and review recent advances in metabolic engineering strategies that enhance organic acid production. We also propose further improvements for the production of organic acids with systems and synthetic biology-guided metabolic engineering strategies. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. mRNA levels of enzymes and receptors implicated in arachidonic acid metabolism in gliomas.

    PubMed

    De Armas, Rafael; Durand, Karine; Guillaudeau, Angélique; Weinbreck, Nicolas; Robert, Sandrine; Moreau, Jean-Jacques; Caire, François; Acosta, Gisela; Pebet, Matias; Chaunavel, Alain; Marin, Benoît; Labrousse, François; Denizot, Yves

    2010-07-01

    Gliomas are tumors of the central nervous system derived from glial cells. They show cellular heterogeneity and lack specific diagnostic markers. Although a possible role for the eicosanoid cascade has been suggested in glioma tumorigenesis, the relationship between enzymes and receptors implicated in arachidonic acid metabolism, with histological tumor type has not yet been determined. Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure and compare transcript levels of enzymes and receptors implicated in both lipoxygenase and cyclooxygenase pathways between oligodendrogliomas, astrocytomas, glioblastomas and mixed oligoastrocytomas. Arachidonic acid metabolism-related enzymes and receptor transcripts (i) were underexpressed in classical oligodendrogliomas compared to astrocytomas and/or glioblastomas, (ii) differed between astrocytomas and glioblastomas and (iii) had an intermediate expression in mixed oligoastrocytomas. mRNA levels of enzymes and receptors implicated both in lipoxygenase and cyclooxygenase pathways differed significantly in gliomas according to the histological type. Copyright 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  3. Sulfuric acid-sulfur heat storage cycle

    DOEpatents

    Norman, John H.

    1983-12-20

    A method of storing heat is provided utilizing a chemical cycle which interconverts sulfuric acid and sulfur. The method can be used to levelize the energy obtained from intermittent heat sources, such as solar collectors. Dilute sulfuric acid is concentrated by evaporation of water, and the concentrated sulfuric acid is boiled and decomposed using intense heat from the heat source, forming sulfur dioxide and oxygen. The sulfur dioxide is reacted with water in a disproportionation reaction yielding dilute sulfuric acid, which is recycled, and elemental sulfur. The sulfur has substantial potential chemical energy and represents the storage of a significant portion of the energy obtained from the heat source. The sulfur is burned whenever required to release the stored energy. A particularly advantageous use of the heat storage method is in conjunction with a solar-powered facility which uses the Bunsen reaction in a water-splitting process. The energy storage method is used to levelize the availability of solar energy while some of the sulfur dioxide produced in the heat storage reactions is converted to sulfuric acid in the Bunsen reaction.

  4. Developmental roles of tyrosine metabolism enzymes in the blood-sucking insect Rhodnius prolixus

    PubMed Central

    Oliveira, Pedro L.

    2017-01-01

    The phenylalanine/tyrosine degradation pathway is frequently described as a catabolic pathway that funnels aromatic amino acids into citric acid cycle intermediates. Previously, we demonstrated that the accumulation of tyrosine generated during the hydrolysis of blood meal proteins in Rhodnius prolixus is potentially toxic, a harmful outcome that is prevented by the action of the first two enzymes in the tyrosine degradation pathway. In this work, we further evaluated the relevance of all other enzymes involved in phenylalanine/tyrosine metabolism in the physiology of this insect. The knockdown of most of these enzymes produced a wide spectrum of distinct phenotypes associated with reproduction, development and nymph survival, demonstrating a highly pleiotropic role of tyrosine metabolism. The phenotypes obtained for two of these enzymes, homogentisate dioxygenase and fumarylacetoacetase, have never before been described in any arthropod. To our knowledge, this report is the first comprehensive gene-silencing analysis of an amino acid metabolism pathway in insects. Amino acid metabolism is exceptionally important in haematophagous arthropods due to their particular feeding behaviour. PMID:28469016

  5. Developmental roles of tyrosine metabolism enzymes in the blood-sucking insect Rhodnius prolixus.

    PubMed

    Sterkel, Marcos; Oliveira, Pedro L

    2017-05-17

    The phenylalanine/tyrosine degradation pathway is frequently described as a catabolic pathway that funnels aromatic amino acids into citric acid cycle intermediates. Previously, we demonstrated that the accumulation of tyrosine generated during the hydrolysis of blood meal proteins in Rhodnius prolixus is potentially toxic, a harmful outcome that is prevented by the action of the first two enzymes in the tyrosine degradation pathway. In this work, we further evaluated the relevance of all other enzymes involved in phenylalanine/tyrosine metabolism in the physiology of this insect. The knockdown of most of these enzymes produced a wide spectrum of distinct phenotypes associated with reproduction, development and nymph survival, demonstrating a highly pleiotropic role of tyrosine metabolism. The phenotypes obtained for two of these enzymes, homogentisate dioxygenase and fumarylacetoacetase, have never before been described in any arthropod. To our knowledge, this report is the first comprehensive gene-silencing analysis of an amino acid metabolism pathway in insects. Amino acid metabolism is exceptionally important in haematophagous arthropods due to their particular feeding behaviour. © 2017 The Author(s).

  6. Efficient production of free fatty acids from ionic liquid-based acid- or enzyme-catalyzed bamboo hydrolysate.

    PubMed

    Mi, Le; Qin, Dandan; Cheng, Jie; Wang, Dan; Li, Sha; Wei, Xuetuan

    2017-03-01

    Two engineered Escherichia coli strains, DQ101 (MG1655 fadD - )/pDQTES and DQ101 (MG1655 fadD - )/pDQTESZ were constructed to investigate the free fatty acid production using ionic liquid-based acid- or enzyme-catalyzed bamboo hydrolysate as carbon source in this study. The plasmid, pDQTES, carrying an acyl-ACP thioesterase 'TesA of E. coli in pTrc99A was constructed firstly, and then (3R)-hydroxyacyl-ACP dehydratase was ligated after the TesA to give the plasmid pDQTESZ. These two strains exhibited efficient fatty acid production when glucose was used as the sole carbon source, with a final concentration of 2.45 and 3.32 g/L, respectively. The free fatty acid production of the two strains on xylose is not as efficient as that on glucose, which was 2.32 and 2.96 g/L, respectively. For mixed sugars, DQ101 (MG1655 fadD - )-based strains utilized glucose and pentose sequentially under the carbon catabolite repression (CCR) regulation. The highest total FFAs concentration from the mixed sugar culture reached 2.81 g/L by DQ101 (MG1655 fadD - )/pDQTESZ. Furthermore, when ionic liquid-based enzyme-catalyzed bamboo hydrolysate was used as the carbon source, the strain DQ101 (MG1655 fadD - )/pDQTESZ could produce 1.23 g/L FFAs with a yield of 0.13 g/g, and while it just produced 0.65 g/L free fatty acid with the ionic liquid-based acid-catalyzed bamboo hydrolysate as the feedstock. The results suggested that enzymatic catalyzed bamboo hydrolysate with ionic liquid pretreatment could serve as an efficient feedstock for free fatty acid production.

  7. Geobiochemistry of metabolism: Standard state thermodynamic properties of the citric acid cycle

    NASA Astrophysics Data System (ADS)

    Canovas, Peter A.; Shock, Everett L.

    2016-12-01

    Integrating microbial metabolism into geochemical modeling allows assessments of energy and mass transfer between the geosphere and the microbial biosphere. Energy and power supplies and demands can be assessed from analytical geochemical data given thermodynamic data for compounds involved in catabolism and anabolism. Results are reported here from a critique of the available standard state thermodynamic data for organic acids and acid anions involved in the citric acid cycle (also known as the tricarboxylic acid cycle or the Krebs cycle). The development of methods for estimating standard state data unavailable from experiments is described, together with methods to predict corresponding values at elevated temperatures and pressures using the revised Helgeson-Kirkham-Flowers (HKF) equation of state for aqueous species. Internal consistency is maintained with standard state thermodynamic data for organic and inorganic aqueous species commonly used in geochemical modeling efforts. Standard state data and revised-HKF parameters are used to predict equilibrium dissociation constants for the organic acids in the citric acid cycle, and to assess standard Gibbs energies of reactions for each step in the cycle at elevated temperatures and pressures. The results presented here can be used with analytical data from natural and experimental systems to assess the energy and power demands of microorganisms throughout the habitable ranges of pressure and temperature, and to assess the consequences of abiotic organic compound alteration processes at conditions of subsurface aquifers, sedimentary basins, hydrothermal systems, meteorite parent bodies, and ocean worlds throughout the solar system.

  8. Sulfuric acid on Europa and the radiolytic sulfur cycle.

    PubMed

    Carlson, R W; Johnson, R E; Anderson, M S

    1999-10-01

    A comparison of laboratory spectra with Galileo data indicates that hydrated sulfuric acid is present and is a major component of Europa's surface. In addition, this moon's visually dark surface material, which spatially correlates with the sulfuric acid concentration, is identified as radiolytically altered sulfur polymers. Radiolysis of the surface by magnetospheric plasma bombardment continuously cycles sulfur between three forms: sulfuric acid, sulfur dioxide, and sulfur polymers, with sulfuric acid being about 50 times as abundant as the other forms. Enhanced sulfuric acid concentrations are found in Europa's geologically young terrains, suggesting that low-temperature, liquid sulfuric acid may influence geological processes.

  9. Structural analysis of Bacillus pumilus phenolic acid decarboxylase, a lipocalin-fold enzyme

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matte, Allan; Grosse, Stephan; Bergeron, Hélène

    The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavoring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a -barrel structure and two -helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally relatedmore » proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the -barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site.« less

  10. Thermostable Lipoxygenase, a Key Enzyme in the Conversion of Linoleic Acid into Thrihydroxy-octadecenoic Acid by Pseudomonas aeruginosa PR3

    USDA-ARS?s Scientific Manuscript database

    Lipoxygenases (LOX) constitute a family of lipid-peroxidizing enzymes catalyzing the oxidation of unsaturated fatty acid with (1Z,4Z)-pentadiene structural unit, leading to formation of the conjugated (Z,E)-hydroperoxydienoic acid. LOXs have been known to be widely distributed in plants and animals...

  11. Correlation between citric acid and nitrate metabolisms during CAM cycle in the atmospheric bromeliad Tillandsia pohliana.

    PubMed

    Freschi, Luciano; Rodrigues, Maria Aurineide; Tiné, Marco Aurélio Silva; Mercier, Helenice

    2010-12-15

    Crassulacean acid metabolism (CAM) confers crucial adaptations for plants living under frequent environmental stresses. A wide metabolic plasticity can be found among CAM species regarding the type of storage carbohydrate, organic acid accumulated at night and decarboxylating system. Consequently, many aspects of the CAM pathway control are still elusive while the impact of this photosynthetic adaptation on nitrogen metabolism has remained largely unexplored. In this study, we investigated a possible link between the CAM cycle and the nitrogen assimilation in the atmospheric bromeliad Tillandsia pohliana by simultaneously characterizing the diel changes in key enzyme activities and metabolite levels of both organic acid and nitrate metabolisms. The results revealed that T. pohliana performed a typical CAM cycle in which phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase phosphorylation seemed to play a crucial role to avoid futile cycles of carboxylation and decarboxylation. Unlike all other bromeliads previously investigated, almost equimolar concentrations of malate and citrate were accumulated at night. Moreover, a marked nocturnal depletion in the starch reservoirs and an atypical pattern of nitrate reduction restricted to the nighttime were also observed. Since reduction and assimilation of nitrate requires a massive supply of reducing power and energy and considering that T. pohliana lives overexposed to the sunlight, we hypothesize that citrate decarboxylation might be an accessory mechanism to increase internal CO₂ concentration during the day while its biosynthesis could provide NADH and ATP for nocturnal assimilation of nitrate. Therefore, besides delivering photoprotection during the day, citrate might represent a key component connecting both CAM pathway and nitrogen metabolism in T. pohliana; a scenario that certainly deserves further study not only in this species but also in other CAM plants that nocturnally accumulate citrate

  12. The catalytic cycle of nitrous oxide reductase - The enzyme that catalyzes the last step of denitrification.

    PubMed

    Carreira, Cíntia; Pauleta, Sofia R; Moura, Isabel

    2017-12-01

    The reduction of the potent greenhouse gas nitrous oxide requires a catalyst to overcome the large activation energy barrier of this reaction. Its biological decomposition to the inert dinitrogen can be accomplished by denitrifiers through nitrous oxide reductase, the enzyme that catalyzes the last step of the denitrification, a pathway of the biogeochemical nitrogen cycle. Nitrous oxide reductase is a multicopper enzyme containing a mixed valence CuA center that can accept electrons from small electron shuttle proteins, triggering electron flow to the catalytic sulfide-bridged tetranuclear copper "CuZ center". This enzyme has been isolated with its catalytic center in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S), proven to be spectroscopic and structurally different. In the last decades, it has been a challenge to characterize the properties of this complex enzyme, due to the different oxidation states observed for each of its centers and the heterogeneity of its preparations. The substrate binding site in those two "CuZ center" forms and which is the active form of the enzyme is still a matter of debate. However, in the last years the application of different spectroscopies, together with theoretical calculations have been useful in answering these questions and in identifying intermediate species of the catalytic cycle. An overview of the spectroscopic, kinetics and structural properties of the two forms of the catalytic "CuZ center" is given here, together with the current knowledge on nitrous oxide reduction mechanism by nitrous oxide reductase and its intermediate species. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. [Effects of two phenolic acids on root zone soil nutrients, soil enzyme activities and pod yield of peanut].

    PubMed

    Li, Qing Kai; Liu, Ping; Tang, Zhao Hui; Zhao, Hai Jun; Wang, Jiang Tao; Song, Xiao Zong; Yang, Li; Wan, Shu Bo

    2016-04-22

    In order to investigate the relationship between the accumulation of phenolic acids in peanut continuous cropping soil and the continuous cropping obstacle of peanut, the effects of p-hydroxy benzoic acid and cinnamic acid on peanut root zone soil nutrients, soil enzyme activities and yield of peanut were studied by pot experiment at three stages of peanut, i.e. the pegging stage of peanut (45 days after seedling), the early podding (75 days after seedling) and the end of podding (105 days after seedling) stages. The results showed that the peanut root zone soil nutrients and enzyme activities changed obviously under the two phenolic acids treatment, especially at the pegging stage of peanut. The soil alkali-hydrolyzable nitrogen, available phosphorus, available potassium, and soil enzyme activities (urease, sucrose, neutral phosphatase) were decreased significantly. At the early and end of podding stages of peanut, the effects of the two phenolic acids on peanut root zone soil nutrients and soil enzyme activities were under a weakening trend. The allelopathy of cinnamic acid was stronger than that of p-hydroxy benzoic acid at the same initial content. The pod yield per pot was reduced by 45.9% and 52.8%, while the pod number of per plant was reduced by 46.2% and 48.9% at higher concentration (80 mg·kg -1 dry soil) of p-hydroxy benzoic acid and cinnamic acid treatments, respectively.

  14. Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway

    PubMed Central

    2016-01-01

    SUMMARY Although the structure of lipoic acid and its role in bacterial metabolism were clear over 50 years ago, it is only in the past decade that the pathways of biosynthesis of this universally conserved cofactor have become understood. Unlike most cofactors, lipoic acid must be covalently bound to its cognate enzyme proteins (the 2-oxoacid dehydrogenases and the glycine cleavage system) in order to function in central metabolism. Indeed, the cofactor is assembled on its cognate proteins rather than being assembled and subsequently attached as in the typical pathway, like that of biotin attachment. The first lipoate biosynthetic pathway determined was that of Escherichia coli, which utilizes two enzymes to form the active lipoylated protein from a fatty acid biosynthetic intermediate. Recently, a more complex pathway requiring four proteins was discovered in Bacillus subtilis, which is probably an evolutionary relic. This pathway requires the H protein of the glycine cleavage system of single-carbon metabolism to form active (lipoyl) 2-oxoacid dehydrogenases. The bacterial pathways inform the lipoate pathways of eukaryotic organisms. Plants use the E. coli pathway, whereas mammals and fungi probably use the B. subtilis pathway. The lipoate metabolism enzymes (except those of sulfur insertion) are members of PFAM family PF03099 (the cofactor transferase family). Although these enzymes share some sequence similarity, they catalyze three markedly distinct enzyme reactions, making the usual assignment of function based on alignments prone to frequent mistaken annotations. This state of affairs has possibly clouded the interpretation of one of the disorders of human lipoate metabolism. PMID:27074917

  15. Early lignin pathway enzymes and routes to chlorogenic acid in switchgrass (Panicum virgatum L.).

    PubMed

    Escamilla-Treviño, Luis L; Shen, Hui; Hernandez, Timothy; Yin, Yanbin; Xu, Ying; Dixon, Richard A

    2014-03-01

    Studying lignin biosynthesis in Panicum virgatum (switchgrass) has provided a basis for generating plants with reduced lignin content and increased saccharification efficiency. Chlorogenic acid (CGA, caffeoyl quinate) is the major soluble phenolic compound in switchgrass, and the lignin and CGA biosynthetic pathways potentially share intermediates and enzymes. The enzyme hydroxycinnamoyl-CoA: quinate hydroxycinnamoyltransferase (HQT) is responsible for CGA biosynthesis in tobacco, tomato and globe artichoke, but there are no close orthologs of HQT in switchgrass or in other monocotyledonous plants with complete genome sequences. We examined available transcriptomic databases for genes encoding enzymes potentially involved in CGA biosynthesis in switchgrass. The protein products of two hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) genes (PvHCT1a and PvHCT2a), closely related to lignin pathway HCTs from other species, were characterized biochemically and exhibited the expected HCT activity, preferring shikimic acid as acyl acceptor. We also characterized two switchgrass coumaroyl shikimate 3'-hydroxylase (C3'H) enzymes (PvC3'H1 and PvC3'H2); both of these cytochrome P450s had the capacity to hydroxylate 4-coumaroyl shikimate or 4-coumaroyl quinate to generate caffeoyl shikimate or CGA. Another switchgrass hydroxycinnamoyl transferase, PvHCT-Like1, is phylogenetically distant from HCTs or HQTs, but exhibits HQT activity, preferring quinic acid as acyl acceptor, and could therefore function in CGA biosynthesis. The biochemical features of the recombinant enzymes, the presence of the corresponding activities in plant protein extracts, and the expression patterns of the corresponding genes, suggest preferred routes to CGA in switchgrass.

  16. Tips on the analysis of phosphatidic acid by the fluorometric coupled enzyme assay.

    PubMed

    Hassaninasab, Azam; Han, Gil-Soo; Carman, George M

    2017-06-01

    The fluorometric coupled enzyme assay to measure phosphatidic acid (PA) involves the solubilization of extracted lipids in Triton X-100, deacylation, and the oxidation of PA-derived glycerol-3-phosphate to produce hydrogen peroxide for conversion of Amplex Red to resorufin. The enzyme assay is sensitive, but plagued by high background fluorescence from the peroxide-containing detergent and incomplete heat inactivation of lipoprotein lipase. These problems affecting the assay reproducibility were obviated by the use of highly pure Triton X-100 and by sufficient heat inactivation of the lipase enzyme. The enzyme assay could accurately measure the PA content from the subcellular fractions of yeast cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Transgenic Production of Epoxy Fatty Acids by Expression of a Cytochrome P450 Enzyme from Euphorbia lagascae Seed

    PubMed Central

    Cahoon, Edgar B.; Ripp, Kevin G.; Hall, Sarah E.; McGonigle, Brian

    2002-01-01

    Seed oils of a number of Asteraceae and Euphorbiaceae species are enriched in 12-epoxyoctadeca-cis-9-enoic acid (vernolic acid), an unusual 18-carbon Δ12-epoxy fatty acid with potential industrial value. It has been previously demonstrated that the epoxy group of vernolic acid is synthesized by the activity of a Δ12-oleic acid desaturase-like enzyme in seeds of the Asteraceae Crepis palaestina and Vernonia galamensis. In contrast, results from metabolic studies have suggested the involvement of a cytochrome P450 enzyme in vernolic acid synthesis in seeds of the Euphorbiaceae species Euphorbia lagascae. To clarify the biosynthetic origin of vernolic acid in E. lagascae seed, an expressed sequence tag analysis was conducted. Among 1,006 randomly sequenced cDNAs from developing E. lagascae seeds, two identical expressed sequence tags were identified that encode a cytochrome P450 enzyme classified as CYP726A1. Consistent with the seed-specific occurrence of vernolic acid in E. lagascae, mRNA corresponding to the CYP726A1 gene was abundant in developing seeds, but was not detected in leaves. In addition, expression of the E. lagascae CYP726A1 cDNA in Saccharomyces cerevisiae was accompanied by production of vernolic acid in cultures supplied with linoleic acid and an epoxy fatty acid tentatively identified as 12-epoxyoctadeca-9,15-dienoic acid (12-epoxy-18:2Δ9,15) in cultures supplied with α-linolenic acid. Consistent with this, expression of CYP726A1 in transgenic tobacco (Nicotiana tabacum) callus or somatic soybean (Glycine max) embryos resulted in the accumulation of vernolic acid and 12-epoxy-18:2Δ9,15. Overall, these results conclusively demonstrate that Asteraceae species and the Euphorbiaceae E. lagascae have evolved structurally unrelated enzymes to generate the Δ12-epoxy group of vernolic acid. PMID:11842164

  18. A futile cycle, formed between two ATP-dependant gamma-glutamyl cycle enzymes, gamma-glutamyl cysteine synthetase and 5-oxoprolinase: the cause of cellular ATP depletion in nephrotic cystinosis?

    PubMed

    Kumar, Akhilesh; Bachhawat, Anand Kumar

    2010-03-01

    Cystinosis, an inherited disease caused by a defect in the lysosomal cystine transporter (CTNS), is characterized by renal proximal tubular dysfunction. Adenosine triphosphate (ATP) depletion appears to be a key event in the pathophysiology of the disease, even though the manner in which ATP depletion occurs is still a puzzle. We present a model that explains how a futile cycle that is generated between two ATP-utilizing enzymes of the gamma-glutamyl cycle leads to ATP depletion. The enzyme gamma-glutamyl cysteine synthetase (gamma-GCS), in the absence of cysteine, forms 5-oxoproline (instead of the normal substrate, gamma-glutamyl cysteine) and the 5-oxoproline is converted into glutamate by the ATP-dependant enzyme, 5-oxoprolinase. Thus, in cysteine-limiting conditions, glutamate is cycled back into glutamate via 5-oxoproline at the cost of two ATP molecules without production of glutathione and is the cause of the decreased levels of glutathione synthesis, as well as the ATP depletion observed in these cells. The model is also compatible with the differences seen in the human patients and the mouse model of cystinosis, where renal failure is not observed.

  19. Characterization of two Streptomyces enzymes that convert ferulic acid to vanillin.

    PubMed

    Yang, Wenwen; Tang, Hongzhi; Ni, Jun; Wu, Qiulin; Hua, Dongliang; Tao, Fei; Xu, Ping

    2013-01-01

    Production of flavors from natural substrates by microbial transformation has become a growing and expanding field of study over the past decades. Vanillin, a major component of vanilla flavor, is a principal flavoring compound used worldwide. Streptomyces sp. strain V-1 is known to be one of the most promising microbial producers of natural vanillin from ferulic acid. Although identification of the microbial genes involved in the biotransformation of ferulic acid to vanillin has been previously reported, purification and detailed characterization of the corresponding enzymes with important functions have rarely been studied. In this study, we isolated and identified 2 critical genes, fcs and ech, encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively, which are involved in the vanillin production from ferulic acid. Both genes were heterologously expressed in Escherichia coli, and the resting cell reactions for converting ferulic acid to vanillin were performed. The corresponding crucial enzymes, Fcs and Ech, were purified for the first time and the enzymatic activity of each purified protein was studied. Furthermore, Fcs was comprehensively characterized, at an optimal pH of 7.0 and temperature of 30°C. Kinetic constants for Fcs revealed the apparent Km, kcat, and Vmax values to be 0.35 mM, 67.7 s(-1), and 78.2 U mg(-1), respectively. The catalytic efficiency (kcat/Km) value of Fcs was 193.4 mM(-1) s(-1) for ferulic acid. The characterization of Fcs and Ech may be helpful for further research in the field of enzymatic engineering and metabolic regulation.

  20. MARINE SULFUR CYCLE. Identification of the algal dimethyl sulfide-releasing enzyme: A missing link in the marine sulfur cycle.

    PubMed

    Alcolombri, Uria; Ben-Dor, Shifra; Feldmesser, Ester; Levin, Yishai; Tawfik, Dan S; Vardi, Assaf

    2015-06-26

    Algal blooms produce large amounts of dimethyl sulfide (DMS), a volatile with a diverse signaling role in marine food webs that is emitted to the atmosphere, where it can affect cloud formation. The algal enzymes responsible for forming DMS from dimethylsulfoniopropionate (DMSP) remain unidentified despite their critical role in the global sulfur cycle. We identified and characterized Alma1, a DMSP lyase from the bloom-forming algae Emiliania huxleyi. Alma1 is a tetrameric, redox-sensitive enzyme of the aspartate racemase superfamily. Recombinant Alma1 exhibits biochemical features identical to the DMSP lyase in E. huxleyi, and DMS released by various E. huxleyi isolates correlates with their Alma1 levels. Sequence homology searches suggest that Alma1 represents a gene family present in major, globally distributed phytoplankton taxa and in other marine organisms. Copyright © 2015, American Association for the Advancement of Science.

  1. Cell cycle effect on the activity of deoxynucleoside analogue metabolising enzymes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fyrberg, Anna; Albertioni, Freidoun; Lotfi, Kourosh

    Deoxynucleoside analogues (dNAs) are cytotoxic towards both replicating and indolent malignancies. The impact of fluctuations in the metabolism of dNAs in relation to cell cycle could have strong implications regarding the activity of dNAs. Deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) are important enzymes for phosphorylation/activation of dNAs. These drugs can be dephosphorylated/deactivated by 5'-nucleotidases (5'-NTs) and elevated activities of 5'-NTs and decreased dCK and/or dGK activities represent resistance mechanisms towards dNAs. The activities of dCK, dGK, and three 5'-NTs were investigated in four human leukemic cell lines in relationship to cell cycle progression and cytotoxicity of dNAs. Synchronization ofmore » cell cultures to arrest in G0/G1 by serum-deprivation was performed followed by serum-supplementation for cell cycle progression. The activities of dCK and dGK increased up to 3-fold in CEM, HL60, and MOLT-4 cells as they started to proliferate, while the activity of cytosolic nucleotidase I was reduced in proliferating cells. CEM, HL60, and MOLT-4 cells were also more sensitive to cladribine, cytarabine, 9-{beta}-D-arabinofuranosylguanine and clofarabine than K562 cells which demonstrated lower levels and less alteration of these enzymes and were least susceptible to the cytotoxic effects of most dNAs. The results suggest that, in the cell lines studied, the proliferation process is associated with a general shift in the direction of activation of dNAs by inducing activities of dCK/dGK and reducing the activity of cN-I which is favourable for the cytotoxic effects of cladribine, cytarabine and, 9-{beta}-D-arabinofuranosylguanine. These results emphasize the importance of cellular proliferation and dNA metabolism by both phosphorylation and dephosphorylation for susceptibility to dNAs. It underscores the need to understand the mechanisms of action and resistance to dNAs in order to increase efficacy of dNAs treatment by new

  2. The synthesis of glutamic acid in the absence of enzymes: Implications for biogenesis

    NASA Technical Reports Server (NTRS)

    Morowitz, Harold; Peterson, Eta; Chang, Sherwood

    1995-01-01

    This paper reports on the non-enzymatic aqueous phase synthesis of amino acids from keto acids, ammonia and reducing agents. The facile synthesis of key metabolic intermediates, particularly in the glycolytic pathway, the citric acid cycle, and the first step of amino acid synthesis, lead to new ways of looking at the problem of biogenesis.

  3. Enzyme dynamics in paddy soils of the rice district (NE Italy) under different cropping patterns

    NASA Astrophysics Data System (ADS)

    Bini, Claudio; Nadimi-Goki, Mandana; Kato, Yoichi; Fornasier, Flavio; Wahsha, Mohammad; Spiandorello, Massimo

    2014-05-01

    The recent widespread interest on soil enzymes is due to the need to develop sensitive indicators of soil quality that reflect the effects of land management on soil and assist land managers in promoting long-term sustainability of terrestrial ecosystems. The activities of six important enzymes involved in C, N, P, and S cycling were investigated in a paddy soil from the Veneto region, Italy, in four different rotation systems (rice-rice-rice: R-R-R; soya-rice-rice: S-R-R; fallow-rice: F-R; pea-soya-rice: P-S-R) with three replications in April (after field preparation, field moist condition), June (after seedling, waterlogged soil condition), August (after tillering stage of rice, waterlogged soil condition) and October (after rice harvesting, drained soil condition) over the 2012 growing season. Our results demonstrated that enzyme activities varied with rotation systems and growth stages in paddy soil. Compared with field moist soil, drained soil condition resulted in a significant increase (P < 0.05) of β-glucosidase, arylsulfatase, alkaline and acid phosphatases, leucine aminopeptidase (except of fallow-rice), and chitinase activities in all rotations, while compared with drained soil, early waterlogging (in month of June) significantly decreased (P moist soil> late waterlogged>early waterlogged. There was an inhibitory effect of waterlogging (except P-S-R rotation) for both alkaline and acid phosphatases due to high pH and redox conditions. However, the response of enzymes to waterlogging differed with the chemical species and the cropping pattern. The best rotation system for chitinase, leucine aminopeptidase and β-glucosidase activity (C and N cycles) proved R-R-R, while for arylsulfatase, alkaline and acid phosphatases (P and S cycles) it was the S-R-R. Key Words: enzyme activity, paddy soil, Crop Rotation System, Italy __ Corresponding Author: Mandana Nadimi-Goki, Tel.: +39 3891356251 E-mail address: mandy.nadimi@gmail.com

  4. Effect of enzyme addition on the nutritive value of high oleic acid sunflower seeds in chicken diets.

    PubMed

    Brenes, A; Centeno, C; Viveros, A; Arija, I

    2008-11-01

    Two experiments were conducted to evaluate the effects of enzyme addition in chicken diets containing high oleic acid sunflower seeds (HOASS). In the first experiment (4 to 21 d of age), enzyme addition (lipase, phospholipase, and a combination of these) was used at the inclusion level of 1 g/kg in diets containing HOASS (250 g/kg) compared with a control corn-soybean diet. Weight gain, feed consumption, relative liver weight, fat digestibility, and amylase, lipase, serum lactate dehydrogenase (LDH), and creatine phosphokinase (CPK) activities were reduced, and feed conversion, relative duodenum, jejunum, ileum, and ceca lengths, plasma uric acid, cholesterol, and glucose concentrations were increased in the unsupplemented HOASS diet compared with the control diet. The addition of enzymes to the HOASS diet increased weight gain, feed consumption, relative pancreas and liver weights, fat digestibility, amylase and lipase activities, plasma uric acid, calcium, serum LDH and CPK, and total protein concentration and reduced feed conversion, relative spleen weight, relative duodenum, jejunum, ileum, and ceca lengths, plasma cholesterol, and glucose compared with the unsupplemented HOASS diet. In the second experiment (0 to 21 d of age), the same enzymes (0.5 g/kg each) were included in diets containing 150 g/kg of HOASS compared with a conventional sunflower meal diet (150 g/kg). The HOASS diet did not affect performance but reduced relative pancreas and abdominal fat weights and relative duodenum and ceca lengths, and increased crude fat, CP, and essential and nonessential amino acid digestibilities (except Ser, which was reduced) compared with the control diet. The addition of enzymes in the HOASS diet increased weight gain, feed consumption, and relative pancreas weight and reduced feed conversion, CP, and essential and nonessential amino acid digestibilities compared with the unsupplemented HOASS diet. In conclusion, the addition of 250 g of HOASS/kg in the diets

  5. Genipin Cross-Linked Glucose Oxidase and Catalase Multi-enzyme for Gluconic Acid Synthesis.

    PubMed

    Cui, Caixia; Chen, Haibin; Chen, Biqiang; Tan, Tianwei

    2017-02-01

    In this work, glucose oxidase (GOD) and catalase (CAT) were used simultaneously to produce gluconic acid from glucose. In order to reduce the distance between the two enzymes, and therefore improve efficiency, GOD and CAT were cross-linked together using genipin. Improvements in gluconic acid production were due to quick removal of harmful intermediate hydrogen peroxide by CAT. GOD activity was significantly affected by the proportion of CAT in the system, with GOD activity in the cross-linked multi-enzyme (CLME) being 10 times higher than that in an un-cross-linked GOD/CAT mixture. The glucose conversion rate after 15 h using 15 % glucose was also 10 % higher using the CLME than was measured using a GOD/CAT mixture.

  6. Thermodynamic framework for identifying free energy inventories of enzyme catalytic cycles

    PubMed Central

    Fried, Stephen D.; Boxer, Steven G.

    2013-01-01

    Pauling’s suggestion that enzymes are complementary in structure to the activated complexes of the reactions they catalyze has provided the conceptual basis to explain how enzymes obtain their fantastic catalytic prowess, and has served as a guiding principle in drug design for over 50 y. However, this model by itself fails to predict the magnitude of enzymes’ rate accelerations. We construct a thermodynamic framework that begins with the classic concept of differential binding but invokes additional terms that are needed to account for subtle effects in the catalytic cycle’s proton inventory. Although the model presented can be applied generally, this analysis focuses on ketosteroid isomerase (KSI) as an example, where recent experiments along with a large body of kinetic and thermodynamic data have provided strong support for the noncanonical thermodynamic contribution described. The resulting analysis precisely predicts the free energy barrier of KSI’s reaction as determined from transition-state theory using only empirical thermodynamic data. This agreement is suggestive that a complete free energy inventory of the KSI catalytic cycle has been identified. PMID:23840058

  7. Integrating enzyme fermentation in lignocellulosic ethanol production: life-cycle assessment and techno-economic analysis.

    PubMed

    Olofsson, Johanna; Barta, Zsolt; Börjesson, Pål; Wallberg, Ola

    2017-01-01

    Cellulase enzymes have been reported to contribute with a significant share of the total costs and greenhouse gas emissions of lignocellulosic ethanol production today. A potential future alternative to purchasing enzymes from an off-site manufacturer is to integrate enzyme and ethanol production, using microorganisms and part of the lignocellulosic material as feedstock for enzymes. This study modelled two such integrated process designs for ethanol from logging residues from spruce production, and compared it to an off-site case based on existing data regarding purchased enzymes. Greenhouse gas emissions and primary energy balances were studied in a life-cycle assessment, and cost performance in a techno-economic analysis. The base case scenario suggests that greenhouse gas emissions per MJ of ethanol could be significantly lower in the integrated cases than in the off-site case. However, the difference between the integrated and off-site cases is reduced with alternative assumptions regarding enzyme dosage and the environmental impact of the purchased enzymes. The comparison of primary energy balances did not show any significant difference between the cases. The minimum ethanol selling price, to reach break-even costs, was from 0.568 to 0.622 EUR L -1 for the integrated cases, as compared to 0.581 EUR L -1 for the off-site case. An integrated process design could reduce greenhouse gas emissions from lignocellulose-based ethanol production, and the cost of an integrated process could be comparable to purchasing enzymes produced off-site. This study focused on the environmental and economic assessment of an integrated process, and in order to strengthen the comparison to the off-site case, more detailed and updated data regarding industrial off-site enzyme production are especially important.

  8. Enzyme immunoassay for tenuazonic acid in apple and tomato products.

    PubMed

    Gross, Madeleine; Curtui, Valeriu; Ackermann, Yvonne; Latif, Hadri; Usleber, Ewald

    2011-12-14

    The Alternaria mycotoxin tenuazonic acid was derivatized with succinic anhydride and conjugated to keyhole limpet hemocyanin (KLH) and to horseradish peroxidase (HRP), respectively. The KLH conjugate was used to produce polyclonal antibodies in rabbits. A competitive direct enzyme immunoassay (EIA) for tenuazonic acid was established, which was moderately sensitive for tenuazonic acid [50% inhibition concentration (IC(50)): 320 ± 130 ng/mL] but strongly reacted with tenuazonic acid acetate (IC(50): 23.3 ± 7.5 ng/mL). Therefore, an optimized EIA protocol was established, which employed acetylation of standard and sample extract solutions. The mean standard curve detection limit (IC(30)) for tenuazonic acid acetate was 5.4 ± 2.0 ng/mL, enabling detection limits for tenuazonic acid in apple and tomato products of 25-50 ng/g (150 ng/g in tomato paste). Recoveries in a concentration range of 50-2000 ng/g were 60-130% in apple juice and tomato juice and 40-150% in other tomato products. Tenuazonic acid was detected in apple juice and tomato products from German retail shops at levels of 50-200 ng/g. In conclusion, this novel EIA for tenuazonic acid could be useful within a screening program for Alternaria mycotoxins in food.

  9. Characterization of enzymes in the oxidation of 1,2-propanediol to D: -(-)-lactic acid by Gluconobacter oxydans DSM 2003.

    PubMed

    Wei, Liujing; Yang, Xuepeng; Gao, Keliang; Lin, Jinping; Yang, Shengli; Hua, Qiang; Wei, Dongzhi

    2010-09-01

    Although Gluconobacter oxydans can convert 1,2-propanediol to D: -(-)-lactic acid, the enzyme(s) responsible for the conversion has remain unknown. In this study, the membrane-bound alcohol dehydrogenase (ADH) of Gluconobacter oxydans DSM 2003 was purified and confirmed to be essential for the process of D: -(-)-lactic acid production by gene knockout and complementation studies. A 25 percent decrease in D: -(-)-lactic acid production was found for the aldehyde dehydrogenase (ALDH) deficient strain of G. oxydans DSM 2003, indicating that this enzyme is involved in the reaction but not necessary. It is the first report that reveals the function of ADH and ALDH in the biooxidation of 1,2-propanediol to D: -(-)-lactic acid by G. oxydans DSM 2003.

  10. Nanoarmoring of Enzymes by Interlocking in Cellulose Fibers With Poly(Acrylic Acid).

    PubMed

    Riccardi, Caterina M; Kasi, Rajeswari M; Kumar, Challa V

    2017-01-01

    A simple method for interlocking glucose oxidase (GOx) and horseradish peroxidase (HRP) in cellulose fibers using poly(acrylic acid) (PAA) as an armor around the enzyme, without any need for activation of the cellulose support, is reported here. The resulting enzyme paper is an inexpensive, stable, simple, wearable, and washable biosensor. PAA functions as a multifunctional tether to interlock the enzyme molecules around the paper fibers so that the enzymes are protected against thermal/chemical denaturation and not released from the paper when washed with a detergent. The decreased conformational entropy of the interlocked enzyme protected by the nanoarmor is likely responsible for increased enzyme stability to heat and chemical denaturants (retained ≥70 percent enzyme activity after washing with urea or SDS for 30min), and the polymer protects the enzyme against inactivation by proteases, bacteria, inhibitors, etc. The kinetics of the interlocked enzyme were similar to that of the enzyme in solution. The V max was 6(±0.5)mM per minute before washing, then increased slightly to 9(±1.4)mM per minute after washing with water. The K m was 22(±6.4mM), which was slightly higher compared to GOx in solution (25-27mM). Because the surface area of the paper does not limit the enzyme loading, about 20% of enzyme was successfully loaded onto the paper (0.2g enzyme per gram of paper), and ≥95% of the enzyme was retained after washing. Interlocking works with other enzymes such as laccase, where ≥60% of the enzyme activity is retained. This novel methodology provides a low cost, simple, modular approach of achieving high enzyme loadings in ordinary filter paper, not limited by cellulose surface area, and there has been no need for complex methods of enzyme engineering or toxic methods of activation of the solid support to prepare highly active biocatalysts. © 2017 Elsevier Inc. All rights reserved.

  11. Contrasting features of urea cycle disorders in human patients and knockout mouse models.

    PubMed

    Deignan, Joshua L; Cederbaum, Stephen D; Grody, Wayne W

    2008-01-01

    The urea cycle exists for the removal of excess nitrogen from the body. Six separate enzymes comprise the urea cycle, and a deficiency in any one of them causes a urea cycle disorder (UCD) in humans. Arginase is the only urea cycle enzyme with an alternate isoform, though no known human disorder currently exists due to a deficiency in the second isoform. While all of the UCDs usually present with hyperammonemia in the first few days to months of life, most disorders are distinguished by a characteristic profile of plasma amino acid alterations that can be utilized for diagnosis. While enzyme assay is possible, an analysis of the underlying mutation is preferable for an accurate diagnosis. Mouse models for each of the urea cycle disorders exist (with the exception of NAGS deficiency), and for almost all of them, their clinical and biochemical phenotypes rather closely resemble the phenotypes seen in human patients. Consequently, all of the current mouse models are highly useful for future research into novel pharmacological and dietary treatments and gene therapy protocols for the management of urea cycle disorders.

  12. Characterization of Two Streptomyces Enzymes That Convert Ferulic Acid to Vanillin

    PubMed Central

    Yang, Wenwen; Tang, Hongzhi; Ni, Jun; Wu, Qiulin; Hua, Dongliang; Tao, Fei; Xu, Ping

    2013-01-01

    Production of flavors from natural substrates by microbial transformation has become a growing and expanding field of study over the past decades. Vanillin, a major component of vanilla flavor, is a principal flavoring compound used worldwide. Streptomyces sp. strain V-1 is known to be one of the most promising microbial producers of natural vanillin from ferulic acid. Although identification of the microbial genes involved in the biotransformation of ferulic acid to vanillin has been previously reported, purification and detailed characterization of the corresponding enzymes with important functions have rarely been studied. In this study, we isolated and identified 2 critical genes, fcs and ech, encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively, which are involved in the vanillin production from ferulic acid. Both genes were heterologously expressed in Escherichia coli, and the resting cell reactions for converting ferulic acid to vanillin were performed. The corresponding crucial enzymes, Fcs and Ech, were purified for the first time and the enzymatic activity of each purified protein was studied. Furthermore, Fcs was comprehensively characterized, at an optimal pH of 7.0 and temperature of 30°C. Kinetic constants for Fcs revealed the apparent K m, k cat, and V max values to be 0.35 mM, 67.7 s−1, and 78.2 U mg−1, respectively. The catalytic efficiency (k cat/K m) value of Fcs was 193.4 mM−1 s−1 for ferulic acid. The characterization of Fcs and Ech may be helpful for further research in the field of enzymatic engineering and metabolic regulation. PMID:23840666

  13. Experiment K-7-21: Effect of Microgravity on 1: Metabolic Enzymes of Type 1 and Type 2 Muscle Fibers, and on 2: Metabolic Enzymes, Neurotransmitter Amino Acids, and Neurotransmitter Associated Enzymes in Selected Regions of the Central Nervous System. Part 2; The Distribution of Selected Enzymes and Amino Acids in the Hippocampal Formation

    NASA Technical Reports Server (NTRS)

    Lowry, O. H.; Krasnov, I.; Ilyina-Kakueva, E. I.; Nemeth, P. M.; McDougal, D. B., Jr.; Choksi, R.; Carter, J. G.; Chi, M. M. Y.; Manchester, J. K.; Pusateri, M. E.

    1994-01-01

    Six key metabolic enzymes plus glutaminase and glutamate decarboxylase, as well as glutamate, aspartate and GABA, were measured in 11 regions of the hippocampal formation of synchronous, flight and tail suspension rats. Major differences were observed in the normal distribution patterns of each enzyme and amino acid, but no substantive effects of either microgravity or tail suspension on these patterns were clearly demonstrated.

  14. Immbolization of uricase enzyme in Langmuir and Langmuir-Blodgett films of fatty acids: possible use as a uric acid sensor.

    PubMed

    Zanon, Nathaly C M; Oliveira, Osvaldo N; Caseli, Luciano

    2012-05-01

    Preserving the enzyme structure in solid films is key for producing various bioelectronic devices, including biosensors, which has normally been performed with nanostructured films that allow for control of molecular architectures. In this paper, we investigate the adsorption of uricase onto Langmuir monolayers of stearic acid (SA), and their transfer to solid supports as Langmuir-Blodgett (LB) films. Structuring of the enzyme in β-sheets was preserved in the form of 1-layer LB film, which was corroborated with a higher catalytic activity than for other uricase-containing LB film architectures where the β-sheets structuring was not preserved. The optimized architecture was also used to detect uric acid within a range covering typical concentrations in the human blood. The approach presented here not only allows for an optimized catalytic activity toward uric acid but also permits one to explain why some film architectures exhibit a superior performance. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Improved pretreatment of lignocellulosic biomass using enzymatically-generated peracetic acid.

    PubMed

    Yin, DeLu Tyler; Jing, Qing; AlDajani, Waleed Wafa; Duncan, Shona; Tschirner, Ulrike; Schilling, Jonathan; Kazlauskas, Romas J

    2011-04-01

    Release of sugars from lignocellulosic biomass is inefficient because lignin, an aromatic polymer, blocks access of enzymes to the sugar polymers. Pretreatments remove lignin and disrupt its structure, thereby enhancing sugar release. In previous work, enzymatically generated peracetic acid was used to pretreat aspen wood. This pretreatment removed 45% of the lignin and the subsequent saccharification released 97% of the sugars remaining after pretreatment. In this paper, the amount of enzyme needed is reduced tenfold using first, an improved enzyme variant that makes twice as much peracetic acid and second, a two-phase reaction to generate the peracetic acid, which allows enzyme reuse. In addition, the eight pretreatment cycles are reduced to only one by increasing the volume of peracetic acid solution and increasing the temperature to 60 °C and the reaction time to 6h. For the pretreatment step, the weight ratio of peracetic acid to wood determines the amount of lignin removed. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. A soluble NADH-dependent fumarate reductase in the reductive tricarboxylic acid cycle of Hydrogenobacter thermophilus TK-6.

    PubMed

    Miura, Akane; Kameya, Masafumi; Arai, Hiroyuki; Ishii, Masaharu; Igarashi, Yasuo

    2008-11-01

    Fumarate reductase (FRD) is an enzyme that reduces fumarate to succinate. In many organisms, it is bound to the membrane and uses electron donors such as quinol. In this study, an FRD from a thermophilic chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6, was purified and characterized. FRD activity using NADH as an electron donor was not detected in the membrane fraction but was found in the soluble fraction. The purified enzyme was demonstrated to be a novel type of FRD, consisting of five subunits. One subunit showed high sequence identity to the catalytic subunits of known FRDs. Although the genes of typical FRDs are assembled in a cluster, the five genes encoding the H. thermophilus FRD were distant from each other in the genome. Furthermore, phylogenetic analysis showed that the H. thermophilus FRD was located in a distinct position from those of known soluble FRDs. This is the first report of a soluble NADH-dependent FRD in Bacteria and of the purification of a FRD that operates in the reductive tricarboxylic acid cycle.

  17. Reduction of α-oxo carboxyylic acids by pigeon liver `malic' enzyme

    PubMed Central

    Tang, Chung L.; Hsu, Robert Y.

    1973-01-01

    1. Pigeon liver `malic' enzyme [l-malate–NADP+ oxidoreductase (decarboxylating); EC 1.1.1.40] was shown to catalyse the reductase reaction: [Formula: see text] l-Malate was identified as the reaction product, and was formed in stoicheiometric amount. 2. In addition to oxaloacetate and pyruvate, a number of other α-oxo carboxylic acids were also reduced. PMID:4764261

  18. Identification of enzymes involved in oxidation of phenylbutyrate.

    PubMed

    Palir, Neža; Ruiter, Jos P N; Wanders, Ronald J A; Houtkooper, Riekelt H

    2017-05-01

    In recent years the short-chain fatty acid, 4-phenylbutyrate (PB), has emerged as a promising drug for various clinical conditions. In fact, PB has been Food and Drug Administration-approved for urea cycle disorders since 1996. PB is more potent and less toxic than its metabolite, phenylacetate (PA), and is not just a pro-drug for PA, as was initially assumed. The metabolic pathway of PB, however, has remained unclear. Therefore, we set out to identify the enzymes involved in the β-oxidation of PB. We used cells deficient in specific steps of fatty acid β-oxidation and ultra-HPLC to measure which enzymes were able to convert PB or its downstream products. We show that the first step in PB oxidation is catalyzed solely by the enzyme, medium-chain acyl-CoA dehydrogenase. The second (hydration) step can be catalyzed by all three mitochondrial enoyl-CoA hydratase enzymes, i.e., short-chain enoyl-CoA hydratase, long-chain enoyl-CoA hydratase, and 3-methylglutaconyl-CoA hydratase. Enzymes involved in the third step include both short- and long-chain 3-hydroxyacyl-CoA dehydrogenase. The oxidation of PB is completed by only one enzyme, i.e., long-chain 3-ketoacyl-CoA thiolase. Taken together, the enzymatic characteristics of the PB degradative pathway may lead to better dose finding and limiting the toxicity of this drug. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  19. 2-Hexadecynoic acid inhibits plasmodial FAS-II enzymes and arrests erythrocytic and liver stage Plasmodium infections.

    PubMed

    Tasdemir, Deniz; Sanabria, David; Lauinger, Ina L; Tarun, Alice; Herman, Rob; Perozzo, Remo; Zloh, Mire; Kappe, Stefan H; Brun, Reto; Carballeira, Néstor M

    2010-11-01

    Acetylenic fatty acids are known to display several biological activities, but their antimalarial activity has remained unexplored. In this study, we synthesized the 2-, 5-, 6-, and 9-hexadecynoic acids (HDAs) and evaluated their in vitro activity against erythrocytic (blood) stages of Plasmodium falciparum and liver stages of Plasmodium yoelii infections. Since the type II fatty acid biosynthesis pathway (PfFAS-II) has recently been shown to be indispensable for liver stage malaria parasites, the inhibitory potential of the HDAs against multiple P. falciparum FAS-II (PfFAS-II) elongation enzymes was also evaluated. The highest antiplasmodial activity against blood stages of P. falciparum was displayed by 5-HDA (IC(50) value 6.6 μg/ml), whereas the 2-HDA was the only acid arresting the growth of liver stage P. yoelii infection, in both flow cytometric assay (IC(50) value 2-HDA 15.3 μg/ml, control drug atovaquone 2.5 ng/ml) and immunofluorescence analysis (IC(50) 2-HDA 4.88 μg/ml, control drug atovaquone 0.37 ng/ml). 2-HDA showed the best inhibitory activity against the PfFAS-II enzymes PfFabI and PfFabZ with IC(50) values of 0.38 and 0.58 μg/ml (IC(50) control drugs 14 and 30 ng/ml), respectively. Enzyme kinetics and molecular modeling studies revealed valuable insights into the binding mechanism of 2-HDA on the target enzymes. All HDAs showed in vitro activity against Trypanosoma brucei rhodesiense (IC(50) values 3.7-31.7 μg/ml), Trypanosoma cruzi (only 2-HDA, IC(50) 20.2 μg/ml), and Leishmania donovani (IC(50) values 4.1-13.4 μg/ml) with generally low or no significant toxicity on mammalian cells. This is the first study to indicate therapeutic potential of HDAs against various parasitic protozoa. It also points out that the malarial liver stage growth inhibitory effect of the 2-HDA may be promoted via PfFAS-II enzymes. The lack of cytotoxicity, lipophilic nature, and calculated pharmacokinetic properties suggests that 2-HDA could be a useful compound to

  20. 2-Hexadecynoic Acid Inhibits Plasmodial FAS-II Enzymes and Arrest Erythrocytic and Liver Stage Plasmodium Infections

    PubMed Central

    Tasdemir, Deniz; Sanabria, David; Lauinger, Ina L.; Tarun, Alice; Herman, Rob; Perozzo, Remo; Zloh, Mire; Kappe, Stefan H.; Brun, Reto; Carballeira, Néstor M.

    2010-01-01

    Acetylenic fatty acids are known to display several biological activities, but their antimalarial activity has remained unexplored. In this study, we synthesized the 2-, 5-, 6-, and 9-hexadecynoic acids (HDAs) and evaluated their in vitro activity against erythrocytic (blood) stages of Plasmodium falciparum and liver stages of P. yoelii infections. Since the type II fatty acid biosynthesis pathway (PfFAS-II) has recently been shown to be indispensable for liver stage malaria parasites, the inhibitory potential of the HDAs against multiple P. falciparum FAS-II (PfFAS-II) elongation enzymes was also evaluated. The highest antiplasmodial activity against blood stages of P. falciparum was displayed by 5-HDA (IC50 value 6.6. μg/ml), whereas the 2-HDA was the only acid arresting the growth of liver stage P. yoelii infection, in both flow cytometric assay (IC50 value 2-HDA 15.3 μg/ml, control drug atovaquone 2.5 ng/ml) and immunofluorescense analysis (IC50 2-HDA 4.88 μg/ml, control drug atovaquone 0.37 ng/ml). 2-HDA showed the best inhibitory against the PfFAS-II enzymes PfFabI and PfFabZ with IC50 values of 0.38 and 0.58 μg/ml (IC50 control drugs 14 and 30 ng/ml) respectively. Enzyme kinetics and molecular modeling studies revealed valuable insights into the binding mechanism of 2-HDA on the target enzymes. All HDAs showed in vitro activity against Trypanosoma brucei rhodesiense (IC50 values 3.7–31.7 μg/ml), Trypanosoma cruzi (only 2-HDA, IC50 20.2 μg/ml), and Leishmania donovani (IC50 values 4.1–13.4 μg/ml) with generally low or no significant toxicity on mammalian cells. This is the first study to indicate therapeutic potential of HDAs against various parasitic protozoa. It also points out that the malarial liver stage growth inhibitory effect of the 2-HDA may be promoted via PfFAS-II enzymes. The lack of cytotoxicity, lipophilic nature and calculated pharmacokinetic properties suggest that 2-HDA could be a useful compound to study the interaction of fatty

  1. Phosphate starvation promoted the accumulation of phenolic acids by inducing the key enzyme genes in Salvia miltiorrhiza hairy roots.

    PubMed

    Liu, Lin; Yang, DongFeng; Liang, TongYao; Zhang, HaiHua; He, ZhiGui; Liang, ZongSuo

    2016-09-01

    Phosphate starvation increased the production of phenolic acids by inducing the key enzyme genes in a positive feedback pathway in Saliva miltiorrhiza hairy roots. SPX may be involved in this process. Salvia miltiorrhiza is a wildly popular traditional Chinese medicine used for the treatment of coronary heart diseases and inflammation. Phosphate is an essential plant macronutrient that is often deficient, thereby limiting crop yield. In this study, we investigated the effects of phosphate concentration on the biomass and accumulation of phenolic acid in S. miltiorrhiza. Results show that 0.124 mM phosphate was favorable for plant growth. Moreover, 0.0124 mM phosphate was beneficial for the accumulation of phenolic acids, wherein the contents of danshensu, caffeic acid, rosmarinic acid, and salvianolic acid B were, respectively, 2.33-, 1.02-, 1.68-, and 2.17-fold higher than that of the control. By contrast, 12.4 mM phosphate inhibited the accumulation of phenolic acids. The key enzyme genes in the phenolic acid biosynthesis pathway were investigated to elucidate the mechanism of phosphate starvation-induced increase of phenolic acids. The results suggest that phosphate starvation induced the gene expression from the downstream pathway to the upstream pathway, i.e., a feedback phenomenon. In addition, phosphate starvation response gene SPX (SYG1, Pho81, and XPR1) was promoted by phosphate deficiency (0.0124 mM). We inferred that SPX responded to phosphate starvation, which then affected the expression of later responsive key enzyme genes in phenolic acid biosynthesis, resulting in the accumulation of phenolic acids. Our findings provide a resource-saving and environmental protection strategy to increase the yield of active substance in herbal preparations. The relationship between SPX and key enzyme genes and the role they play in phenolic acid biosynthesis during phosphate deficiency need further studies.

  2. Investigation on uric acid biosensor model for enzyme layer thickness for the application of arthritis disease diagnosis.

    PubMed

    Parthasarathy, P; Vivekanandan, S

    2018-12-01

    Uric acid biosensors for arthritis disease has been developed for the specific selection of uricase enzyme film thickness coated over the TiO 2 -CeO 2 nano-composite matrix is modelled mathematically. This model is purely based on R-diffusion conditions with irreversible first-order catalytic reactions. By arithmetical method, the impact of the thickness of enzyme layer on the current response of the biosensor was explored. This article displays a structure for choice of the enzyme layer thickness, guaranteeing the adequately stable sensitivity of a biosensor in a required extent of the maximal enzymatic rate. The numerical outcomes showed subjective and sensible quantitative information for oxidation current due to uric acid also shows the maximum change in the biosensor current response due to the change in membrane thickness, which will be more suitable for uric acid biosensor for the application of arthritis disease diagnosis.

  3. Isolation and Compositional Analysis of a CP12-Associated Complex of Calvin Cycle Enzymes from Nicotiana tabacum

    USDA-ARS?s Scientific Manuscript database

    CP12 is a small intrinsically unstructured protein that forms a multiprotein complex with two Calvin Cycle enzymes, phosphoribulokinase (PRK) and NAD(P)-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The complex can be reconstituted in vitro from recombinant proteins under conditions t...

  4. SURFACE DEGRADATION OF COMPOSITE RESINS BY ACIDIC MEDICINES AND pH-CYCLING

    PubMed Central

    Valinoti, Ana Carolina; Neves, Beatriz Gonçalves; da Silva, Eduardo Moreira; Maia, Lucianne Cople

    2008-01-01

    This study evaluated the effects of acidic medicines (Dimetapp® and Claritin®), under pH-cycling conditions, on the surface degradation of four composite resins (microhybrid: TPH, Concept, Opallis and Nanofilled: Supreme). Thirty disc-shaped specimens (Ø = 5.0 mm / thickness = 2.0 mm) of each composite were randomly assigned to 3 groups (n = 10): a control and two experimental groups, according to the acidic medicines evaluated. The specimens were finished and polished with aluminum oxide discs, and the surface roughness was measured by using a profilometer. After the specimens were submitted to a pH-cycling regimen and immersion in acidic medicines for 12 days, the surface roughness was measured again. Two specimens for each material and group were analyzed by scanning electron microscopy (SEM) before and after pH-cycling. Data were analyzed by the Student's-t test, ANOVA, Duncan's multiple range test and paired t-test (α=0.05). Significant increase in roughness was found only for TPH in the control group and TPH and Supreme immersed in Claritin® (p<0.05). SEM analyses showed that the 4 composite resins underwent erosion and surface degradation after being subjected to the experimental conditions. In conclusion, although the roughness was slightly affected, the pH-cycling and acidic medicines caused surface degradation of the composite resins evaluated. Titratable acidity seemed to play a more crucial role on surface degradation of composite resins than pH. PMID:19089257

  5. Unfolding and inactivation during thermal denaturation of an enzyme that exhibits phytase and acid phosphatase activities.

    PubMed

    Wang, Xiao-Yun; Meng, Fan-Guo; Zhou, Hai-Meng

    2004-03-01

    The thermostability of an enzyme that exhibits phytase and acid phosphatase activities was studied. Kinetics of inactivation and unfolding during thermal denaturation of the enzyme were compared. The loss of phytase activity on thermal denaturation is most suggestive of a reversible process. As for acid phosphatase activities, an interesting phenomenon was observed; there are two phases in thermal inactivation: when the temperature was between 45 and 50 degrees C, the thermal inactivation could be characterized as an irreversible inactivation which had some residual activity and when the temperature was above 55 degrees C, the thermal inactivation could be characterized as an irreversible process which had no residual activity. The microscopic rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method [Adv. Enzymol. Relat. Areas Mol. Biol. 61 (1988) 381]. Fluorescence analyses indicate that when the enzyme was treated at temperatures below 60 degrees C for 60 min, the conformation of the enzyme had no detectable change; when the temperatures were above 60 degrees C, some fluorescence red-shift could be observed with a decrease in emission intensity. The inactivation rates (k(+0)) of free enzymes were faster than those of conformational changes during thermal denaturation at the same temperature. The rapid inactivation and slow conformational changes of phytase during thermal denaturation suggest that inactivation occurs before significant conformational changes of the enzyme, and the active site of this enzyme is situated in a relatively fragile region which makes the active site more flexible than the molecule as a whole.

  6. Comparison of the effects of gemfibrozil and clofibric acid on peroxisomal enzymes and cholesterol synthesis of rat hepatocytes.

    PubMed

    Hashimoto, F; Taira, S; Hayashi, H

    1998-11-01

    We studied whether the peroxisomal proliferation, induction of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and activation of cholesterol synthesis by gemfibrozil shown in whole body (Hashimoto F., Ishikawa T., Hamada S. and Hayashi H., Biochemical. Pharm., 49, 1213-1221 (1995)) is also detected at a culture cell level, and we made a comparative analysis of the effects of clofibric acid. Gemfibrozil at 0.25 mM increased the activity of some peroxisomal enzymes (catalase and the cyanide-insensitive fatty acyl-CoA oxidizing system) after incubation for 72 h. However, contrary to whole body experiments, gemfibrozil decreased the activity of HMG-CoA reductase and cholesterol synthesis from [14C]acetate. At 1 mM, gemfibrozil decreased not only the activity of HMG-CoA reductase and cholesterol synthesis, but also the protein content of the cells and peroxisomal enzyme activity, indicating nonspecific inhibition at this concentration. Clofibric acid (0.25 and 1 mM) increased the activity of peroxisomal enzymes, but decreased the activity of HMG-CoA reductase and cholesterol synthesis. With respect to the direct effect on HMG-CoA reductase in the cell homogenate, gemfibrozil at 0.25 mm did not affect the activity, but it clearly inhibited the activity at 2 mM and above. Clofibric acid at 2 mM hardly affected the activity, but it clearly decreased the activity at 5 mM and over. That is, gemfibrozil directly inhibited the activity more strongly than clofibric acid. The direct inhibition of the enzyme itself required higher concentrations of both agents than did inhibition at the culture cell level. These results suggest that the cytotoxicity of gemfibrozil is greater than that of clofibric acid, and that gemfibrozil, as well as clofibric acid, can induce peroxisomal enzymes in the culture cell level. In contrast to whole body results, gemfibrozil may suppress cholesterol synthesis from [14C]acetate through the inhibition of HMG-CoA reductase at the culture

  7. Induction of Shikimic Acid Pathway Enzymes by Light in Suspension Cultured Cells of Parsley (Petroselinum crispum) 1

    PubMed Central

    McCue, Kent F.; Conn, Eric E.

    1990-01-01

    Light treatment of suspension cultured cells of parsley (Petroselinum crispum) was shown to increase the activity of the shikimic acid pathway enzyme, 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) synthase (EC 4.1.2.15). DAHP synthase activity was assayed for two isoforms, DS-Mn and DS-Co (RJ Ganson, TA d'Amato, RA Jensen [1986] Plant Physiol 82: 203-210). Light increased the enzymatic activity of the plastidic isoform DS-Mn as much as 2-fold, averaging 1.6-fold with >95% confidence. The cytosolic isoform DS-Co was unaffected. Cycloheximide and actinomycin D, translational and transcriptional inhibitors, respectively, both reversed induction of DS-Mn by light suggesting transcriptional regulation of the gene. Chorismate mutase activity was assayed for the two isoforms CM I and CM II (BK Singh, JA Connelly, EE Conn [1985] Arch Biochem Biophys 243: 374-384). Treatment by light did not significantly affect either chorismate mutase isoform. The ratio of the two chorismate mutase isoforms changed during the growth cycle, with an increase in the ratio of plastidic to cytosolic isoforms occurring towards the end of logarithmic growth. PMID:16667741

  8. Enzymatic production of ferulic acid from defatted rice bran by using a combination of bacterial enzymes.

    PubMed

    Uraji, Misugi; Kimura, Masayo; Inoue, Yosikazu; Kawakami, Kayoko; Kumagai, Yuya; Harazono, Koichi; Hatanaka, Tadashi

    2013-11-01

    Ferulic acid (FA), which is present in the cell walls of some plants, is best known for its antioxidant property. By combining a commercial enzyme that shows FA esterase activity with several Streptomyces carbohydrate-hydrolyzing enzymes, we succeeded in enhancing the enzymatic production of FA from defatted rice bran. In particular, the combination of three xylanases, an α-L-arabinofuranosidase, and an acetyl xylan esterase from Streptomyces spp. produced the highest increase in the amount of released FAs among all the enzymes in the Streptomyces enzymes library. This enzyme combination also had an effect on FA production from other biomasses, such as raw rice bran, wheat bran, and corncob.

  9. An update on the Enzyme Portal: an integrative approach for exploring enzyme knowledge

    PubMed Central

    Onwubiko, J.; Zaru, R.; Rosanoff, S.; Antunes, R.; Bingley, M.; Watkins, X.; O'Donovan, C.; Martin, M. J.

    2017-01-01

    Abstract Enzymes are a key part of life processes and are increasingly important for various areas of research such as medicine, biotechnology, bioprocessing and drug research. The goal of the Enzyme Portal is to provide an interface to all European Bioinformatics Institute (EMBL-EBI) data about enzymes (de Matos, P., et al., (2013), BMC Bioinformatics, 14 (1), 103). These data include enzyme function, sequence features and family classification, protein structure, reactions, pathways, small molecules, diseases and the associated literature. The sources of enzyme data are: the UniProt Knowledgebase (UniProtKB) (UniProt Consortium, 2015), the Protein Data Bank in Europe (PDBe), (Valenkar, S., et al., Nucleic Acids Res.2016; 44, D385–D395) Rhea—a database of enzyme-catalysed reactions (Morgat, A., et al., Nucleic Acids Res. 2015; 43, D459-D464), Reactome—a database of biochemical pathways (Fabregat, A., et al., Nucleic Acids Res. 2016; 44, D481–D487), IntEnz—a resource with enzyme nomenclature information (Fleischmann, A., et al., Nucleic Acids Res. 2004 32, D434–D437) and ChEBI (Hastings, J., et al., Nucleic Acids Res. 2013) and ChEMBL (Bento, A. P., et al., Nucleic Acids Res. 201442, 1083–1090)—resources which contain information about small-molecule chemistry and bioactivity. This article describes the redesign of Enzyme Portal and the increased functionality added to maximise integration and interpretation of these data. Use case examples of the Enzyme Portal and the versatile workflows its supports are illustrated. We welcome the suggestion of new resources for integration. PMID:28158609

  10. An update on the Enzyme Portal: an integrative approach for exploring enzyme knowledge.

    PubMed

    Pundir, S; Onwubiko, J; Zaru, R; Rosanoff, S; Antunes, R; Bingley, M; Watkins, X; O'Donovan, C; Martin, M J

    2017-03-01

    Enzymes are a key part of life processes and are increasingly important for various areas of research such as medicine, biotechnology, bioprocessing and drug research. The goal of the Enzyme Portal is to provide an interface to all European Bioinformatics Institute (EMBL-EBI) data about enzymes (de Matos, P., et al. , (2013), BMC Bioinformatics , (1), 103). These data include enzyme function, sequence features and family classification, protein structure, reactions, pathways, small molecules, diseases and the associated literature. The sources of enzyme data are: the UniProt Knowledgebase (UniProtKB) (UniProt Consortium, 2015), the Protein Data Bank in Europe (PDBe), (Valenkar, S., et al ., Nucleic Acids Res. 2016; , D385-D395) Rhea-a database of enzyme-catalysed reactions (Morgat, A., et al .,  Nucleic Acids Res.  2015; , D459-D464), Reactome-a database of biochemical pathways (Fabregat, A., et al ., Nucleic Acids Res. 2016;  , D481-D487), IntEnz-a resource with enzyme nomenclature information (Fleischmann, A., et al ., Nucleic Acids Res.  2004 , D434-D437) and ChEBI (Hastings, J., et al .,  Nucleic Acids Res. 2013) and ChEMBL (Bento, A. P., et al ., Nucleic Acids Res.  2014 , 1083-1090)-resources which contain information about small-molecule chemistry and bioactivity. This article describes the redesign of Enzyme Portal and the increased functionality added to maximise integration and interpretation of these data. Use case examples of the Enzyme Portal and the versatile workflows its supports are illustrated. We welcome the suggestion of new resources for integration. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

  11. The Effects of Subacute Exposure of Peracetic Acid on Lipid Peroxidation and Hepatic Enzymes in Wistar Rats

    PubMed Central

    Marjani, Abdoljalal; Golalipour, Mohammad J.; Gharravi, Anneh M.

    2010-01-01

    Objectives This study was undertaken to determine the effect of subacute exposure of peracetic acid on lipid peroxidation and hepatic enzymes in Wistar rats. Methods 48 male animals in Treatment Group I, II and III received 0.2%, 2% and 20% peracetic acid daily for 2 and 4 weeks. Results Serum malondialdehyde increased and Alanine Transaminase and Aspartate Transaminase decreased significantly in groups 2 and 3, compared to the control group. The malondialdehyde, Alanine Transaminase and Aspartate Transaminase with 0.2% and 2% doses of peracetic acid for 2 weeks do not lead to the alteration of malondialdehyde and enzyme activities. Conclusion This study demonstrated that the enhancement of malondialdehyde could provide an oxidative damage induced by disinfectant peroxidation at 20% and 2% doses at 2 and 4 weeks. The consumption of peroxidation with 20% for 2 weeks and 2% for 4 weeks can cause the increase of malondialdehyde and the decrease of enzyme activities, respectively. PMID:22043353

  12. Aedes aegypti juvenile hormone acid methyl transferase, the ultimate enzyme in the biosynthetic pathway of juvenile hormone III, exhibits substrate control

    USDA-ARS?s Scientific Manuscript database

    We report on the cloning, sequencing, characterization, 3D modeling and docking of Aedes aegypti juvenile hormone acid methyl transferase (AeaJHAMT), the enzyme that converts juvenile hormone acid (JHA) into juvenile hormone (JH). Purified recombinant AeaJHAMT was extensively characterized for enzym...

  13. Oxaloacetate-to-malate conversion by mineral photoelectrochemistry: implications for the viability of the reductive tricarboxylic acid cycle in prebiotic chemistry

    NASA Astrophysics Data System (ADS)

    Guzman, Marcelo I.; Martin, Scot T.

    2008-10-01

    The carboxylic acids produced by the reductive tricarboxylic acid (rTCA) cycle are possibly a biosynthetic core of initial life, although several steps such as the reductive kinetics of oxaloacetate (OAA) to malate (MA) are problematic by conventional chemical routes. In this context, we studied the kinetics of this reaction as promoted by ZnS mineral photoelectrochemistry. The quantum efficiency φMA of MA production from the photoelectrochemical reduction of OAA followed φMA=0.13 [OAA] (2.1×10-3+[OAA])-1 and was independent of temperature (5 to 50°C). To evaluate the importance of this forward rate under a prebiotic scenario, we also studied the temperature-dependent rate of the backward thermal decarboxylation of OAA to pyruvate (PA), which followed an Arrhenius behavior as log (k-2)=11.74 4956/T, where k-2 is in units of s-1. These measured rates were employed in conjunction with the indirectly estimated carboxylation rate of PA to OAA to assess the possible importance of mineral photoelectrochemistry in the conversion of OAA to MA under several scenarios of prebiotic conditions on early Earth. As an example, our analysis shows that there is 90% efficiency with a forward velocity of 3 yr/cycle for the OAA→MA step of the rTCA cycle at 280 K. Efficiency and velocity both decrease for increasing temperature. These results suggest high viability for mineral photoelectrochemistry as an enzyme-free engine to drive the rTCA cycle through the early aeons of early Earth, at least for the investigated OAA→MA step.

  14. Contrasting Features of Urea Cycle Disorders in Human Patients and Knockout Mouse Models

    PubMed Central

    Deignan, Joshua L.; Cederbaum, Stephen D.; Grody, Wayne W.

    2009-01-01

    The urea cycle exists for the removal of excess nitrogen from the body. Six separate enzymes comprise the urea cycle, and a deficiency in any one of them causes a urea cycle disorder (UCD) in humans. Arginase is the only urea cycle enzyme with an alternate isoform, though no known human disorder currently exists due to a deficiency in the second isoform. While all of the UCDs usually present with hyperammonemia in the first few days to months of life, most disorders are distinguished by a characteristic profile of plasma amino acid alterations that can be utilized for diagnosis. While enzyme assay is possible, an analysis of the underlying mutation is preferable for an accurate diagnosis. Mouse models for each of the urea cycle disorders exist (with the exception of NAGS deficiency), and for almost all of them, their clinical and biochemical phenotypes rather closely resemble the phenotypes seen in human patients. Consequently, all of the current mouse models are highly useful for future research into novel pharmacological and dietary treatments and gene therapy protocols for the management of urea cycle disorders. PMID:17933574

  15. Problem-Based Test: Replication of Mitochondrial DNA during the Cell Cycle

    ERIC Educational Resources Information Center

    Setalo, Gyorgy, Jr.

    2013-01-01

    Terms to be familiar with before you start to solve the test: cell cycle, generation time, S-phase, cell culture synchronization, isotopic pulse-chase labeling, density labeling, equilibrium density-gradient centrifugation, buoyant density, rate-zonal centrifugation, nucleoside, nucleotide, kinase enzymes, polymerization of nucleic acids,…

  16. A Soluble NADH-Dependent Fumarate Reductase in the Reductive Tricarboxylic Acid Cycle of Hydrogenobacter thermophilus TK-6▿

    PubMed Central

    Miura, Akane; Kameya, Masafumi; Arai, Hiroyuki; Ishii, Masaharu; Igarashi, Yasuo

    2008-01-01

    Fumarate reductase (FRD) is an enzyme that reduces fumarate to succinate. In many organisms, it is bound to the membrane and uses electron donors such as quinol. In this study, an FRD from a thermophilic chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6, was purified and characterized. FRD activity using NADH as an electron donor was not detected in the membrane fraction but was found in the soluble fraction. The purified enzyme was demonstrated to be a novel type of FRD, consisting of five subunits. One subunit showed high sequence identity to the catalytic subunits of known FRDs. Although the genes of typical FRDs are assembled in a cluster, the five genes encoding the H. thermophilus FRD were distant from each other in the genome. Furthermore, phylogenetic analysis showed that the H. thermophilus FRD was located in a distinct position from those of known soluble FRDs. This is the first report of a soluble NADH-dependent FRD in Bacteria and of the purification of a FRD that operates in the reductive tricarboxylic acid cycle. PMID:18757546

  17. Chopper-controlled discharge life cycling studies on lead-acid batteries

    NASA Technical Reports Server (NTRS)

    Kraml, J. J.; Ames, E. P.

    1982-01-01

    State-of-the-art 6 volt lead-acid golf car batteries were tested. A daily charge/discharge cycling to failure points under various chopper controlled pulsed dc and continuous current load conditions was undertaken. The cycle life and failure modes were investigated for depth of discharge, average current chopper frequency, and chopper duty cycle. It is shown that battery life is primarily and inversely related to depth of discharge and discharge current. Failure mode is characterized by a gradual capacity loss with consistent evidence of cell element aging.

  18. Delta-aminolevulinic acid dehydratase enzyme activity in blood, brain, and liver of lead-dosed ducks

    USGS Publications Warehouse

    Dieter, M.P.; Finley, M.T.

    1979-01-01

    Mallard ducks were dosed with a single shotgun pellet (ca. 200 mg lead). After 1 month there was about 1 ppm lead in blood, 2.5 in liver, and 0.5 in brain. Lead-induced inhibition of delta-aminolevulinic acid dehydratase enzyme in blood and cerebellum was much greater than in cerebral hemisphere or liver and was strongly correlated with the lead concentration in these tissues. The cerebellar portion of the brain was more sensitive to delta-aminolevulinic acid dehydratase enzyme inhibition by lead than were the other tissues examined. There was also a greater increase in the glial cell marker enzyme, butyrylcholinesterase, in cerebellum than in cerebral hemisphere, suggesting that nonregenerating neuronal cells were destroyed by lead and replaced by glial cells in that portion of the brain. Even partial loss of cerebellar tissue is severely debilitating in waterfowl, because functions critical to survival such as visual, auditory, motor, and reflex responses are integrated at this brain center.

  19. The dogfish shark (Squalus acanthias) increases both hepatic and extrahepatic ornithine urea cycle enzyme activities for nitrogen conservation after feeding.

    PubMed

    Kajimura, Makiko; Walsh, Patrick J; Mommsen, Thomas P; Wood, Chris M

    2006-01-01

    Urea not only is utilized as a major osmolyte in marine elasmobranchs but also constitutes their main nitrogenous waste. This study investigated the effect of feeding, and thus elevated nitrogen intake, on nitrogen metabolism in the Pacific spiny dogfish Squalus acanthias. We determined the activities of ornithine urea cycle (O-UC) and related enzymes in liver and nonhepatic tissues. Carbamoyl phosphate synthetase III (the rate-limiting enzyme of the O-UC) activity in muscle is high compared with liver, and the activities in both tissues increased after feeding. The contribution of muscle to urea synthesis in the dogfish body appears to be much larger than that of liver when body mass is considered. Furthermore, enhanced activities of the O-UC and related enzymes (glutamine synthetase, ornithine transcarbamoylase, arginase) were seen after feeding in both liver and muscle and were accompanied by delayed increases in plasma urea, trimethylamine oxide, total free amino acids, alanine, and chloride concentrations, as well as in total osmolality. The O-UC and related enzymes also occurred in the intestine but showed little change after feeding. Feeding did not change the rate of urea excretion, indicating strong N retention after feeding. Ammonia excretion, which constituted only a small percentage of total N excretion, was raised in fed fish, while plasma ammonia did not change, suggesting that excess ammonia in plasma is quickly ushered into synthesis of urea or protein. In conclusion, we suggest that N conservation is a high priority in this elasmobranch and that feeding promotes ureogenesis and growth. Furthermore, exogenous nitrogen from food is converted into urea not only by the liver but also by the muscle and to a small extent by the intestine.

  20. Impact of repeated dry-wet cycles on soil greenhouse gas emissions, extracellular enzyme activity and nutrient cycling in a temperate forest

    NASA Astrophysics Data System (ADS)

    Leitner, Sonja; Zimmermann, Michael; Bockholt, Jan; Schartner, Markus; Brugner, Paul; Holtermann, Christian; Zechmeister-Boltenstern, Sophie

    2014-05-01

    Climate change research predicts that both frequency and intensity of weather extremes such as long drought periods and heavy rainfall events will increase in mid Europe over the next decades. Soil moisture is one of the major factors controlling microbial soil processes, and it has been widely agreed that feedback effects between altered precipitation and changed soil fluxes of the greenhouse gases CO2, CH4 and N2O could intensify climate change. In a field experiment in an Austrian beech forest, we established a precipitation manipulation experiment, which will be conducted for 3 years. We use roofs to exclude rainfall from reaching the forest soil and simulate drought periods, and a sprinkler system to simulate heavy rainfall events. We applied repeated dry-wet cycles in two intensities: one treatment received 6 cycles of 1 month drought followed by 75mm irrigation within 2 hours, and a parallel treatment received 3 cycles of 2 months drought followed by 150mm irrigation within 3 hours. We took soil samples 1 day before, 1 day after and 1 week after rewetting events and analyzed them for soil nutrients and extracellular enzyme activities. Soil fluxes of CO2, N2O and CH4 were constantly monitored with an automated flux chamber system, and environmental parameters were recorded via dataloggers. In addition, we determined fluxes and nutrient concentrations of bulk precipitation, throughfall, stemflow, litter percolate and soil water. Next we plan to analyze soil microbial community composition via PLFAs to investigate microbial stress resistance and resilience, and we will use ultrasonication to measure soil aggregate stability and protection of soil organic matter in stressed and control plots. The results of the first year show that experimental rainfall manipulation has influenced soil extracellular enzymes. Potential phenoloxidase activity was significantly reduced in stressed treatments compared to control plots. All measured hydrolytic enzymes (cellulase

  1. Levels of Arabidopsis thaliana Leaf Phosphatidic Acids, Phosphatidylserines, and Most Trienoate-Containing Polar Lipid Molecular Species Increase during the Dark Period of the Diurnal Cycle.

    PubMed

    Maatta, Sara; Scheu, Brad; Roth, Mary R; Tamura, Pamela; Li, Maoyin; Williams, Todd D; Wang, Xuemin; Welti, Ruth

    2012-01-01

    Previous work has demonstrated that plant leaf polar lipid fatty acid composition varies during the diurnal (dark-light) cycle. Fatty acid synthesis occurs primarily during the light, but fatty acid desaturation continues in the absence of light, resulting in polyunsaturated fatty acids reaching their highest levels toward the end of the dark period. In this work, Arabidopsis thaliana were grown at constant (21°C) temperature with 12-h light and 12-h dark periods. Collision induced dissociation time-of-flight mass spectrometry (MS) demonstrated that 16:3 and 18:3 fatty acid content in membrane lipids of leaves are higher at the end of the dark than at the end of the light period, while 16:1, 16:2, 18:0, and 18:1 content are higher at the end of the light period. Lipid profiling of membrane galactolipids, phospholipids, and lysophospholipids by electrospray ionization triple quadrupole MS indicated that the monogalactosyldiacylglycerol, phosphatidylglycerol, and phosphatidylcholine classes include molecular species whose levels are highest at end of the light period and others that are highest at the end of the dark period. The levels of phosphatidic acid (PA) and phosphatidylserine classes were higher at the end of the dark period, and molecular species within these classes either followed the class pattern or were not significantly changed in the diurnal cycle. Phospholipase D (PLD) is a family of enzymes that hydrolyzes phospholipids to produce PA. Analysis of several PLD mutant lines suggests that PLDζ2 and possibly PLDα1 may contribute to diurnal cycling of PA. The polar lipid compositional changes are considered in relation to recent data that demonstrate phosphatidylcholine acyl editing.

  2. Structural characterization of the Mycobacterium tuberculosis biotin biosynthesis enzymes 7,8-diaminopelargonic acid synthase and dethiobiotin synthetase .

    PubMed

    Dey, Sanghamitra; Lane, James M; Lee, Richard E; Rubin, Eric J; Sacchettini, James C

    2010-08-10

    Mycobacterium tuberculosis (Mtb) depends on biotin synthesis for survival during infection. In the absence of biotin, disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest, an unusual phenotype for an Mtb auxotroph. Humans lack the enzymes for biotin production, making the proteins of this essential Mtb pathway promising drug targets. To this end, we have determined the crystal structures of the second and third enzymes of the Mtb biotin biosynthetic pathway, 7,8-diaminopelargonic acid synthase (DAPAS) and dethiobiotin synthetase (DTBS), at respective resolutions of 2.2 and 1.85 A. Superimposition of the DAPAS structures bound either to the SAM analogue sinefungin or to 7-keto-8-aminopelargonic acid (KAPA) allowed us to map the putative binding site for the substrates and to propose a mechanism by which the enzyme accommodates their disparate structures. Comparison of the DTBS structures bound to the substrate 7,8-diaminopelargonic acid (DAPA) or to ADP and the product dethiobiotin (DTB) permitted derivation of an enzyme mechanism. There are significant differences between the Mtb enzymes and those of other organisms; the Bacillus subtilis DAPAS, presented here at a high resolution of 2.2 A, has active site variations and the Escherichia coli and Helicobacter pylori DTBS have alterations in their overall folds. We have begun to exploit the unique characteristics of the Mtb structures to design specific inhibitors against the biotin biosynthesis pathway in Mtb.

  3. Bioaccessibility and inhibitory effects on digestive enzymes of carnosic acid in sage and rosemary.

    PubMed

    Ercan, Pınar; El, Sedef Nehir

    2018-04-28

    In this study, the aim was to determine the bioaccessibilities of carnosic acid in sage and rosemary and in vitro inhibitory effects of these samples on lipid and starch digestive enzymes by evaluating the lipase, α-amylase and α-glucosidase enzyme inhibition activities. The content of carnosic acid in rosemary (18.72 ± 0.33 mg/g) was found to be higher than that content of that in sage (3.76 ± 0.13 mg/g) (p < 0.05). The carnosic acid bioaccessibilities were found as 45.10 ± 1.88% and 38.32 ± 0.21% in sage and rosemary, respectively. The tested sage and rosemary showed inhibitory activity against α-glucosidase (Concentration of inhibitor required to produce a 50% inhibition of the initial rate of reaction - IC 50 88.49 ± 2.35, 76.80 ± 1.68 μg/mL, respectively), α-amylase (IC 50 107.65 ± 12.64, 95.65 ± 2.73 μg/mL, respectively) and lipase (IC 50 6.20 ± 0.63, 4.31 ± 0.62 μg/mL, respectively). Furthermore, to the best of our knowledge, this is the first work that carnosic acid standard equivalent inhibition capacities (CAEIC 50 ) for these food samples were determined and these values were in agreement with the IC 50 values. These results show that sage and rosemary are potent inhibitors of lipase, α-amylase and α-glucosidase digestive enzymes. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Involvement of a lipoxygenase-like enzyme in abscisic Acid biosynthesis.

    PubMed

    Creelman, R A; Bell, E; Mullet, J E

    1992-07-01

    Several lines of evidence indicate that abscisic acid (ABA) is derived from 9'-cis-neoxanthin or 9'-cis-violaxanthin with xanthoxin as an intermediate. (18)O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11', 12') double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties.

  5. The shikimate pathway: review of amino acid sequence, function and three-dimensional structures of the enzymes.

    PubMed

    Mir, Rafia; Jallu, Shais; Singh, T P

    2015-06-01

    The aromatic compounds such as aromatic amino acids, vitamin K and ubiquinone are important prerequisites for the metabolism of an organism. All organisms can synthesize these aromatic metabolites through shikimate pathway, except for mammals which are dependent on their diet for these compounds. The pathway converts phosphoenolpyruvate and erythrose 4-phosphate to chorismate through seven enzymatically catalyzed steps and chorismate serves as a precursor for the synthesis of variety of aromatic compounds. These enzymes have shown to play a vital role for the viability of microorganisms and thus are suggested to present attractive molecular targets for the design of novel antimicrobial drugs. This review focuses on the seven enzymes of the shikimate pathway, highlighting their primary sequences, functions and three-dimensional structures. The understanding of their active site amino acid maps, functions and three-dimensional structures will provide a framework on which the rational design of antimicrobial drugs would be based. Comparing the full length amino acid sequences and the X-ray crystal structures of these enzymes from bacteria, fungi and plant sources would contribute in designing a specific drug and/or in developing broad-spectrum compounds with efficacy against a variety of pathogens.

  6. Palladium alpha-lipoic acid complex formulation enhances activities of Krebs cycle dehydrogenases and respiratory complexes I-IV in the heart of aged rats.

    PubMed

    Sudheesh, N P; Ajith, T A; Janardhanan, K K; Krishnan, C V

    2009-08-01

    Age-related decline in the capacity to withstand stress, such as ischemia and reperfusion, results in congestive heart failure. Though the mechanisms underlying cardiac decay are not clear, age dependent somatic damages to mitochondrial DNA (mtDNA), loss of mitochondrial function, and a resultant increase in oxidative stress in heart muscle cells may be responsible for the increased risk for cardiovascular diseases. The effect of a safe nutritional supplement, POLY-MVA, containing the active ingredient palladium alpha-lipoic acid complex, was evaluated on the activities of the Krebs cycle enzymes such as isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase as well as mitochondrial complexes I, II, III, and IV in heart mitochondria of aged male albino rats of Wistar strain. Administration of 0.05 ml/kg of POLY-MVA (which is equivalent to 0.38 mg complexed alpha-lipoic acid/kg, p.o), once daily for 30 days, was significantly (p<0.05) effective to enhance the Krebs cycle dehydrogenases, and mitochondrial electron transport chain complexes. The unique electronic and redox properties of palladium alpha-lipoic acid complex appear to be a key to this physiological effectiveness. The results strongly suggest that this formulation might be effective to protect the aging associated risk of cardiovascular and neurodegenerative diseases.

  7. Introduction to the Glutamate-Glutamine Cycle.

    PubMed

    Sonnewald, Ursula; Schousboe, Arne

    2016-01-01

    The term 'glutamate-glutamine cycle' was coined several decades ago based on the observation that using certain 14 C-labeled precursors for studies of brain metabolism the specific radioactivity of glutamine generated from glutamate was higher than that of glutamate, its immediate precursor. This is metabolically impossible unless it is assumed that at least two distinct pools of these amino acids exist. This combined with the finding that the enzyme synthesizing glutamine from glutamate was expressed in astrocytes but not in neurons formed the basis of the notion that a cycle must exist in which glutamate released from neurons is transported into astrocytes, converted to glutamine which is subsequently returned to neurons and converted to glutamate by an enzyme the activity of which is much higher in neurons than in astrocytes. Originally this cycle was supposed to function in a stoichiometric fashion but more recent research has seriously questioned this.This volume of Advances in Neurobiology is intended to provide a detailed discussion of recent developments in research aimed at delineating the functional roles of the cycle taking into account that in order for this system to work there must be a tight coupling between metabolism of glutamate in astrocytes, transfer of glutamine to neurons and de novo synthesis of glutamine in astrocytes. To understand this, knowledge about the activity and regulation of the enzymes and transporters involved in these processes is required and as can be seen from the table of contents these issues will be dealt with in detail in the individual chapters of the book.

  8. Evaluation of endogenous nitric oxide synthesis in congenital urea cycle enzyme defects.

    PubMed

    Nagasaka, Hironori; Tsukahara, Hirokazu; Yorifuji, Tohru; Miida, Takashi; Murayama, Kei; Tsuruoka, Tomoko; Takatani, Tomozumi; Kanazawa, Masaki; Kobayashi, Kunihiko; Okano, Yoshiyuki; Takayanagi, Masaki

    2009-03-01

    Nitric oxide (NO) is synthesized from arginine and O(2) by nitric oxide synthase (NOS). Citrulline, which is formed as a by-product of the NOS reaction, can be recycled to arginine by the 2 enzymes acting in the urea cycle: argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Although the complete urea cycle is expressed only in the liver, ASS and ASL are expressed in other organs including the kidney and vascular endothelium. To examine possible alterations of the NO pathway in urea cycle defects, we measured plasma concentrations of arginine and citrulline and serum concentrations of nitrite/nitrate (NOx(-), stable NO metabolites) and asymmetric dimethylarginine (ADMA, an endogenous NOS inhibitor) in patients with congenital urea cycle disorders of 3 types: ornithine transcarbamylase (OTC) deficiency, ASS deficiency, and ASL deficiency. All were receiving oral arginine replacement at the time of this study. The same parameters were also measured in healthy subjects, who participated as controls. The OTC-deficient patients had significantly high NOx(-) and nonsignificantly high ADMA concentrations. Their NOx(-) was significantly positively correlated with arginine. The ASS-deficient patients had significantly low NOx(-) and significantly high ADMA concentrations. The ASL-deficient patients had normal NOx(-) and nonsignificantly high ADMA concentrations. In ASS-deficient and ASL-deficient patients, the NOx(-) was significantly inversely correlated with citrulline. These results suggest that NO synthesis is enhanced in OTC-deficient patients while receiving arginine but that NO synthesis remains low in ASS-deficient patients despite receiving arginine. They also suggest that endogenous NO synthesis is negatively affected by citrulline and ADMA in ASS-deficient and ASL-deficient patients. Although the molecular mechanisms remain poorly understood, we infer that the NO pathway might play a role in the pathophysiology related to congenital urea cycle

  9. Combined Effects of Lanthanum (III) and Acid Rain on Antioxidant Enzyme System in Soybean Roots.

    PubMed

    Zhang, Xuanbo; Du, Yuping; Wang, Lihong; Zhou, Qing; Huang, Xiaohua; Sun, Zhaoguo

    2015-01-01

    Rare earth element pollution (REEs) and acid rain (AR) pollution simultaneously occur in many regions, which resulted in a new environmental issue, the combined pollution of REEs and AR. The effects of the combined pollution on the antioxidant enzyme system of plant roots have not been reported. Here, the combined effects of lanthanum ion (La3+), one type of REE, and AR on the antioxidant enzyme system of soybean roots were investigated. In the combined treatment of La3+ (0.08 mM) and AR, the cell membrane permeability and the peroxidation of cell membrane lipid of soybean roots increased, and the superoxide dismutase, catalase, peroxidase and reduced ascorbic acid served as scavengers of reactive oxygen species. In other combined treatments of La3+ (0.40 mM, 1.20 mM) and AR, the membrane permeability, malonyldialdehyde content, superoxide dismutase activity, peroxidase activity and reduced ascorbic acid content increased, while the catalase activity decreased. The increased superoxide dismutase activity, peroxidase activity and reduced ascorbic acid content were inadequate to scavenge the excess hydrogen peroxide and superoxide, leading to the damage of the cell membrane, which was aggravated with the increase in the concentration of La3+ and the level of AR. The deleterious effects of the combined treatment of La3+ and AR were stronger than those of the single treatment of La3+ or AR. Moreover, the activity of antioxidant enzyme system in the combined treatment group was affected directly and indirectly by mineral element content in soybean plants.

  10. Combined Effects of Lanthanum (III) and Acid Rain on Antioxidant Enzyme System in Soybean Roots

    PubMed Central

    Zhang, Xuanbo; Du, Yuping; Wang, Lihong; Zhou, Qing; Huang, Xiaohua; Sun, Zhaoguo

    2015-01-01

    Rare earth element pollution (REEs) and acid rain (AR) pollution simultaneously occur in many regions, which resulted in a new environmental issue, the combined pollution of REEs and AR. The effects of the combined pollution on the antioxidant enzyme system of plant roots have not been reported. Here, the combined effects of lanthanum ion (La3+), one type of REE, and AR on the antioxidant enzyme system of soybean roots were investigated. In the combined treatment of La3+ (0.08 mM) and AR, the cell membrane permeability and the peroxidation of cell membrane lipid of soybean roots increased, and the superoxide dismutase, catalase, peroxidase and reduced ascorbic acid served as scavengers of reactive oxygen species. In other combined treatments of La3+ (0.40 mM, 1.20 mM) and AR, the membrane permeability, malonyldialdehyde content, superoxide dismutase activity, peroxidase activity and reduced ascorbic acid content increased, while the catalase activity decreased. The increased superoxide dismutase activity, peroxidase activity and reduced ascorbic acid content were inadequate to scavenge the excess hydrogen peroxide and superoxide, leading to the damage of the cell membrane, which was aggravated with the increase in the concentration of La3+ and the level of AR. The deleterious effects of the combined treatment of La3+ and AR were stronger than those of the single treatment of La3+ or AR. Moreover, the activity of antioxidant enzyme system in the combined treatment group was affected directly and indirectly by mineral element content in soybean plants. PMID:26230263

  11. Short-Chain Fatty Acids Enhance the Lipid Accumulation of 3T3-L1 Cells by Modulating the Expression of Enzymes of Fatty Acid Metabolism.

    PubMed

    Yu, Haining; Li, Ran; Huang, Haiyong; Yao, Ru; Shen, Shengrong

    2018-01-01

    Short-chain fatty acids (SCFA) such as acetic acid, propionic acid, and butyric acid are produced by fermentation by gut microbiota. In this paper, we investigate the effects of SCFA on 3T3-L1 cells and the underlying molecular mechanisms. The cells were treated with acetic acid, propionic acid, or butyric acid when cells were induced to differentiate into adipocytes. MTT assay was employed to detect the viability of 3T3-L1 cells. Oil Red O staining was used to visualize the lipid content in 3T3-L1 cells. A triglyceride assay kit was used to detect the triacylglycerol content in 3T3-L1 cells. qRT-PCR and Western blot were used to evaluate the expression of metabolic enzymes. MTT results showed that safe concentrations of acetic acid, propionic acid, and butyric acid were less than 6.4, 3.2, and 0.8 mM, respectively. Oil Red O staining and triacylglycerols detection results showed that treatment with acetic acid, propionic acid, and butyric acid accelerated the 3T3-L1 adipocyte differentiation. qRT-PCR and Western blot results showed that the expressions of lipoprotein lipase (LPL), adipocyte fatty acid binding protein 4 (FABP4), fatty acid transporter protein 4 (FATP4), and fatty acid synthase (FAS) were significantly increased by acetic acid, propionic acid, and butyric acid treatment during adipose differentiation (p < 0.05). In conclusion, SCFA promoted lipid accumulation by modulating the expression of enzymes of fatty acid metabolism. © 2018 AOCS.

  12. Immobilization of BSA, enzymes and cells of Bacillus stearothermophilus onto cellulose polygalacturonic acid and starch based graft copolymers containing maleic arhydride

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Beddows, C.G.; Gil, M.H.; Guthrie, J.T.

    1986-01-01

    Poly(maleic anhydride styrene) graft copolymers of cellulose, pectin polygalacturonic acid salt, calcium polygalacturonate, and starch were prepared and used to immobilize proteins. The cellulose grafts coupled quite appreciable quantities of acid phosphatase, glucose oxidase, and trypsin. However, the general retention of activity was somewhat disappointing. Further investigation with acid phosphatase showed that the amount of enzyme immobilized increased as the amount of anhydride in the graft copolymer increased but no such relationship existed for the enzymic activity. The cellulose graft copolymers were hydrolyzed and it appeared that the carboxyl group aided adsorption of the enzyme. Attempts to couple acid phosphatasemore » using CMC through the free carboxyl groups, created by hydrolysis, gave only a small increase in the extent of protein coupling. However, the unhydrolyzed system gave a useful degree of immobilization of cells of Bacillus stearothermophilus, as did a poly(maleic anhydride/styrene)-cocellulose system. Attempts to improve the activity by using grafts based on other polysaccharide supports met with mixed success. Pectin products were soluble. Polygalacturonic acid products were partially soluble and extremely high levels of enzymic activity were obtained. This was probably due in part to the hydrophilic nature of the system, which also encouraged absorption of the enzyme. Attempts were made to reduce the solubility by using the calcium pectinate salt. Immobilization of acid phosphatase and trypsin resulted in increased protein coupling but relatively poor activities were attained. Calcium polygalacturonate was used to prepare an insoluble graft copolymeric system containing acrylonitrile-comaleic anhydride. The resulting gels gave excellent coupling with acid phosphatase which had a very good retention of activity.« less

  13. Studies on the Growth Effects of the Canaline-Urea Cycle Amino Acids with Lemna minor L. 1

    PubMed Central

    Rosenthal, Gerald A.; Gulati, Dushyant K.; Sabharwal, P. S.

    1975-01-01

    The aquatic microphyte, Lemna minor L., was utilized to assess the relative toxicity and general growth effects of canavanine, canaline, ureidohomoserine (UHS), and canavaninosuccinate (CSA). These amino acids are constituents of the canaline-urea cycle and structural analogues of the ornithine-urea cycle amino acids. Comparative growth studies with L. minor revealed that the canaline-urea cycle amino acids are potent antimetabolites. With the exception of CSA, they are extremely toxic at a concentration of 5 μm. Over a concentration range of 1 to 4 μm, canavanine is the most growth-inhibiting of the canaline-urea cycle amino acids. At or above 5 μm, canavanine and canaline possess comparable toxicity. UHS is less growth-inhibiting than canavanine or canaline, and CSA is the least toxic of the canaline-urea cycle intermediates. PMID:16659316

  14. Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

    PubMed Central

    Rumpho, Mary E.; Pochareddy, Sirisha; Worful, Jared M.; Summer, Elizabeth J.; Bhattacharya, Debashish; Pelletreau, Karen N.; Tyler, Mary S.; Lee, Jungho; Manhart, James R.; Soule, Kara M.

    2009-01-01

    Phosphoribulokinase (PRK), a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction (Calvin) cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc (sea slug) Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclear-encoded proteins that are directed to complex (i.e. four membrane-bound) algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V. litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V. litorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months. PMID:19995736

  15. Establishment of a Simple and Convenient Method for Folic Acid Enzyme Chemiluminescence Immunoassay

    NASA Astrophysics Data System (ADS)

    Liu, Ting; Zeng, Ling; Yu, Zhengwei; Yang, Yanfei; Qu, Yunhuan

    2018-01-01

    The enzyme chemiluminescence new immunoassay for folic acid (FA) was established by competition model. Add FA samples to a microtiter plate precoated with the goat anti-mouse IgG firstly, then add enzyme abled FA and FA monoclonal antibody (McAb). The values of CLIA were measured to reflect the quantity of FA. The limit of detection(LOD) of assay is 0.37ng/mL. The assay shows good correlation during 1∼30 ng/mL with correlation coefficient 0.9976. The intra- and inter-assay coefficients of variation are 4.8 % ∼ 7.3 % and 6.1 % ∼ 12.2 %, respectively. The recovery of folic acid in serum is 90.4 %∼113.2 %. Compared with determine value clinically in chemiluminescence immunoassay kit from Roche company, the correlative equation is y = 0.9689x + 0.0228, and correlation coefficient is 0.9780. Various components and kit overall show good stabilities. This method is simple and convenient, and has low LOD value. The method has overcome the shortcomings of the present references. It is easy to apply and has broad clinical application prospect. It lays an experimental foundation for the preparation of Mc Ab against folic acid and the development of domestic kit.

  16. Enzyme Sorption onto Soil and Biocarbon Amendments Alters Catalytic Capacity and Depends on the Specific Protein and pH

    NASA Astrophysics Data System (ADS)

    Foster, E.; Fogle, E. J.; Cotrufo, M. F.

    2017-12-01

    Enzymes catalyze biogeochemical reactions in soils and play a key role in nutrient cycling in agricultural systems. Often, to increase soil nutrients, agricultural managers add organic amendments and have recently experimented with charcoal-like biocarbon products. These amendments can enhance soil water and nutrient holding capacity through increasing porosity. However, the large surface area of the biocarbon has the potential to sorb nutrients and other organic molecules. Does the biocarbon decrease nutrient cycling through sorption of enzymes? In a laboratory setting, we compared the interaction of two purified enzymes β-glucosidase and acid phosphatase with a sandy clay loam and two biocarbons. We quantified the sorbed enzymes at three different pHs using a Bradford protein assay and then measured the activity of the sorbed enzyme via high-throughput fluorometric analysis. Both sorption and activity depended upon the solid phase, pH, and specific enzyme. Overall the high surface area biocarbon impacted the catalytic capacity of the enzymes more than the loam soil, which may have implications for soil nutrient management with these organic amendments.

  17. Enzymic synthesis of indole-3-acetyl-1-O-beta-d-glucose. II. Metabolic characteristics of the enzyme

    NASA Technical Reports Server (NTRS)

    Leznicki, A. J.; Bandurski, R. S.

    1988-01-01

    The synthesis of indole-3-acetyl-1-O-beta-D-glucose from indole-3-acetic acid (IAA) and uridine diphosphoglucose (UDPG) has been shown to be a reversible reaction with the equilibrium away from ester formation and toward formation of IAA. The enzyme occurs primarily in the liquid endosperm of the corn kernel but some activity occurs in the embryo. It is relatively specific showing no glucose ester formation with oxindole-3-acetic acid or 7-hydroxy-oxindole-3-acetic acid, and low activity with phenylpropene acids, such as rho-coumaric acid. The enzyme is also specific for the nucleotide sugar showing no activity with UDPGalactose or UDPXylose. The enzyme is inhibited by inorganic pyrophosphate, by phosphate esters and by phospholipids, particularly phosphatidyl ethanolamine. The enzyme is inhibited by zeatin, by 2,4-dichlorophenoxy-acetic acid, by IAA-myo-inositol and IAA-glucan, but not by zeatin riboside, and only weakly by gibberellic acid, abscisic acid and kinetin. The reaction is slightly stimulated by both calcium and calmodulin and, in some cases, by thiol compounds. The role of this enzyme in the homeostatic control of indole-3-acetic acid levels in Zea mays is discussed.

  18. Alterations in Krebs cycle enzyme activities and carbohydrate catabolism in two strains of Trypanosoma brucei during in vitro differentiation of their bloodstream to procyclic stages.

    PubMed

    Durieux, P O; Schütz, P; Brun, R; Köhler, P

    1991-03-01

    A rapid switch from a fermentative to a primarily oxidative type of glucose utilization was observed during in vitro differentiation of Trypanosoma brucei STIB348 and EATRO1244 bloodstream to procyclic trypomastigotes. In accordance with previously published reports bloodstream populations produced pyruvate as the major end product of glucose catabolism, together with very small amounts of CO2, succinate and glycerol. During differentiation pyruvate excretion decreased within 48 h to the low levels produced by 28-day procyclic stages. Concomitant with the decline in pyruvate formation, acetate appeared as a new product and the rates of respiratory CO2 increased considerably. The amount of carbon released with these compounds could account for nearly all of the glucose carbon consumed. Rates of glucose utilization and formation of acetate and CO2 in cells differentiated for 48 h were essentially the same as those found in 28-day procyclics. Succinate and glycerol excretion remained low during the entire transformation process, and no significant difference in the pattern and quantities of end products were found between the two trypanosome strains. During trypanosome differentiation the changes in metabolism were associated with marked alterations in enzyme activity levels. Activities of the tricarboxylic acid (TCA) cycle enzymes citrate synthase, isocitrate dehydrogenase (NAD+), succinate dehydrogenase and fumarase were not detectable in bloodstream trypomastigotes but appeared upon differentiation for 24 h. An exception was citrate synthase whose activity was not demonstrable until 48 h postinoculation into culture. After 48 h the majority of the TCA cycle enzyme activities continued to increase steadily until day 28. Pyruvate kinase activity decreased in differentiating cells after 48 h to about 25% of the level found in bloodstream trypomastigotes.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Purification and preliminary characterization of (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid synthase, an enzyme involved in biosynthesis of the antitumor agent sparsomycin.

    PubMed

    Parry, R J; Hoyt, J C

    1997-02-01

    Sparsomycin is an antitumor antibiotic produced by Streptomyces sparsogenes. Biosynthetic experiments have previously demonstrated that one component of sparsomycin is derived from L-tryptophan via the intermediacy of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid and (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid. An enzyme which catalyzes the conversion of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid to (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid has been purified 740-fold to homogeneity from S. sparsogenes. The molecular mass of the native and denatured enzyme was 87 kDa, indicating that the native enzyme is monomeric. The enzyme required NAD+ for activity but lacked rigid substrate specificity, since analogs of both NAD+ and 3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid could serve as substrates. The enzyme was very weakly inhibited by mycophenolic acid. Monovalent cations were required for activity, with potassium ions being the most effective. The enzyme exhibited sensitivity toward diethylpyrocarbonate and some thiol-directed reagents, and it was irreversibly inhibited by 6-chloropurine. The properties of the enzyme suggest it is mechanistically related to inosine-5'-monophosphate dehydrogenase.

  20. Evolution of the biosynthesis of the branched-chain amino acids

    NASA Technical Reports Server (NTRS)

    Keefe, Anthony D.; Lazcano, Antonio; Miller, Stanley L.

    1995-01-01

    The origins of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threomine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from alpha-ketoisovalerc acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use fo the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.

  1. The relationship between leaf rolling and ascorbate-glutathione cycle enzymes in apoplastic and symplastic areas of Ctenanthe setosa subjected to drought stress.

    PubMed

    Saruhan, Neslihan; Terzi, Rabiye; Saglam, Aykut; Kadioglu, Asim

    2009-01-01

    The ascorbate-glutathione (ASC-GSH) cycle has an important role in defensive processes against oxidative damage generated by drought stress. In this study, the changes that take place in apoplastic and symplastic ASC-GSH cycle enzymes of the leaf and petiole were investigated under drought stress causing leaf rolling in Ctenanthe setosa (Rose.) Eichler (Marantaceae). Apoplastic and symplastic extractions of leaf and petiole were performed at different visual leaf rolling scores from 1 to 4 (1 is unrolled, 4 is tightly rolled and the others are intermediate forms). Glutathione reductase (GR), a key enzyme in the GSH regeneration cycle, and ascorbate (ASC) were present in apoplastic spaces of the leaf and petiole, whereas dehydroascorbate reductase (DHAR), which uses glutathione as reductant, monodehydroascorbate reductase (MDHAR), which uses NAD(P)H as reductant, and glutathione were absent. GR, DHAR and MDHAR activities increased in the symplastic and apoplastic areas of the leaf. Apoplastic and symplastic ASC and dehydroascorbate (DHA), the oxidized form of ascorbate, rose at all scores except score 4 of symplastic ASC in the leaf. On the other hand, while reduced glutathione (GSH) content was enhanced, oxidized glutathione (GSSG) content decreased in the leaf during rolling. As for the petiole, GR activity increased in the apoplastic area but decreased in the symplastic area. DHAR and MDHAR activities increased throughout all scores, but decreased to the score 1 level at score 4. The ASC content of the apoplast increased during leaf rolling. Conversely, symplastic ASC content increased at score 2, however decreased at the later scores. While the apoplastic DHA content declined, symplastic DHA rose at score 2, but later was down to the level of score 1. While GSH content enhanced during leaf rolling, GSSG content did not change except at score 2. As well, there were good correlations between leaf rolling and ASC-GSH cycle enzyme activities in the leaf (GR and DHAR

  2. Differential Sensitivities of Fast- and Slow-Cycling Cancer Cells to Inosine Monophosphate Dehydrogenase 2 Inhibition by Mycophenolic Acid

    PubMed Central

    Chen, Kan; Cao, Wanlu; Li, Juan; Sprengers, Dave; Hernanda, Pratika Y; Kong, Xiangdong; van der Laan, Luc JW; Man, Kwan; Kwekkeboom, Jaap; Metselaar, Herold J; Peppelenbosch, Maikel P; Pan, Qiuwei

    2015-01-01

    As uncontrolled cell proliferation requires nucleotide biosynthesis, inhibiting enzymes that mediate nucleotide biosynthesis constitutes a rational approach to the management of oncological diseases. In practice, however, results of this strategy are mixed and thus elucidation of the mechanisms by which cancer cells evade the effect of nucleotide biosynthesis restriction is urgently needed. Here we explored the notion that intrinsic differences in cancer cell cycle velocity are important in the resistance toward inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). In short-term experiments, MPA treatment of fast-growing cancer cells effectively elicited G0/G1 arrest and provoked apoptosis, thus inhibiting cell proliferation and colony formation. Forced expression of a mutated IMPDH2, lacking a binding site for MPA but retaining enzymatic activity, resulted in complete resistance of cancer cells to MPA. In nude mice subcutaneously engrafted with HeLa cells, MPA moderately delayed tumor formation by inhibiting cell proliferation and inducing apoptosis. Importantly, we developed a lentiviral vector–based Tet-on label-retaining system that enables to identify, isolate and functionally characterize slow-cycling or so-called label-retaining cells (LRCs) in vitro and in vivo. We surprisingly found the presence of LRCs in fast-growing tumors. LRCs were superior in colony formation, tumor initiation and resistance to MPA as compared with fast-cycling cells. Thus, the slow-cycling compartment of cancer seems predominantly responsible for resistance to MPA. PMID:26467706

  3. Caffeic and chlorogenic acids inhibit key enzymes linked to type 2 diabetes (in vitro): a comparative study.

    PubMed

    Oboh, Ganiyu; Agunloye, Odunayo M; Adefegha, Stephen A; Akinyemi, Ayodele J; Ademiluyi, Adedayo O

    2015-03-01

    Chlorogenic acid is a major phenolic compound that forms a substantial part of plant foods and is an ester of caffeic acid and quinic acid. However, the effect of the structures of both chlorogenic and caffeic acids on their antioxidant and antidiabetic potentials have not been fully understood. Thus, this study sought to investigate and compare the interaction of caffeic acid and chlorogenic acid with α-amylase and α-glucosidase (key enzymes linked to type 2 diabetes) activities in vitro. The inhibitory effect of the phenolic acids on α-amylase and α-glucosidase activities was evaluated. Thereafter, their antioxidant activities as typified by their 1,1-diphenyl-2 picrylhydrazyl radical scavenging ability and ferric reducing antioxidant properties were determined. The results revealed that both phenolic acids inhibited α-amylase and α-glucosidase activities in a dose-dependent manner (2-8 μg/mL). However, caffeic acid had a significantly (p<0.05) higher inhibitory effect on α-amylase [IC50 (concentration of sample causing 50% enzyme inhibition)=3.68 μg/mL] and α-glucosidase (IC50=4.98 μg/mL) activities than chlorogenic acid (α-amylase IC50=9.10 μg/mL and α-glucosidase IC50=9.24 μg/mL). Furthermore, both phenolic acids exhibited high antioxidant properties, with caffeic acid showing higher effects. The esterification of caffeic acid with quinic acid, producing chlorogenic acid, reduces their ability to inhibit α-amylase and α-glucosidase activities. Thus, the inhibition of α-amylase and α-glucosidase activities by the phenolic acids could be part of the possible mechanism by which the phenolic acids exert their antidiabetic effects.

  4. In Vivo Roles of Fatty Acid Biosynthesis Enzymes in Biosynthesis of Biotin and α-Lipoic Acid in Corynebacterium glutamicum

    PubMed Central

    Nagashima, Takashi; Nakamura, Eri; Kato, Ryosuke; Ohshita, Masakazu; Hayashi, Mikiro; Takeno, Seiki

    2017-01-01

    ABSTRACT For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI. Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA. These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicum. IMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis, which use an individual nonaggregating type II fatty

  5. Catalyzed enzyme electrodes

    DOEpatents

    Zawodzinski, Thomas A.; Wilson, Mahlon S.; Rishpon, Judith; Gottesfeld, Shimshon

    1993-01-01

    An enzyme electrode is prepared with a composite coating on an electrical conductor. The composite coating is formed from a casting solution of a perfluorosulfonic acid polymer, an enzyme, and a carbon supported catalyst. The solution may be cast directly on the conductor surface or may be formed as a membrane and applied to the surface. The perfluorosulfonic acid ionomer formed from the casting solution provides an insoluble biocompatible protective matrix for the enzyme and acts to retain the enzyme for long term availability in the electrode structure. The carbon supported catalyst provides catalytic sites throughout the layer for the oxidation of hydrogen peroxide from the enzyme reactions. The carbon support then provides a conductive path for establishing an electrical signal to the electrical conductor. In one embodiment, the electrical conductor is a carbon cloth that permits oxygen or other gas to be introduced to the perfluorosulfonic polymer to promote the enzyme reaction independent of oxygen in the solution being tested.

  6. Differential expression of glucose-metabolizing enzymes in multiple sclerosis lesions.

    PubMed

    Nijland, Philip G; Molenaar, Remco J; van der Pol, Susanne M A; van der Valk, Paul; van Noorden, Cornelis J F; de Vries, Helga E; van Horssen, Jack

    2015-12-04

    Demyelinated axons in multiple sclerosis (MS) lesions have an increased energy demand in order to maintain conduction. However, oxidative stress-induced mitochondrial dysfunction likely alters glucose metabolism and consequently impairs neuronal function in MS. Imaging and pathological studies indicate that glucose metabolism is altered in MS, although the underlying mechanisms and its role in neurodegeneration remain elusive. We investigated expression patterns of key enzymes involved in glycolysis, tricarboxylic acid (TCA) cycle and lactate metabolism in well-characterized MS tissue to establish which regulators of glucose metabolism are involved in MS and to identify underlying mechanisms. Expression levels of glycolytic enzymes were increased in active and inactive MS lesions, whereas expression levels of enzymes involved in the TCA cycle were upregulated in active MS lesions, but not in inactive MS lesions. We observed reduced expression and production capacity of mitochondrial α-ketoglutarate dehydrogenase (αKGDH) in demyelinated axons, which correlated with signs of axonal dysfunction. In inactive lesions, increased expression of lactate-producing enzymes was observed in astrocytes, whereas lactate-catabolising enzymes were mainly detected in axons. Our results demonstrate that the expression of various enzymes involved in glucose metabolism is increased in both astrocytes and axons in active MS lesions. In inactive MS lesions, we provide evidence that astrocytes undergo a glycolytic shift resulting in enhanced astrocyte-axon lactate shuttling, which may be pivotal for the survival of demyelinated axons. In conclusion, we show that key enzymes involved in energy metabolism are differentially expressed in active and inactive MS lesions. Our findings imply that, in addition to reduced oxidative phosphorylation activity, other bioenergetic pathways are affected as well, which may contribute to ongoing axonal degeneration in MS.

  7. Genetic alterations in Krebs cycle and its impact on cancer pathogenesis.

    PubMed

    Sajnani, Karishma; Islam, Farhadul; Smith, Robert Anthony; Gopalan, Vinod; Lam, Alfred King-Yin

    2017-04-01

    Cancer cells exhibit alterations in many cellular processes, including oxygen sensing and energy metabolism. Glycolysis in non-oxygen condition is the main energy production process in cancer rather than mitochondrial respiration as in benign cells. Genetic and epigenetic alterations of Krebs cycle enzymes favour the shift of cancer cells from oxidative phosphorylation to anaerobic glycolysis. Mutations in genes encoding aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarate hydratase, and citrate synthase are noted in many cancers. Abnormalities of Krebs cycle enzymes cause ectopic production of Krebs cycle intermediates (oncometabolites) such as 2-hydroxyglutarate, and citrate. These oncometabolites stabilize hypoxia inducible factor 1 (HIF1), nuclear factor like 2 (Nrf2), inhibit p53 and prolyl hydroxylase 3 (PDH3) activities as well as regulate DNA/histone methylation, which in turn activate cell growth signalling. They also stimulate increased glutaminolysis, glycolysis and production of reactive oxygen species (ROS). Additionally, genetic alterations in Krebs cycle enzymes are involved with increased fatty acid β-oxidations and epithelial mesenchymal transition (EMT) induction. These altered phenomena in cancer could in turn promote carcinogenesis by stimulating cell proliferation and survival. Overall, epigenetic and genetic changes of Krebs cycle enzymes lead to the production of oncometabolite intermediates, which are important driving forces of cancer pathogenesis and progression. Understanding and applying the knowledge of these mechanisms opens new therapeutic options for patients with cancer. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  8. Biochemical characterization and structural insight into aliphatic β-amino acid adenylation enzymes IdnL1 and CmiS6.

    PubMed

    Cieślak, Jolanta; Miyanaga, Akimasa; Takaku, Ryoma; Takaishi, Makoto; Amagai, Keita; Kudo, Fumitaka; Eguchi, Tadashi

    2017-07-01

    Macrolactam antibiotics such as incednine and cremimycin possess an aliphatic β-amino acid as a starter unit of their polyketide chain. In the biosynthesis of incednine and cremimycin, unique stand-alone adenylation enzymes IdnL1 and CmiS6 select and activate the proper aliphatic β-amino acid as a starter unit. In this study, we describe the enzymatic characterization and the structural basis of substrate specificity of IdnL1 and CmiS6. Functional analysis revealed that IdnL1 and CmiS6 recognize 3-aminobutanoic acid and 3-aminononanoic acid, respectively. We solved the X-ray crystal structures of IdnL1 and CmiS6 to understand the recognition mechanism of these aliphatic β-amino acids. These structures revealed that IdnL1 and CmiS6 share a common recognition motif that interacts with the β-amino group of the substrates. However, the hydrophobic side-chains of the substrates are accommodated differently in the two enzymes. IdnL1 has a bulky Leu220 located close to the terminal methyl group of 3-aminobutanoate of the trapped acyl-adenylate intermediate to construct a shallow substrate-binding pocket. In contrast, CmiS6 possesses Gly220 at the corresponding position to accommodate 3-aminononanoic acid. This structural observation was supported by a mutational study. Thus, the size of amino acid residue at the 220 position is critical for the selection of an aliphatic β-amino acid substrate in these adenylation enzymes. Proteins 2017; 85:1238-1247. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  9. Mitochondrial Probe Methyltriphenylphosphonium (TPMP) Inhibits the Krebs Cycle Enzyme 2-Oxoglutarate Dehydrogenase.

    PubMed

    Elkalaf, Moustafa; Tůma, Petr; Weiszenstein, Martin; Polák, Jan; Trnka, Jan

    2016-01-01

    Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70-4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes.

  10. Mitochondrial Probe Methyltriphenylphosphonium (TPMP) Inhibits the Krebs Cycle Enzyme 2-Oxoglutarate Dehydrogenase

    PubMed Central

    Elkalaf, Moustafa; Tůma, Petr; Weiszenstein, Martin; Polák, Jan

    2016-01-01

    Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70–4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes. PMID:27537184

  11. Recombinant human glutathione S-transferases catalyse enzymic isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro.

    PubMed Central

    Chen, H; Juchau, M R

    1998-01-01

    The steric conversion of 13-cis-retinoic acid (13-cRA) to all-trans-retinoic acid (t-RA) has been proposed as an activation mechanism for the observed therapeutic and teratogenic activities of 13-cRA. Here we have investigated the catalysis of isomerization of 13-cRA to t-RA by recombinant human glutathione S-transferases (GSTs). Substrate was incubated with GST in 0.1 M sodium phosphate buffer, pH 7.5, at 37 degrees C in total darkness. The t-RA generated was measured quantitatively by HPLC. Under the reaction conditions used, GSTP1-1 was far more effective than human GSTM1-1 or human GSTA1-1 in catalysing the isomerization reaction. The reaction catalysed by GSTP1-1 showed substrate saturation and the Km and Vmax values for the reaction were approx. 7 microM and 650 pmol/min per nmol respectively. The reaction rate increased linearly with increasing enzyme concentration. The reaction was inhibited both by heat treatment and by S-decylglutathione (a potent inhibitor of transferase activity associated with GST). Additions of polyclonal rabbit antiserum for human GSTP1-1 to the reaction resulted in a significant decrease in generation of t-RA (70-80%). In addition, ethacrynic acid, a selective substrate for Pi isoforms of GST, also inhibited the isomerization of 13-cRA to t-RA catalysed by GSTP1-1. Under the same reaction conditions, GSTP1-1 was much less effective in catalysing the steric conversion of 9-cis-retinoic acid to t-RA, indicating that the enzyme was stereospecific for the conversion of 13-cRA to t-RA. These observations suggest that enzymic catalysis was the primary mechanism for the GSTP1-1-dependent conversion of 13-cRA to t-RA. Reactions catalysed by a purified rat hepatic GST Pi isoenzyme proceeded more slowly than reactions catalysed by human GSTP1-1. Comparative studies also showed that there were marked species differences in catalytic activities between various purified mammalian hepatic GST mixtures. PMID:9806904

  12. Recombinant human glutathione S-transferases catalyse enzymic isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro.

    PubMed

    Chen, H; Juchau, M R

    1998-11-15

    The steric conversion of 13-cis-retinoic acid (13-cRA) to all-trans-retinoic acid (t-RA) has been proposed as an activation mechanism for the observed therapeutic and teratogenic activities of 13-cRA. Here we have investigated the catalysis of isomerization of 13-cRA to t-RA by recombinant human glutathione S-transferases (GSTs). Substrate was incubated with GST in 0.1 M sodium phosphate buffer, pH 7.5, at 37 degrees C in total darkness. The t-RA generated was measured quantitatively by HPLC. Under the reaction conditions used, GSTP1-1 was far more effective than human GSTM1-1 or human GSTA1-1 in catalysing the isomerization reaction. The reaction catalysed by GSTP1-1 showed substrate saturation and the Km and Vmax values for the reaction were approx. 7 microM and 650 pmol/min per nmol respectively. The reaction rate increased linearly with increasing enzyme concentration. The reaction was inhibited both by heat treatment and by S-decylglutathione (a potent inhibitor of transferase activity associated with GST). Additions of polyclonal rabbit antiserum for human GSTP1-1 to the reaction resulted in a significant decrease in generation of t-RA (70-80%). In addition, ethacrynic acid, a selective substrate for Pi isoforms of GST, also inhibited the isomerization of 13-cRA to t-RA catalysed by GSTP1-1. Under the same reaction conditions, GSTP1-1 was much less effective in catalysing the steric conversion of 9-cis-retinoic acid to t-RA, indicating that the enzyme was stereospecific for the conversion of 13-cRA to t-RA. These observations suggest that enzymic catalysis was the primary mechanism for the GSTP1-1-dependent conversion of 13-cRA to t-RA. Reactions catalysed by a purified rat hepatic GST Pi isoenzyme proceeded more slowly than reactions catalysed by human GSTP1-1. Comparative studies also showed that there were marked species differences in catalytic activities between various purified mammalian hepatic GST mixtures.

  13. Kinetics and spatial distribution of enzymes of carbon, nitrogen and phosphorus cycles in earthworm biopores

    NASA Astrophysics Data System (ADS)

    Hoang Thi Thu, Duyen; Razavi, Bahar S.

    2016-04-01

    Earthworms boost microbial activities and consequently form hotspots in soil. The distribution of enzyme activities inside the earthworm biopores is completely unknown. For the first time, we analyzed enzyme kinetics and visualized enzyme distribution inside and outside biopores by in situ soil zymography. Kinetic parameters (Vmax and Km) of 6 enzymes β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) were determined in biopores formed by Lumbricus terrestris L.. The spatial distributions of GLU, NAG and APT become visible via zymograms in comparison between earthworm-inhabited and earthworm-free soil. Zymography showed heterogeneous distribution of hotspots in the rhizosphere and biopores. The hotspot areas were 2.4 to 14 times larger in the biopores than in soil without earthworms. The significantly higher Vmax values for GLU, CBH, XYL, NAG and APT in biopores confirmed the stimulation of enzyme activities by earthworms. For CBH, XYL and NAG, the 2- to 3-fold higher Km values in biopores indicated different enzyme systems with lower substrate affinity compared to control soil. The positive effects of earthworms on Vmax were cancelled by the Km increase for CBH, XYL and NAG at a substrate concentration below 20 μmol g-1 soil. The change of enzyme systems reflected a shift in dominant microbial populations toward species with lower affinity to holo-celluloses and to N-acetylglucosamine, and with higher affinity to proteins as compared to the biopores-free soil. We conclude that earthworm biopores are microbial hotspots with much higher and dense distribution of enzyme activities compared to bulk soil. References Spohn M, Kuzyakov Y. (2014) Spatial and temporal dynamics of hotspots of enzyme activity in soil as affected by living and dead roots - a soil zymography analysis, Plant Soil 379: 67-77. Blagodatskaya, E., Kuzyakov, Y., 2013. Review paper: Active microorganisms in soil

  14. Alternative carbohydrate reserves used in the daily cycle of crassulacean acid metabolism

    Treesearch

    C.C. Black; J.-Q. Chen; R.L. Doong; M.N. Angelov; Shi-Jean S. Sung

    1996-01-01

    Each day a massive interlocked biochemical cycle occurs in the green tissues of crassulacean acid metabolism plants.The function of this interlocked cycle, in its simplest context, is to furnish most of the CO2 for CAM plant photosynthesis.In this unified presentation our aims are (1) to divide CAM plants into two metabolic groups, (2) to...

  15. Salicylic acid antagonizes abscisic acid inhibition of shoot growth and cell cycle progression in rice

    NASA Astrophysics Data System (ADS)

    Meguro, Ayano; Sato, Yutaka

    2014-04-01

    We analysed effects of abscisic acid (ABA, a negative regulatory hormone), alone and in combination with positive or neutral hormones, including salicylic acid (SA), on rice growth and expression of cell cycle-related genes. ABA significantly inhibited shoot growth and induced expression of OsKRP4, OsKRP5, and OsKRP6. A yeast two-hybrid assay showed that OsKRP4, OsKRP5, and OsKRP6 interacted with OsCDKA;1 and/or OsCDKA;2. When SA was simultaneously supplied with ABA, the antagonistic effect of SA completely blocked ABA inhibition. SA also blocked ABA inhibition of DNA replication and thymidine incorporation in the shoot apical meristem. These results suggest that ABA arrests cell cycle progression by inducing expression of OsKRP4, OsKRP5, and OsKRP6, which inhibit the G1/S transition, and that SA antagonizes ABA by blocking expression of OsKRP genes.

  16. Structural Basis for the Recognition of Mycolic Acid Precursors by KasA, a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis *

    PubMed Central

    Schiebel, Johannes; Kapilashrami, Kanishk; Fekete, Agnes; Bommineni, Gopal R.; Schaefer, Christin M.; Mueller, Martin J.; Tonge, Peter J.; Kisker, Caroline

    2013-01-01

    The survival of Mycobacterium tuberculosis depends on mycolic acids, very long α-alkyl-β-hydroxy fatty acids comprising 60–90 carbon atoms. However, despite considerable efforts, little is known about how enzymes involved in mycolic acid biosynthesis recognize and bind their hydrophobic fatty acyl substrates. The condensing enzyme KasA is pivotal for the synthesis of very long (C38–42) fatty acids, the precursors of mycolic acids. To probe the mechanism of substrate and inhibitor recognition by KasA, we determined the structure of this protein in complex with a mycobacterial phospholipid and with several thiolactomycin derivatives that were designed as substrate analogs. Our structures provide consecutive snapshots along the reaction coordinate for the enzyme-catalyzed reaction and support an induced fit mechanism in which a wide cavity is established through the concerted opening of three gatekeeping residues and several α-helices. The stepwise characterization of the binding process provides mechanistic insights into the induced fit recognition in this system and serves as an excellent foundation for the development of high affinity KasA inhibitors. PMID:24108128

  17. Involvement of a Lipoxygenase-Like Enzyme in Abscisic Acid Biosynthesis 1

    PubMed Central

    Creelman, Robert A.; Bell, Erin; Mullet, John E.

    1992-01-01

    Several lines of evidence indicate that abscisic acid (ABA) is derived from 9′-cis-neoxanthin or 9′-cis-violaxanthin with xanthoxin as an intermediate. 18O-labeling experiments show incorporation primarily into the side chain carboxyl group of ABA, suggesting that oxidative cleavage occurs at the 11, 12 (11′, 12′) double bond of xanthophylls. Carbon monoxide, a strong inhibitor of heme-containing P-450 monooxygenases, did not inhibit ABA accumulation, suggesting that the oxygenase catalyzing the carotenoid cleavage step did not contain heme. This observation, plus the ability of lipoxygenase to make xanthoxin from violaxanthin, suggested that a lipoxygenase-like enzyme is involved in ABA biosynthesis. To test this idea, the ability of several soybean (Glycine max L.) lipoxygenase inhibitors (5,8,11-eicosatriynoic acid, 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and naproxen) to inhibit stress-induced ABA accumulation in soybean cell culture and soybean seedlings was determined. All lipoxygenase inhibitors significantly inhibited ABA accumulation in response to stress. These results suggest that the in vivo oxidative cleavage reaction involved in ABA biosynthesis requires activity of a nonheme oxygenase having lipoxygenase-like properties. PMID:16668998

  18. OPHIDIAN L-AMINO ACID OXIDASE. THE NATURE OF THE ENZYME-SUBSTRATE COMPLEXES.

    PubMed

    ZELLER, E A; RAMACHANDER, G; FLEISHER, G A; ISHIMARU, T; ZELLER, V

    1965-04-01

    1. To investigate the kinetics of ophidian l-amino acid oxidase, V and K(m) were determined for phenylalanines that were substituted in every ring position with groups of various size and reactivity, and for a few ring-substituted tryptophans and histidines. The venom of one representative from each of three major classes of poisonous snakes, Naja melanoleuca, Vipera russelli and Crotalus adamanteus, served as a source of the ophidian l-amino acid oxidase. Both crude and crystalline enzyme from the venom of C. adamanteus were tested. 2. The introduction of a benzene ring into glycine and alanine caused some increase of V and a very marked depression of K(m). 3. With the exception of fluorine, residues in the ortho position of phenylalanine led to a decrease of V. The rates induced by various substitutions follow the pattern: meta >/= para >/= ortho. Within the halogen series, the effects become more pronounced with increasing atomic number. 4. Ring substitution in heterocyclic amino acids also affected the V values markedly. For methyl-substituted tryptophans the pattern was: 5-methyl >/= 6-methyl >/= 4-methyl. In a few instances ring substitution accounts for a considerable elevation of V, as shown for beta-quinol-4-ylalanine and its 6-methoxy derivative. 5. The kinetic constants appear to be unaffected by relatively high concentrations of the corresponding d-amino acids. 6. A general principle that permits a uniform interpretation of a vast body of information is suggested. It is based on the assumption that most substrates form not only eutopic but also dystopic complexes with the enzyme. The latter, in contrast with the former, do not permit the formation of reaction products. K values for eutopic and dystopic complexes are computed. Similar concepts have been presented to elucidate the action of alpha-chymotrypsin (Hein & Niemann, 1962) and of monoamine oxidase.

  19. Biological Monitoring of 3-Phenoxybenzoic Acid in Urine by an Enzyme -Linked Immunosorbent Assay

    EPA Science Inventory

    An enzyme-linked immunosorbent assay (ELISA) method was employed for determination of the pyrethroid biomarker, 3-phenoxybenzoic acid (3-PBA) in human urine samples. The optimized coating antigen concentration was 0.5 ng/mL with a dilution of 1:4000 for the 3-PBA antibody and 1:6...

  20. Halogenated methanesulfonic acids: A new class of organic micropollutants in the water cycle.

    PubMed

    Zahn, Daniel; Frömel, Tobias; Knepper, Thomas P

    2016-09-15

    Mobile and persistent organic micropollutants may impact raw and drinking waters and are thus of concern for human health. To identify such possible substances of concern nineteen water samples from five European countries (France, Switzerland, The Netherlands, Spain and Germany) and different compartments of the water cycle (urban effluent, surface water, ground water and drinking water) were enriched with mixed-mode solid phase extraction. Hydrophilic interaction liquid chromatography - high resolution mass spectrometry non-target screening of these samples led to the detection and structural elucidation of seven novel organic micropollutants. One structure could already be confirmed by a reference standard (trifluoromethanesulfonic acid) and six were tentatively identified based on experimental evidence (chloromethanesulfonic acid, dichloromethanesulfonic acid, trichloromethanesulfonic acid, bromomethanesulfonic acid, dibromomethanesulfonic acid and bromochloromethanesulfonic acid). Approximated concentrations for these substances show that trifluoromethanesulfonic acid, a chemical registered under the European Union regulation REACH with a production volume of more than 100 t/a, is able to spread along the water cycle and may be present in concentrations up to the μg/L range. Chlorinated and brominated methanesulfonic acids were predominantly detected together which indicates a common source and first experimental evidence points towards water disinfection as a potential origin. Halogenated methanesulfonic acids were detected in drinking waters and thus may be new substances of concern. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Altered Expression of Urea Cycle Enzymes in Amyloid-β Protein Precursor Overexpressing PC12 Cells and in Sporadic Alzheimer's Disease Brain.

    PubMed

    Jęśko, Henryk; Lukiw, Walter J; Wilkaniec, Anna; Cieślik, Magdalena; Gąssowska-Dobrowolska, Magdalena; Murawska, Emilia; Hilgier, Wojciech; Adamczyk, Agata

    2018-01-01

    Urea cycle enzymes may play important yet poorly characterized roles in Alzheimer's disease (AD). Our previous results showed that amyloid-β (Aβ) affects urea cycle enzymes in rat pheochromocytoma (PC12) cells. The aim of the present study was to investigate the changes in arginases, other urea cycle enzymes, and nitric oxide synthases (NOSs) in PC12 cells transfected with AβPP bearing the double 'Swedish' mutation (APPsw, K670M/N671L) and in postmortem sporadic AD brain hippocampus; the mutation intensifies Aβ production and strongly associates with AD neuropathology. mRNA expression was analyzed using real-time PCR in cell cultures and DNA microarrays in hippocampal CA1 area of human AD brains. Arginase activity was measured spectrophotometrically, and arginine, ornithine, and citrulline levels by high-performance liquid chromatography. Our data demonstrated that the expression and activity of arginases (Arg1 and Arg2), as well as the expression of argininosuccinate synthase (Ass) were significantly reduced in APPsw cells compared to control. However, argininosuccinate lyase (Asl) was upregulated in APPsw cells. Real-time PCR analysis revealed significant elevation of neuronal nitric oxide synthase (Nnos) mRNA in APPsw cells, without changes in the endothelial Enos, whereas inducible Inos was undetectable. The changes were found to follow closely those observed in the human hippocampal CA1 region of sporadic AD brains. The changes in enzyme expression were accompanied in APPsw cells by significantly elevated citrulline, ornithine, and arginine. Our findings demonstrate that AβPP/Aβ alters arginine metabolism and induces a shift of cellular homeostasis that may support the oxidative/nitrosative stress observed in AD.

  2. Identification of the algal dimethyl sulfide-releasing enzyme: A missing link in the marine sulfur cycle

    NASA Astrophysics Data System (ADS)

    Alcolombri, Uria; Ben-Dor, Shifra; Feldmesser, Ester; Levin, Yishai; Tawfik, Dan S.; Vardi, Assaf

    2015-06-01

    Algal blooms produce large amounts of dimethyl sulfide (DMS), a volatile with a diverse signaling role in marine food webs that is emitted to the atmosphere, where it can affect cloud formation. The algal enzymes responsible for forming DMS from dimethylsulfoniopropionate (DMSP) remain unidentified despite their critical role in the global sulfur cycle. We identified and characterized Alma1, a DMSP lyase from the bloom-forming algae Emiliania huxleyi. Alma1 is a tetrameric, redox-sensitive enzyme of the aspartate racemase superfamily. Recombinant Alma1 exhibits biochemical features identical to the DMSP lyase in E. huxleyi, and DMS released by various E. huxleyi isolates correlates with their Alma1 levels. Sequence homology searches suggest that Alma1 represents a gene family present in major, globally distributed phytoplankton taxa and in other marine organisms.

  3. Submolecular regulation of cell transformation by deuterium depleting water exchange reactions in the tricarboxylic acid substrate cycle.

    PubMed

    Boros, László G; D'Agostino, Dominic P; Katz, Howard E; Roth, Justine P; Meuillet, Emmanuelle J; Somlyai, Gábor

    2016-02-01

    The naturally occurring isotope of hydrogen ((1)H), deuterium ((2)H), could have an important biological role. Deuterium depleted water delays tumor progression in mice, dogs, cats and humans. Hydratase enzymes of the tricarboxylic acid (TCA) cycle control cell growth and deplete deuterium from redox cofactors, fatty acids and DNA, which undergo hydride ion and hydrogen atom transfer reactions. A model is proposed that emphasizes the terminal complex of mitochondrial electron transport chain reducing molecular oxygen to deuterium depleted water (DDW); this affects gluconeogenesis as well as fatty acid oxidation. In the former, the DDW is thought to diminish the deuteration of sugar-phosphates in the DNA backbone, helping to preserve stability of hydrogen bond networks, possibly protecting against aneuploidy and resisting strand breaks, occurring upon exposure to radiation and certain anticancer chemotherapeutics. DDW is proposed here to link cancer prevention and treatment using natural ketogenic diets, low deuterium drinking water, as well as DDW production as the mitochondrial downstream mechanism of targeted anti-cancer drugs such as Avastin and Glivec. The role of (2)H in biology is a potential missing link to the elusive cancer puzzle seemingly correlated with cancer epidemiology in western populations as a result of excessive (2)H loading from processed carbohydrate intake in place of natural fat consumption. Published by Elsevier Ltd.

  4. Production of Caproic Acid from Mixed Organic Waste: An Environmental Life Cycle Perspective

    PubMed Central

    2017-01-01

    Caproic acid is an emerging platform chemical with diverse applications. Recently, a novel biorefinery process, that is, chain elongation, was developed to convert mixed organic waste and ethanol into renewable caproic acids. In the coming years, this process may become commercialized, and continuing to improve on the basis of numerous ongoing technological and microbiological studies. This study aims to analyze the environmental performance of caproic acid production from mixed organic waste via chain elongation at this current, early stage of technological development. To this end, a life cycle assessment (LCA) was performed to evaluate the environmental impact of producing 1 kg caproic acid from organic waste via chain elongation, in both a lab-scale and a pilot-scale system. Two mixed organic waste were used as substrates: the organic fraction of municipal solid waste (OFMSW) and supermarket food waste (SFW). Ethanol use was found to be the dominant cause of environmental impact over the life cycle. Extraction solvent recovery was found to be a crucial uncertainty that may have a substantial influence on the life-cycle impacts. We recommend that future research and industrial producers focus on the reduction of ethanol use in chain elongation and improve the recovery efficiency of the extraction solvent. PMID:28513150

  5. Development of an enzyme-linked immunosorbent assay and immunoaffinity chromatography for glycyrrhizic acid using an anti-glycyrrhizic acid monoclonal antibody.

    PubMed

    Zhang, Yue; Qu, Huihua; Zeng, Wenhao; Zhao, Yan; Shan, Wenchao; Wang, Xueqian; Wang, Qingguo; Zhao, Yan

    2015-07-01

    In this work, a new monoclonal antibody specific for glycyrrhizic acid was prepared and characterized. A hybridoma secreting an anti-glycyrrhizic acid monoclonal antibody was produced by fusing splenocytes from a mouse immunized against a glycyrrhizic acid-bovine serum albumin conjugate with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line (Sp2/0-Ag14). Subsequently, an indirect, competitive enzyme-linked immunosorbent assay for glycyrrhizic acid was developed using the monoclonal antibody. In this assay, we detected an effective measuring range of 78.12-2500 ng/mL. Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10%. In addition, glycyrrhizic acid levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. We then successfully developed a reliable immunoaffinity chromatography to separate glycyrrhizic acid completely from its parent medicine. These methods will contribute to further research investigations to better understand the interactions of glycyrrhizic acid with other drugs in the complex system of traditional Chinese medicine. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Application of ionic liquids based enzyme-assisted extraction of chlorogenic acid from Eucommia ulmoides leaves.

    PubMed

    Liu, Tingting; Sui, Xiaoyu; Li, Li; Zhang, Jie; Liang, Xin; Li, Wenjing; Zhang, Honglian; Fu, Shuang

    2016-01-15

    A new approach for ionic liquid based enzyme-assisted extraction (ILEAE) of chlorogenic acid (CGA) from Eucommia ulmoides is presented in which enzyme pretreatment was used in ionic liquids aqueous media to enhance extraction yield. For this purpose, the solubility of CGA and the activity of cellulase were investigated in eight 1-alkyl-3-methylimidazolium ionic liquids. Cellulase in 0.5 M [C6mim]Br aqueous solution was found to provide better performance in extraction. The factors of ILEAE procedures including extraction time, extraction phase pH, extraction temperatures and enzyme concentrations were investigated. Moreover, the novel developed approach offered advantages in term of yield and efficiency compared with other conventional extraction techniques. Scanning electronic microscopy of plant samples indicated that cellulase treated cell wall in ionic liquid solution was subjected to extract, which led to more efficient extraction by reducing mass transfer barrier. The proposed ILEAE method would develope a continuous process for enzyme-assisted extraction including enzyme incubation and solvent extraction process. In this research, we propose a novel view for enzyme-assisted extraction of plant active component, besides concentrating on enzyme facilitated cell wall degradation, focusing on improvement of bad permeability of ionic liquids solutions. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Highly sensitive and selective microRNA detection based on DNA-bio-bar-code and enzyme-assisted strand cycle exponential signal amplification.

    PubMed

    Dong, Haifeng; Meng, Xiangdan; Dai, Wenhao; Cao, Yu; Lu, Huiting; Zhou, Shufeng; Zhang, Xueji

    2015-04-21

    Herein, a highly sensitive and selective microRNA (miRNA) detection strategy using DNA-bio-bar-code amplification (BCA) and Nb·BbvCI nicking enzyme-assisted strand cycle for exponential signal amplification was designed. The DNA-BCA system contains a locked nucleic acid (LNA) modified DNA probe for improving hybridization efficiency, while a signal reported molecular beacon (MB) with an endonuclease recognition site was designed for strand cycle amplification. In the presence of target miRNA, the oligonucleotides functionalized magnetic nanoprobe (MNP-DNA) and gold nanoprobe (AuNP-DNA) with numerous reported probes (RP) can hybridize with target miRNA, respectively, to form a sandwich structure. After sandwich structures were separated from the solution by the magnetic field, the RP were released under high temperature to recognize the MB and cleaved the hairpin DNA to induce the dissociation of RP. The dissociated RP then triggered the next strand cycle to produce exponential fluorescent signal amplification for miRNA detection. Under optimized conditions, the exponential signal amplification system shows a good linear range of 6 orders of magnitude (from 0.3 pM to 3 aM) with limit of detection (LOD) down to 52.5 zM, while the sandwich structure renders the system with high selectivity. Meanwhile, the feasibility of the proposed strategy for cell miRNA detection was confirmed by analyzing miRNA-21 in HeLa lysates. Given the high-performance for miRNA analysis, the strategy has a promising application in biological detection and in clinical diagnosis.

  8. Clinical benefit using sperm hyaluronic acid binding technique in ICSI cycles: a systematic review and meta-analysis.

    PubMed

    Beck-Fruchter, Ronit; Shalev, Eliezer; Weiss, Amir

    2016-03-01

    The human oocyte is surrounded by hyaluronic acid, which acts as a natural selector of spermatozoa. Human sperm that express hyaluronic acid receptors and bind to hyaluronic acid have normal shape, minimal DNA fragmentation and low frequency of chromosomal aneuploidies. Use of hyaluronic acid binding assays in intracytoplasmic sperm injection (ICSI) cycles to improve clinical outcomes has been studied, although none of these studies had sufficient statistical power. In this systematic review and meta-analysis, electronic databases were searched up to June 2015 to identify studies of ICSI cycles in which spermatozoa able to bind hyaluronic acid was selected. The main outcomes were fertilization rate and clinical pregnancy rate. Secondary outcomes included cleavage rate, embryo quality, implantation rate, spontaneous abortion and live birth rate. Seven studies and 1437 cycles were included. Use of hyaluronic acid binding sperm selection technique yielded no improvement in fertilization and pregnancy rates. A meta-analysis of all available studies showed an improvement in embryo quality and implantation rate; an analysis of prospective studies only showed an improvement in embryo quality. Evidence does not support routine use of hyaluronic acid binding assays in all ICSI cycles. Identification of patients that might benefit from this technique needs further study. Copyright © 2015 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  9. Evaluation of Krebs cycle enzymes in the brain of rats after chronic administration of antidepressants.

    PubMed

    Scaini, Giselli; Santos, Patricia M; Benedet, Joana; Rochi, Natália; Gomes, Lara M; Borges, Lislaine S; Rezin, Gislaine T; Pezente, Daiana P; Quevedo, João; Streck, Emilio L

    2010-05-31

    Several works report brain impairment of metabolism as a mechanism underlying depression. Citrate synthase and succinate dehydrogenase are enzymes localized within cells in the mitochondrial matrix and are important steps of Krebs cycle. In addition, citrate synthase has been used as a quantitative enzyme marker for the presence of intact mitochondria. Thus, we investigated citrate synthase and succinate dehydrogenase activities from rat brain after chronic administration of paroxetine, nortriptiline and venlafaxine. Adult male Wistar rats received daily injections of paroxetine (10mg/kg), nortriptiline (15mg/kg), venlafaxine (10mg/kg) or saline in 1.0mL/kg volume for 15 days. Twelve hours after the last administration, the rats were killed by decapitation, the hippocampus, striatum and prefrontal cortex were immediately removed, and activities of citrate synthase and succinate dehydrogenase were measured. We verified that chronic administration of paroxetine increased citrate synthase activity in the prefrontal cortex, hippocampus, striatum and cerebral cortex of adult rats; cerebellum was not affected. Chronic administration of nortriptiline and venlafaxine did not affect the enzyme activity in these brain areas. Succinate dehydrogenase activity was increased by chronic administration of paroxetine and nortriptiline in the prefrontal cortex, hippocampus, striatum and cerebral cortex of adult rats; cerebellum was not affected either. Chronic administration of venlafaxine increased succinate dehydrogenase activity in prefrontal cortex, but did not affect the enzyme activity in cerebellum, hippocampus, striatum and cerebral cortex. Considering that metabolism impairment is probably involved in the pathophysiology of depressive disorders, an increase in these enzymes by antidepressants may be an important mechanism of action of these drugs. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  10. Inactivation of thiol-dependent enzymes by hypothiocyanous acid: role of sulfenyl thiocyanate and sulfenic acid intermediates

    PubMed Central

    Barrett, Tessa J.; Pattison, David I.; Leonard, Stephen E.; Carroll, Kate S.; Davies, Michael J.; Hawkins, Clare L.

    2012-01-01

    Myeloperoxidase (MPO) forms reactive oxidants including hypochlorous and hypothiocyanous acids (HOCl and HOSCN) under inflammatory conditions. HOCl causes extensive tissue damage and plays a role in the progression of many inflammatory-based diseases. Although HOSCN is a major MPO oxidant, particularly in smokers, who have elevated plasma thiocyanate, the role of this oxidant in disease is poorly characterized. HOSCN induces cellular damage by targeting thiols. However, the specific targets and mechanisms involved in this process are not well defined. We show that exposure of macrophages to HOSCN results in the inactivation of intracellular enzymes, including creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In each case, the active-site thiol residue is particularly sensitive to oxidation, with evidence for reversible inactivation and the formation of sulfenyl thiocyanate and sulfenic acid intermediates, on treatment with HOSCN (less than fivefold molar excess). Experiments with DAz-2, a cell-permeable chemical trap for sulfenic acids, demonstrate that these intermediates are formed on many cellular proteins, including GAPDH and CK, in macrophages exposed to HOSCN. This is the first direct evidence for the formation of protein sulfenic acids in HOSCN-treated cells and highlights the potential of this oxidant to perturb redox signaling processes. PMID:22248862

  11. Recovery of Phenolic Acid and Enzyme Production from Corn Silage Biologically Treated by Trametes versicolor.

    PubMed

    Bucić-Kojić, Ana; Šelo, Gordana; Zelić, Bruno; Planinić, Mirela; Tišma, Marina

    2017-03-01

    Corn silage is used as high-energy forage for dairy cows and more recently for biogas production in a process of anaerobic co-digestion with cow manure. In this work, fresh corn silage after the harvest was used as a substrate in solid-state fermentations with T. versicolor with the aim of phenolic acid recovery and enzyme (laccase and manganese peroxidase) production. During 20 days of fermentation, 10.4-, 3.4-, 3.0-, and 1.8-fold increments in extraction yield of syringic acid, vanillic acid, p-hydroxybenzoic acid, and caffeic acid, respectively, were reached when compared to biologically untreated corn silage. Maximal laccase activity was gained on the 4th day of fermentation (V.A. = 180.2 U/dm 3 ), and manganese peroxidase activity was obtained after the 3rd day of fermentation (V.A. = 30.1 U/dm 3 ). The addition of copper(II) sulfate as inducer during solid state fermentation resulted in 8.5- and 7-fold enhancement of laccase and manganese peroxidase activities, respectively. Furthermore, the influence of pH and temperature on enzyme activities was investigated. Maximal activity of laccase was obtained at T = 50 °C and pH = 3.0, while manganese peroxidase is active at temperature range T = 45-70 °C with the maximal activity at pH = 4.5.

  12. Liver X receptor agonists augment human islet function through activation of anaplerotic pathways and glycerolipid/free fatty acid cycling.

    PubMed

    Ogihara, Takeshi; Chuang, Jen-Chieh; Vestermark, George L; Garmey, James C; Ketchum, Robert J; Huang, Xiaolun; Brayman, Kenneth L; Thorner, Michael O; Repa, Joyce J; Mirmira, Raghavendra G; Evans-Molina, Carmella

    2010-02-19

    Recent studies in rodent models suggest that liver X receptors (LXRs) may play an important role in the maintenance of glucose homeostasis and islet function. To date, however, no studies have comprehensively examined the role of LXRs in human islet biology. Human islets were isolated from non-diabetic donors and incubated in the presence or absence of two synthetic LXR agonists, TO-901317 and GW3965, under conditions of low and high glucose. LXR agonist treatment enhanced both basal and stimulated insulin secretion, which corresponded to an increase in the expression of genes involved in anaplerosis and reverse cholesterol transport. Furthermore, enzyme activity of pyruvate carboxylase, a key regulator of pyruvate cycling and anaplerotic flux, was also increased. Whereas LXR agonist treatment up-regulated known downstream targets involved in lipogenesis, we observed no increase in the accumulation of intra-islet triglyceride at the dose of agonist used in our study. Moreover, LXR activation increased expression of the genes encoding hormone-sensitive lipase and adipose triglyceride lipase, two enzymes involved in lipolysis and glycerolipid/free fatty acid cycling. Chronically, insulin gene expression was increased after treatment with TO-901317, and this was accompanied by increased Pdx-1 nuclear protein levels and enhanced Pdx-1 binding to the insulin promoter. In conclusion, our data suggest that LXR agonists have a direct effect on the islet to augment insulin secretion and expression, actions that should be considered either as therapeutic or unintended side effects, as these agents are developed for clinical use.

  13. Structural Dissection of the Maltodextrin Disproportionation Cycle of the Arabidopsis Plastidial Disproportionating Enzyme 1 (DPE1)*

    PubMed Central

    O'Neill, Ellis C.; Stevenson, Clare E. M.; Tantanarat, Krit; Latousakis, Dimitrios; Donaldson, Matthew I.; Rejzek, Martin; Nepogodiev, Sergey A.; Limpaseni, Tipaporn; Field, Robert A.; Lawson, David M.

    2015-01-01

    The degradation of transitory starch in the chloroplast to provide fuel for the plant during the night requires a suite of enzymes that generate a series of short chain linear glucans. However, glucans of less than four glucose units are no longer substrates for these enzymes, whereas export from the plastid is only possible in the form of either maltose or glucose. In order to make use of maltotriose, which would otherwise accumulate, disproportionating enzyme 1 (DPE1; a 4-α-glucanotransferase) converts two molecules of maltotriose to a molecule of maltopentaose, which can now be acted on by the degradative enzymes, and one molecule of glucose that can be exported. We have determined the structure of the Arabidopsis plastidial DPE1 (AtDPE1), and, through ligand soaking experiments, we have trapped the enzyme in a variety of conformational states. AtDPE1 forms a homodimer with a deep, long, and open-ended active site canyon contained within each subunit. The canyon is divided into donor and acceptor sites with the catalytic residues at their junction; a number of loops around the active site adopt different conformations dependent on the occupancy of these sites. The “gate” is the most dynamic loop and appears to play a role in substrate capture, in particular in the binding of the acceptor molecule. Subtle changes in the configuration of the active site residues may prevent undesirable reactions or abortive hydrolysis of the covalently bound enzyme-substrate intermediate. Together, these observations allow us to delineate the complete AtDPE1 disproportionation cycle in structural terms. PMID:26504082

  14. In Vivo Roles of Fatty Acid Biosynthesis Enzymes in Biosynthesis of Biotin and α-Lipoic Acid in Corynebacterium glutamicum.

    PubMed

    Ikeda, Masato; Nagashima, Takashi; Nakamura, Eri; Kato, Ryosuke; Ohshita, Masakazu; Hayashi, Mikiro; Takeno, Seiki

    2017-10-01

    For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicum IMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis , which use an individual nonaggregating type II fatty acid synthase

  15. Enzyme-assisted extraction enhancing the umami taste amino acids recovery from several cultivated mushrooms.

    PubMed

    Poojary, Mahesha M; Orlien, Vibeke; Passamonti, Paolo; Olsen, Karsten

    2017-11-01

    In this study, enzyme-assisted extraction was performed to extract umami taste and total free amino acids (FAAs) from the six different mushrooms including shiitake (Lentinus edodes), oyster (Pleurotus ostreatus), tea tree (Agrocybe aegerita) and, white, brown and portobello champignons (Agaricus bisporus). β-Glucanase and Flavourzyme® were used as the enzymes for cell wall and proteins hydrolysis, respectively. It was found that β-glucanase treatment alone did not enhance the extraction efficiency, however in combination, β-glucanase and Flavourzyme® enhanced the extraction efficiency significantly up to 20-fold compared to conventional HCl mediated extraction, depending on the mushroom species. The optimal conditions for the enzyme treatment were: water as extraction solvent (initial pH = 7), enzyme concentration of 5% v/w each of β-glucanase and Flavourzyme®, temperature 50°C and an incubation time of 1h. White and brown champignons were found to be the richest source of umami taste FAAs (26.75±1.07 and 25.6±0.9mg/g DM, respectively). Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Exquisite Enzyme-Fenton Biomimetic Catalysts for Hydroxyl Radical Production by Mimicking an Enzyme Cascade.

    PubMed

    Zhang, Qi; Chen, Shuo; Wang, Hua; Yu, Hongtao

    2018-03-14

    Hydrogen peroxide (H 2 O 2 ) is a key reactant in the Fenton process. As a byproduct of enzymatic reaction, H 2 O 2 can be obtained via catalytical oxidation of glucose using glucose oxidase in the presence of O 2 . Another oxidation product (gluconic acid) can suitably adjust the microenvironmental pH contributing to the Fe 3+ /Fe 2+ cycle in the Fenton reaction. Enzymes are extremely efficient at catalyzing a variety of reactions with high catalytic activity, substrate specificity, and yields in living organisms. Inspired by the multiple functions of natural multienzyme systems, an exquisite nanozyme-modified α-FeOOH/porous carbon (PC) biomimetic catalyst constructed by in situ growth of glucose oxidase-mimicking Au nanoparticles and crystallization of adsorbed ferric ions within carboxyl into hierarchically PC is developed as an efficient enzyme-Fenton catalyst. The products (H 2 O 2 , ∼4.07 mmol·L -1 ) of the first enzymatic reaction are immediately used as substrates for the second Fenton-like reaction to generate the valuable • OH (∼96.84 μmol·L -1 ), thus mimicking an enzyme cascade pathway. α-FeOOH nanocrystals, attached by C-O-Fe bondings, are encapsulated into the mesoporous PC frameworks, facilitating the electron transfer between α-FeOOH and the PC support and greatly suppressing iron leaching. This study paves a new avenue for designing biomimetic enzyme-based Fenton catalysts mimicking a natural system for • OH production.

  17. The enzymic hydrolysis of amygdalin

    PubMed Central

    Haisman, D. R.; Knight, D. J.

    1967-01-01

    Chromatographic examination has shown that the enzymic hydrolysis of amygdalin by an almond β-glucosidase preparation proceeds consecutively: amygdalin was hydrolysed to prunasin and glucose; prunasin to mandelonitrile and glucose; mandelonitrile to benzaldehyde and hydrocyanic acid. Gentiobiose was not formed during the enzymic hydrolysis. The kinetics of the production of mandelonitrile and hydrocyanic acid from amygdalin by the action of the β-glucosidase preparation favour the probability that three different enzymes are involved, each specific for one hydrolytic stage, namely, amygdalin lyase, prunasin lyase and hydroxynitrile lyase. Cellulose acetate electrophoresis of the enzyme preparation showed that it contained a number of enzymically active components. PMID:4291788

  18. 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.

  19. Role of keto acids and reduced-oxygen-scavenging enzymes in the growth of Legionella species.

    PubMed Central

    Pine, L; Hoffman, P S; Malcolm, G B; Benson, R F; Franzus, M J

    1986-01-01

    Keto acids and reduced-oxygen-scavenging enzymes were examined for their roles in supporting the growth of Legionella species and for their potential reactions between the chemical components of the media. When grown in an experimental ACES (2-[(2-amino-2-oxoethyl)-amino] ethanesulfonic acid)-buffered chemically defined (ABCD) broth, the presence of keto acids shortened the lag periods, increased the rates of growth, and gave maximum cell yields. In addition, keto acids affected the specific activities of reduced-oxygen-scavenging enzymes determined during growth. The specific activities of superoxide dismutase of Legionella pneumophila (Knoxville) and L. dumoffii (TEX-KL) were increased three- to eightfold, while that of L. bozemanii (WIGA) was not affected. All strains appeared to be equally sensitive to the effects of superoxide anion (O2-) generated by light-activated riboflavin, and all were equally protected by the presence of keto acids in the ABCD broth. Production of trace amounts of acetate and succinate in pyruvate- and alpha-ketoglutarate-containing media exposed to light suggested that hydrogen peroxide was formed. Pyruvate and alpha-ketoglutarate were products of growth on amino acids, and there was no quantitative evidence that these keto acids were metabolized when they were added to the medium. The rate of cysteine oxidation in ABCD broth was increased by the presence of ferric ion or by exposure to light or by both, and keto acids reduced the rate of this oxidation. ACES buffer was a substrate for the production of O2- in the presence of light, and the combined addition of Fe2+ ions, cysteine, and either keto acid to the medium strongly inhibited the production of O2-. Thus, keto acids inhibited the rate of cysteine oxidation, they stimulated rapid growth by an unknown process, and, in combination with added Fe2+ ions and cysteine, they reversed the toxic effects of light by inhibiting O2- production. PMID:3009529

  20. Differentiated effect of ageing on the enzymes of Krebs' cycle, electron transfer complexes and glutamate metabolism of non-synaptic and intra-synaptic mitochondria from cerebral cortex.

    PubMed

    Villa, R F; Gorini, A; Hoyer, S

    2006-11-01

    The effect of ageing on the activity of enzymes linked to Krebs' cycle, electron transfer chain and glutamate metabolism was studied in three different types of mitochondria of cerebral cortex of 1-year old and 2-year old male Wistar rats. We assessed the maximum rate (V(max)) of the mitochondrial enzyme activities in non-synaptic perikaryal mitochondria, and in two populations of intra-synaptic mitochondria. The results indicated that: (i) in normal, steady-state cerebral cortex the values of the catalytic activities of the enzymes markedly differed in the various populations of mitochondria; (ii) in intra-synaptic mitochondria, ageing affected the catalytic properties of the enzymes linked to Krebs' cycle, electron transfer chain and glutamate metabolism; (iii) these changes were more evident in intra-synaptic "heavy" than "light" mitochondria. These results indicate a different age-related vulnerability of subpopulations of mitochondria in vivo located into synapses than non-synaptic ones.

  1. Changes in sodium and uric acid concentrations in plasma during the menstrual cycle.

    PubMed

    Mira, M; Stewart, P M; Gebski, V; Llewellyn-Jones, D; Abraham, S F

    1984-03-01

    Hormonal changes during the menstrual cycle are well documented, but many other biochemical variables have not been studied. We find that in the luteal phase of the menstrual cycle the concentrations of sodium and uric acid are significantly lower. The changes may be of significance for the determination of the normal reference interval.

  2. Regulation of retinoic acid synthetic enzymes by WT1 and HDAC inhibitors in 293 cells.

    PubMed

    Li, Yifan; Wang, Lei; Ai, Weipeng; He, Nianhui; Zhang, Lin; Du, Jihui; Wang, Yong; Mao, Xingjian; Ren, Junqi; Xu, Dan; Zhou, Bei; Li, Rong; Mai, Liwen

    2017-09-01

    All-trans retinoic acid (atRA), which is mainly generated endogenously via two steps of oxidation from vitamin A (retinol), plays an indispensible role in the development of the kidney and many other organs. Enzymes that catalyze the oxidation of retinol to generate atRA, including aldehyde dehydrogenase 1 family (ALDH1)A1, ALDH1A2 and ALDH1A3, exhibit complex expression patterns at different stages of renal development. However, molecular triggers that control these differential expression levels are poorly understood. In this study, we provide in vitro evidence to demonstrate that Wilms' tumor 1 (WT1) negatively regulates the expression of the atRA synthetic enzymes, ALDH1A1, ALDH1A2 and ALDH1A3, in the 293 cell line, leading to significant blockage of atRA production. Furthermore, we demonstrate that the suppression of ALDH1A1 by WT1 can be markedly attenuated by histone deacetylase inhibitors (HDACis). Taken together, we provide evidence to indicate that WT1 and HDACs are strong regulators of endogenous retinoic acid synthetic enzymes in 293 cells, indicating that they may be involved in the regulation of atRA synthesis.

  3. Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

    PubMed

    Daar, I O; Artymiuk, P J; Phillips, D C; Maquat, L E

    1986-10-01

    Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G X C----C X G transversion in the codon for amino acid-104 and specifies a structurally altered protein in which a glutamate residue is replaced by an aspartate residue. The importance of glutamate-104 to enzyme structure and function is implicated by its conservation in the TPI protein of all species that have been characterized to date. The glutamate-to-aspartate substitution results in a thermolabile enzyme as demonstrated by assays of TPI activity in cultured fibroblasts of each patient and cultured Chinese hamster ovary (CHO) cells that were stably transformed with the mutant alleles. Although this substitution conserves the overall charge of amino acid-104, the x-ray crystal structure of chicken TPI indicates that the loss of a side-chain methylene group (-CH2CH2COO- ---- -CH2COO-) is sufficient to disrupt the counterbalancing of charges that normally exists within a hydrophobic pocket of the native enzyme.

  4. Imbricatolic acid from Juniperus communis L. prevents cell cycle progression in CaLu-6 cells.

    PubMed

    De Marino, Simona; Cattaneo, Fabio; Festa, Carmen; Zollo, Franco; Iaccio, Annalisa; Ammendola, Rosario; Incollingo, Filomena; Iorizzi, Maria

    2011-11-01

    Imbricatolic acid was isolated from the methanolic extract of the fresh ripe berries of Juniperus communis (Cupressaceae) together with sixteen known compounds and a new dihydrobenzofuran lignan glycoside named juniperoside A. Their structures were determined by spectroscopic methods and by comparison with the spectral data reported in literature. Imbricatolic acid was evaluated for its ability to prevent cell cycle progression in p53-null CaLu-6 cells. This compound induces the upregulation of cyclin-dependent kinase inhibitors and their accumulation in the G1 phase of the cell cycle, as well as the degradation of cyclins A, D1, and E1. Furthermore, no significant imbricatolic acid-induced apoptosis was observed. Therefore, this plant-derived compound may play a role in the control of cell cycle. © Georg Thieme Verlag KG Stuttgart · New York.

  5. Chronotherapeutic effect of fisetin on expression of urea cycle enzymes and inflammatory markers in hyperammonaemic rats.

    PubMed

    Subramanian, Perumal; Jayakumar, Murugesan; Jayapalan, Jaime Jacqueline; Hashim, Onn Haji

    2014-12-01

    Elevated blood ammonia leads to hyperammonaemia that affects vital central nervous system (CNS) functions. Fisetin, a naturally occurring flavonoid, exhibits therapeutic benefits, such as anti-cancer, anti-diabetic, anti-oxidant, anti-angiogenic, neuroprotective and neurotrophic effects. In this study, the chronotherapeutic effect of fisetin on ammonium chloride (AC)-induced hyperammonaemic rats was investigated, to ascertain the time point at which the maximum drug effect is achieved. The anti-hyperammonaemic potential of fisetin (50mg/kg b.w. oral) was analysed when administered to AC treated (100mg/kg b.w. i.p.) rats at 06:00, 12:00, 18:00 and 00:00h. Amelioration of pathophysiological conditions by fisetin at different time points was measured by analysing the levels of expression of liver urea cycle enzymes (carbamoyl phosphate synthetase-I (CPS-I), ornithine transcarbamoylase (OTC) and argininosuccinate synthetase (ASS)), nuclear transcription factor kappaB (NF-κB p65), brain glutamine synthetase (GS) and inducible nitric oxide synthase (iNOS) by Western blot analysis. Fisetin increased the expression of CPS-I, OTC, ASS and GS and decreased iNOS and NF-κB p65 in hyperammonaemic rats. Fisetin administration at 00:00h showed more significant effects on the expression of liver and brain markers, compared with other time points. Fisetin could exhibit anti-hyperammonaemic effect owing to its anti-oxidant and cytoprotective influences. The temporal variation in the effect of fisetin could be due to the (i) chronopharmacological, chronopharmacokinetic properties of fisetin and (ii) modulations in the endogenous circadian rhythms of urea cycle enzymes, brain markers, redox enzymes and renal clearance during hyperammonaemia by fisetin. However, future studies in these lines are necessitated. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  6. The effect of propionic acid and valeric acid on the cell cycle in root meristems of Pisum sativum

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tramontano, W.A.; Yang, Shauyu; Delillo, A.R.

    1990-01-01

    Propionic acid and valeric acid at 1mM reduced the mitotic index of root meristem cells of Pisum sativum to < 1% after 12 hr in aerated White's medium. This effect varied with different acid concentrations. After a 12 hr exposure to either acid, seedlings transferred to fresh medium without either acid, resumed their normal mitotic index after 12 hr, with a burst of mitosis 8 hr post-transfer. Exposure of root meristem cells to either acid also inhibited ({sup 3}H)-TdR incorporation. Neither acid significantly altered the distribution of meristematic cells in G1 and G2 after 12 hr. The incorporation of ({supmore » 3}H) - uridine was also unaltered by the addition of either acid. This information suggests that propionic acid and valeric acid, limit progression through the cell cycle by inhibiting DNA synthesis and arresting cells in G1 and G2. These results were consistent with previous data which utilized butyric acid.« less

  7. Lipotoxicity in steatohepatitis occurs despite an increase in tricarboxylic acid cycle activity

    PubMed Central

    Patterson, Rainey E.; Kalavalapalli, Srilaxmi; Williams, Caroline M.; Nautiyal, Manisha; Mathew, Justin T.; Martinez, Janie; Reinhard, Mary K.; McDougall, Danielle J.; Rocca, James R.; Yost, Richard A.; Cusi, Kenneth; Garrett, Timothy J.

    2016-01-01

    The hepatic tricarboxylic acid (TCA) cycle is central to integrating macronutrient metabolism and is closely coupled to cellular respiration, free radical generation, and inflammation. Oxidative flux through the TCA cycle is induced during hepatic insulin resistance, in mice and humans with simple steatosis, reflecting early compensatory remodeling of mitochondrial energetics. We hypothesized that progressive severity of hepatic insulin resistance and the onset of nonalcoholic steatohepatitis (NASH) would impair oxidative flux through the hepatic TCA cycle. Mice (C57/BL6) were fed a high-trans-fat high-fructose diet (TFD) for 8 wk to induce simple steatosis and NASH by 24 wk. In vivo fasting hepatic mitochondrial fluxes were determined by 13C-nuclear magnetic resonance (NMR)-based isotopomer analysis. Hepatic metabolic intermediates were quantified using mass spectrometry-based targeted metabolomics. Hepatic triglyceride accumulation and insulin resistance preceded alterations in mitochondrial metabolism, since TCA cycle fluxes remained normal during simple steatosis. However, mice with NASH had a twofold induction (P < 0.05) of mitochondrial fluxes (μmol/min) through the TCA cycle (2.6 ± 0.5 vs. 5.4 ± 0.6), anaplerosis (9.1 ± 1.2 vs. 16.9 ± 2.2), and pyruvate cycling (4.9 ± 1.0 vs. 11.1 ± 1.9) compared with their age-matched controls. Induction of the TCA cycle activity during NASH was concurrent with blunted ketogenesis and accumulation of hepatic diacylglycerols (DAGs), ceramides (Cer), and long-chain acylcarnitines, suggesting inefficient oxidation and disposal of excess free fatty acids (FFA). Sustained induction of mitochondrial TCA cycle failed to prevent accretion of “lipotoxic” metabolites in the liver and could hasten inflammation and the metabolic transition to NASH. PMID:26814015

  8. Enzyme-free detection and quantification of double-stranded nucleic acids.

    PubMed

    Feuillie, Cécile; Merheb, Maxime Mohamad; Gillet, Benjamin; Montagnac, Gilles; Hänni, Catherine; Daniel, Isabelle

    2012-08-01

    We have developed a fully enzyme-free SERRS hybridization assay for specific detection of double-stranded DNA sequences. Although all DNA detection methods ranging from PCR to high-throughput sequencing rely on enzymes, this method is unique for being totally non-enzymatic. The efficiency of enzymatic processes is affected by alterations, modifications, and/or quality of DNA. For instance, a limitation of most DNA polymerases is their inability to process DNA damaged by blocking lesions. As a result, enzymatic amplification and sequencing of degraded DNA often fail. In this study we succeeded in detecting and quantifying, within a mixture, relative amounts of closely related double-stranded DNA sequences from Rupicapra rupicapra (chamois) and Capra hircus (goat). The non-enzymatic SERRS assay presented here is the corner stone of a promising approach to overcome the failure of DNA polymerase when DNA is too degraded or when the concentration of polymerase inhibitors is too high. It is the first time double-stranded DNA has been detected with a truly non-enzymatic SERRS-based method. This non-enzymatic, inexpensive, rapid assay is therefore a breakthrough in nucleic acid detection.

  9. EVALUATION OF AN ENZYME-LINKED IMMUNOSORBENT ASSAY FOR BIOLOGICAL MONITORING OF 3-PHENOXYBENZOIC ACID IN URINE

    EPA Science Inventory

    Abstract describes the development of an enzyme-linked immunosorbent assay (ELISA) method for monitoring 2,4-dichlorophenoxyacetic acid (2,4-D exposures). The ELISA is compared with a gas chromatograhy/mass spectrometry procedure. ELISA method development steps and comparative ...

  10. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS–PKS hybrid enzyme

    PubMed Central

    Yun, Choong-Soo; Motoyama, Takayuki; Osada, Hiroyuki

    2015-01-01

    Tenuazonic acid (TeA) is a well-known mycotoxin produced by various plant pathogenic fungi. However, its biosynthetic gene has been unknown to date. Here we identify the TeA biosynthetic gene from Magnaporthe oryzae by finding two TeA-inducing conditions of a low-producing strain. We demonstrate that TeA is synthesized from isoleucine and acetoacetyl-coenzyme A by TeA synthetase 1 (TAS1). TAS1 is a unique non-ribosomal peptide synthetase and polyketide synthase (NRPS–PKS) hybrid enzyme that begins with an NRPS module. In contrast to other NRPS/PKS hybrid enzymes, the PKS portion of TAS1 has only a ketosynthase (KS) domain and this domain is indispensable for TAS1 activity. Phylogenetic analysis classifies this KS domain as an independent clade close to type I PKS KS domain. We demonstrate that the TAS1 KS domain conducts the final cyclization step for TeA release. These results indicate that TAS1 is a unique type of NRPS–PKS hybrid enzyme. PMID:26503170

  11. Analysis of 16S rRNA gene lactic acid bacteria (LAB) isolate from Markisa fruit (Passiflora sp.) as a producer of protease enzyme and probiotics

    NASA Astrophysics Data System (ADS)

    Hidayat, Habibi

    2017-03-01

    16S rRNA gene analysis of bacteria lactic acid (LAB) isolate from Markisa Kuning Fruit (Passiflora edulis var. flavicarpa) as a producer of protease enzyme and probiotics has been done. The aim of the study is to determine the protease enzyme activity and 16S rRNA gene amplification using PCR. The calculation procedure was done to M4 isolate bacteria lactic acid (LAB) Isolate which has been resistant to acids with pH 2.0 in the manner of screening protease enzyme activity test result 6.5 to clear zone is 13 mm againts colony diametre is 2 mm. The results of study enzyme activity used spectrophotometer UV-Vis obtainable the regression equation Y=0.02983+0.001312X, with levels of protein M4 isolate is 0.6594 mg/mL and enzyme activity of obtainable is 0.8626 unit/ml while the spesific enzyme activity produced is 1.308 unit/mg. Then, 16S rRNA gene amplificatiom and DNA sequencing has been done. The results of study showed that the bacteria species contained from M4 bacteria lactic acid (LAB) isolate is Weisella cibiria strain II-I-59. Weisella cibiria strain II-I-59 is one of bacteria could be utilized in the digestive tract.

  12. Iron mediates catalysis of nucleic acid processing enzymes: support for Fe(II) as a cofactor before the great oxidation event.

    PubMed

    Okafor, C Denise; Lanier, Kathryn A; Petrov, Anton S; Athavale, Shreyas S; Bowman, Jessica C; Hud, Nicholas V; Williams, Loren Dean

    2017-04-20

    Life originated in an anoxic, Fe2+-rich environment. We hypothesize that on early Earth, Fe2+ was a ubiquitous cofactor for nucleic acids, with roles in RNA folding and catalysis as well as in processing of nucleic acids by protein enzymes. In this model, Mg2+ replaced Fe2+ as the primary cofactor for nucleic acids in parallel with known metal substitutions of metalloproteins, driven by the Great Oxidation Event. To test predictions of this model, we assay the ability of nucleic acid processing enzymes, including a DNA polymerase, an RNA polymerase and a DNA ligase, to use Fe2+ in place of Mg2+ as a cofactor during catalysis. Results show that Fe2+ can indeed substitute for Mg2+ in catalytic function of these enzymes. Additionally, we use calculations to unravel differences in energetics, structures and reactivities of relevant Mg2+ and Fe2+ complexes. Computation explains why Fe2+ can be a more potent cofactor than Mg2+ in a variety of folding and catalytic functions. We propose that the rise of O2 on Earth drove a Fe2+ to Mg2+ substitution in proteins and nucleic acids, a hypothesis consistent with a general model in which some modern biochemical systems retain latent abilities to revert to primordial Fe2+-based states when exposed to pre-GOE conditions. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. [Human drug metabolizing enzymes. II. Conjugation enzymes].

    PubMed

    Vereczkey, L; Jemnitz, K; Gregus, Z

    1998-09-01

    In this review we focus on human conjugation enzymes (UDP-glucuronyltransferases, methyl-trasferases, N-acetyl-transferases, O-acetyl-transferases, Amidases/carboxyesterases, sulfotransferases, Glutation-S-transferases and the enzymes involved in the conjugation with amino acids) that participate in the metabolism of xenobiotics. Although conjugation reactions in most of the cases result in detoxication, more and more publications prove that the reactions catalysed by these enzymes very often lead to activated molecules that may attack macromolecules (proteins, RNAs, DNAs), resulting in toxicity (liver, neuro-, embryotoxicity, allergy, carcinogenecity). We have summarised the data available on these enzymes concerning their catalytic profile and specificity, inhibition, induction properties, their possible role in the generation of toxic compounds, their importance in clinical practice and drug development.

  14. Metabolomics of prematurity: analysis of patterns of amino acids, enzymes, and endocrine markers by categories of gestational age.

    PubMed

    Wilson, Kumanan; Hawken, Steven; Ducharme, Robin; Potter, Beth K; Little, Julian; Thébaud, Bernard; Chakraborty, Pranesh

    2014-02-01

    Prematurity may influence the levels of amino acids, enzymes, and endocrine markers obtained through newborn screening. Identifying which analytes are the most affected by degree of prematurity could provide insight into how prematurity impacts metabolism. Analytes from blood spots assayed by Newborn Screening Ontario between March 2006 and April 2009 were used in this analysis. We examined the associations between the degree of prematurity and the levels of amino acids, enzymes, and endocrine markers in all newborns with and without adjustment for birth weight, feeding status, sample timing, transfusion, and sex. Our analysis included the following cohorts: 373,819 children born at term (>36 wk gestation), 26,483 near-term children (33-36 wk gestation), 4,354 very premature children (28-32 wk gestation), and 1,146 extremely premature children (<28 wk gestation). Of the amino acids showing consistent trends across categories of prematurity, the levels of three amino acids (arginine, leucine, and valine) were at least 50% different between the cohorts of extremely premature and term children. The levels of 17-hydroxyprogesterone increased with increasing prematurity, while thyrotropin-stimulating hormone values consistently decreased with increasing prematurity. None of the three enzyme markers we examined showed a trend in levels across categories of prematurity. This study demonstrates that children at different stages of prematurity are metabolically distinct. Future research should focus on the mechanism by which specific analytes are influenced by prematurity.

  15. Construction of a D-amino acid oxidase reactor based on magnetic nanoparticles modified by a reactive polymer and its application in screening enzyme inhibitors.

    PubMed

    Mu, Xiaoyu; Qiao, Juan; Qi, Li; Liu, Ying; Ma, Huimin

    2014-08-13

    Developing facile and high-throughput methods for exploring pharmacological inhibitors of D-amino acid oxidase (DAAO) has triggered increasing interest. In this work, DAAO was immobilized on the magnetic nanoparticles, which were modified by a biocompatible reactive polymer, poly(glycidyl methacrylate) (PGMA) via an atom transfer radical polymerization technique. Interestingly, the enzyme immobilization process was greatly promoted with the assistance of a lithium perchlorate catalyst. Meanwhile, a new amino acid ionic liquid (AAIL) was successfully synthesized and employed as the efficient chiral ligand in a chiral ligand exchange capillary electrophoresis (CLE-CE) system for chiral separation of amino acids (AAs) and quantitation of methionine, which was selected as the substrate of DAAO. Then, the apparent Michaelis-Menten constants in the enzyme system were determined with the proposed CLE-CE method. The prepared DAAO-PGMA-Fe3O4 nanoparticles exhibited excellent reusability and good stability. Moreover, the enzyme reactor was successfully applied in screening DAAO inhibitors. These results demonstrated that the enzyme could be efficiently immobilized on the polymer-grafted magnetic nanoparticles and that the obtained enzyme reactor has great potential in screening enzyme inhibitors, further offering new insight into monitoring the relevant diseases.

  16. Structural and Mechanistic Insight into the Listeria monocytogenes Two-enzyme Lipoteichoic Acid Synthesis System*

    PubMed Central

    Campeotto, Ivan; Percy, Matthew G.; MacDonald, James T.; Förster, Andreas; Freemont, Paul S.; Gründling, Angelika

    2014-01-01

    Lipoteichoic acid (LTA) is an important cell wall component required for proper cell growth in many Gram-positive bacteria. In Listeria monocytogenes, two enzymes are required for the synthesis of this polyglycerolphosphate polymer. The LTA primase LtaPLm initiates LTA synthesis by transferring the first glycerolphosphate (GroP) subunit onto the glycolipid anchor and the LTA synthase LtaSLm extends the polymer by the repeated addition of GroP subunits to the tip of the growing chain. Here, we present the crystal structures of the enzymatic domains of LtaPLm and LtaSLm. Although the enzymes share the same fold, substantial differences in the cavity of the catalytic site and surface charge distribution contribute to enzyme specialization. The eLtaSLm structure was also determined in complex with GroP revealing a second GroP binding site. Mutational analysis confirmed an essential function for this binding site and allowed us to propose a model for the binding of the growing chain. PMID:25128528

  17. Interaction of ethacrynic acid with control sites of renal glucose metabolism.

    PubMed

    Fúlgraff, G; Dingler-Núnemann, H

    1975-01-01

    Ethacrynic acid stimulates in vitro concentration dependent renal gluconeogenesis from substrates which enter the gluconeogenic pathway at the level of the triosephosphates like glycerol or fructose or from substrates which have to pass the oxaloacetate shuttle like pyruvate or from intermediary products of fatty acid oxydation or citrate cycle. Our results suggest that a site of action of ethacrynic acid in this metabolic aspect is the enzyme system fructose diphosphatase/frutose-6-phosphate kinase and eventually additionally pyruvate carboxylase.

  18. Enzymes and Enzyme Activity Encoded by Nonenveloped Viruses.

    PubMed

    Azad, Kimi; Banerjee, Manidipa; Johnson, John E

    2017-09-29

    Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses-a group of viruses that lack any lipid coating or envelope-play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.

  19. Integrated Analysis of the Transcriptome and Metabolome of Corynebacterium glutamicum during Penicillin-Induced Glutamic Acid Production.

    PubMed

    Hirasawa, Takashi; Saito, Masaki; Yoshikawa, Katsunori; Furusawa, Chikara; Shmizu, Hiroshi

    2018-05-01

    Corynebacterium glutamicum is known for its ability to produce glutamic acid and has been utilized for the fermentative production of various amino acids. Glutamic acid production in C. glutamicum is induced by penicillin. In this study, the transcriptome and metabolome of C. glutamicum is analyzed to understand the mechanism of penicillin-induced glutamic acid production. Transcriptomic analysis with DNA microarray revealed that expression of some glycolysis- and TCA cycle-related genes, which include those encoding the enzymes involved in conversion of glucose to 2-oxoglutaric acid, is upregulated after penicillin addition. Meanwhile, expression of some TCA cycle-related genes, encoding the enzymes for conversion of 2-oxoglutaric acid to oxaloacetic acid, and the anaplerotic reactions decreased. In addition, expression of NCgl1221 and odhI, encoding proteins involved in glutamic acid excretion and inhibition of the 2-oxoglutarate dehydrogenase, respectively, is upregulated. Functional category enrichment analysis of genes upregulated and downregulated after penicillin addition revealed that genes for signal transduction systems are enriched among upregulated genes, whereas those for energy production and carbohydrate and amino acid metabolisms are enriched among the downregulated genes. As for the metabolomic analysis using capillary electrophoresis time-of-flight mass spectrometry, the intracellular content of most metabolites of the glycolysis and the TCA cycle decreased dramatically after penicillin addition. Overall, these results indicate that the cellular metabolism and glutamic acid excretion are mainly optimized at the transcription level during penicillin-induced glutamic acid production by C. glutamicum. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Nickel-based Enzyme Systems*

    PubMed Central

    Ragsdale, Stephen W.

    2009-01-01

    Of the eight known nickel enzymes, all but glyoxylase I catalyze the use and/or production of gases central to the global carbon, nitrogen, and oxygen cycles. Nickel appears to have been selected for its plasticity in coordination and redox chemistry and is able to cycle through three redox states (1+, 2+, 3+) and to catalyze reactions spanning ∼1.5 V. This minireview focuses on the catalytic mechanisms of nickel enzymes, with an emphasis on the role(s) of the metal center. The metal centers vary from mononuclear to complex metal clusters and catalyze simple hydrolytic to multistep redox reactions. PMID:19363030

  1. Human triose-phosphate isomerase deficiency: a single amino acid substitution results in a thermolabile enzyme.

    PubMed Central

    Daar, I O; Artymiuk, P J; Phillips, D C; Maquat, L E

    1986-01-01

    Triose-phosphate isomerase (TPI; D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) deficiency is a recessive disorder that results in hemolytic anemia and neuromuscular dysfunction. To determine the molecular basis of this disorder, a TPI allele from two unrelated patients homozygous for TPI deficiency was compared with an allele from a normal individual. Each disease-associated sequence harbors a G X C----C X G transversion in the codon for amino acid-104 and specifies a structurally altered protein in which a glutamate residue is replaced by an aspartate residue. The importance of glutamate-104 to enzyme structure and function is implicated by its conservation in the TPI protein of all species that have been characterized to date. The glutamate-to-aspartate substitution results in a thermolabile enzyme as demonstrated by assays of TPI activity in cultured fibroblasts of each patient and cultured Chinese hamster ovary (CHO) cells that were stably transformed with the mutant alleles. Although this substitution conserves the overall charge of amino acid-104, the x-ray crystal structure of chicken TPI indicates that the loss of a side-chain methylene group (-CH2CH2COO- ---- -CH2COO-) is sufficient to disrupt the counterbalancing of charges that normally exists within a hydrophobic pocket of the native enzyme. Images PMID:2876430

  2. Effect of exogenous enzymes in maize-based diets varying in nutrient density for young broilers: growth performance and digestibility of energy, minerals and amino acids.

    PubMed

    Cowieson, A J; Ravindran, V

    2008-01-01

    1. A total of 192 male broilers (Cobb 500) were used in a growth and digestibility assay, involving a 2 x 2 factorial arrangement of treatments, to assess the effects of an enzyme cocktail of xylanase, amylase and protease in maize-based diets. 2. The following two diets were formulated: a positive control diet containing adequate nutrient concentrations for broiler starters as per breeder recommendations and a negative control diet to contain approximately 0.63 MJ/kg apparent metabolisable energy (AME) and 3% amino acids less than the positive control. 3. A further two dietary treatments were developed by supplementing each control diet with an enzyme product containing xylanase, amylase and protease. 4. Birds fed on the negative control diet had poorer weight gain and feed efficiency than those given the positive control diet. There was no effect of diet or enzyme on feed intake. The digestibility of nitrogen, calcium, phosphorus and most amino acids were unaffected by dietary nutrient density. 5. Supplementation of both the positive and negative control diets with the enzyme improved weight gain and feed efficiency compared with the unsupplemented diets. In the case of the negative control, supplemental enzyme improved performance to that of the unsupplemented positive control diet. There was no interaction between diet and enzyme for either weight gain or FCR, suggesting similar beneficial responses regardless of the nutrient density of the diet. 6. In both diets, enzyme supplementation improved AME by an average of 3% (0.35 MJ/kg DM) and nitrogen retention by an average of 11.7% (26 g/kg DM intake vs 29 g/kg DM intake). There was no interaction between diet and enzyme for AME or nitrogen retention. 7. Ileal digestibilities of calcium and phosphorus were not influenced by supplemental enzyme, whereas the digestibility of nitrogen and most amino acids was improved by enzyme addition compared with the unsupplemented control diets. There was no interaction between

  3. Knockdown of Both Mitochondrial Isocitrate Dehydrogenase Enzymes In Pancreatic Beta Cells Inhibits Insulin Secretion

    PubMed Central

    MacDonald, Michael J.; Brown, Laura J.; Longacre, Melissa J.; Stoker, Scott W.; Kendrick, Mindy A.; Hasan, Noaman M.

    2013-01-01

    Background There are three isocitrate dehydrogenases (IDHs) in the pancreatic insulin cell; IDH1 (cytosolic) and IDH2 (mitochondrial) use NADP(H). IDH3 is mitochondrial, uses NAD(H) and was believed to be the IDH that supports the citric acid cycle. Methods With shRNAs targeting mRNAs for these enzymes we generated cell lines from INS-1 832/13 cells with severe (80%–90%) knockdown of the mitochondrial IDHs separately and together in the same cell line. Results With knockdown of both mitochondrial IDH’s mRNA, enzyme activity and protein level, but not with knockdown of one mitochondrial IDH, glucose- and BCH (an allosteric activator of glutamate dehydrogenase)-plus-glutamine-stimulated insulin release were inhibited. Cellular levels of citrate, α-ketoglutarate, malate and ATP were altered in patterns consistent with blockage at the mitochondrial IDH reactions. We were able to generate only 50% knockdown of Idh1 mRNA in multiple cell lines (without inhibition of insulin release) possibly because greater knockdown of IDH1 was not compatible with cell line survival. Conclusions The mitochondrial IDHs are redundant for insulin secretion. When both enzymes are severely knocked down, their low activities (possibly assisted by transport of IDH products and other metabolic intermediates from the cytosol into mitochondria) are sufficient for cell growth, but inadequate for insulin secretion when the requirement for intermediates is certainly more rapid. The results also indicate that IDH2 can support the citric acid cycle. General Significance As almost all mammalian cells possess substantial amounts of all three IDH enzymes, the biological principles suggested by these results are probably extrapolatable to many tissues. PMID:23876293

  4. Effect of alternative pathway therapy on branched chain amino acid metabolism in urea cycle disorder patients.

    PubMed

    Scaglia, Fernando; Carter, Susan; O'Brien, William E; Lee, Brendan

    2004-04-01

    Urea cycle disorders (UCDs) are a group of inborn errors of hepatic metabolism caused by the loss of enzymatic activities that mediate the transfer of nitrogen from ammonia to urea. These disorders often result in life-threatening hyperammonemia and hyperglutaminemia. A combination of sodium phenylbutyrate and sodium phenylacetate/benzoate is used in the clinical management of children with urea cycle defects as a glutamine trap, diverting nitrogen from urea synthesis to alternatives routes of excretion. We have observed that patients treated with these compounds have selective branched chain amino acid (BCAA) deficiency despite adequate dietary protein intake. However, the direct effect of alternative therapy on the steady state levels of plasma branched chain amino acids has not been well characterized. We have measured steady state plasma branched chain and other essential non-branched chain amino acids in control subjects, untreated ornithine transcarbamylase deficiency females and treated null activity urea cycle disorder patients in the fed steady state during the course of stable isotope studies. Steady-state leucine levels were noted to be significantly lower in treated urea cycle disorder patients when compared to either untreated ornithine transcarbamylase deficiency females or control subjects (P<0.0001). This effect was reproduced in control subjects who had depressed leucine levels when treated with sodium phenylacetate/benzoate (P<0.0001). Our studies suggest that this therapeutic modality has a substantial impact on the metabolism of branched chain amino acids in urea cycle disorder patients. These findings suggest that better titration of protein restriction could be achieved with branched chain amino acid supplementation in patients with UCDs who are on alternative route therapy.

  5. Characterization of fatty acid modifying enzyme activity in staphylococcal mastitis isolates and other bacteria

    PubMed Central

    2012-01-01

    Background Fatty acid modifying enzyme (FAME) has been shown to modify free fatty acids to alleviate their bactericidal effect by esterifying fatty acids to cholesterol or alcohols. Although it has been shown in previous studies that FAME is required for Staphylococcus aureus survival in skin abscesses, FAME is poorly studied compared to other virulence factors. FAME activity had also been detected in coagulase-negative staphylococci (CNS). However, FAME activity was only surveyed after a bacterial culture was grown for 24 h. Therefore if FAME activity was earlier in the growth phase, it would not have been detected by the assay and those strains would have been labeled as FAME negative. Results Fifty CNS bovine mastitis isolates and several S. aureus, Escherichia coli, and Streptococcus uberis strains were assayed for FAME activity over 24 h. FAME activity was detected in 54% of CNS and 80% S. aureus strains surveyed but none in E. coli or S. uberis. While some CNS strains produced FAME activity comparable to the lab strain of S. aureus, the pattern of FAME activity varied among strains and across species of staphylococci. All CNS that produced FAME activity also exhibited lipase activity. Lipase activity relative to colony forming units of these CNS decreased over the 24 h growth period. No relationship was observed between somatic cell count in the milk and FAME activity in CNS. Conclusions Some staphylococcal species surveyed produced FAME activity, but E. coli and S. uberis strains did not. All FAME producing CNS exhibited lipase activity which may indicate that both these enzymes work in concert to alter fatty acids in the bacterial environment. PMID:22726316

  6. The Photosynthetic Cycle

    DOE R&D Accomplishments Database

    Calvin, Melvin

    1955-03-21

    A cyclic sequence of transformations, including the carboxylation of RuDP (ribulose diphosphate) and its re-formation, has been deduced as the route for the creation of reduced carbon compounds in photosynthetic organisms. With the demonstration of RuDP as substrate for the carboxylation in a cell-free system, each of the reactions has now been carried out independently in vitro. Further purification of this last enzyme system has confirmed the deduction that the carboxylation of RuDP leads directly to the two molecules of PGA (phosphoglyceric acid) involving an internal dismutation and suggesting the name "carboxydismutase" for the enzyme. As a consequence of this knowledge of each of the steps in the photosynthetic CO{sub 2} reduction cycle, it is possible to define the reagent requirements to maintain it. The net requirement for the reduction of one molecule of CO{sub 2} is four equivalents of [H]and three molecules of ATP (adenine triphosphate). These must ultimately be supplied by the photochemical reaction. Some possible ways in which this may be accomplished are discussed.

  7. Sulfur isotopic constraints from a single enzyme on the cellular to global sulfur cycles

    NASA Astrophysics Data System (ADS)

    Sim, M. S.; Adkins, J. F.; Sessions, A. L.; Orphan, V. J.; McGlynn, S.

    2017-12-01

    Since first reported more than a half century ago, sulfur isotope fractionation between sulfate and sulfide has been used as a diagnostic indicator of microbial sulfate reduction, giving added dimensions to the microbial ecological and geochemical studies of the sulfur cycle. A wide range of fractionation has attracted particular attention because it may serve as a potential indicator of environmental or physiological variables such as substrate concentrations or specific respiration rates. In theory, the magnitude of isotope fractionation depends upon the sulfur isotope effect imparted by the involved enzymes and the relative rate of each enzymatic reaction. The former defines the possible range of fractionation quantitatively, while the latter responds to environmental stimuli, providing an underlying rationale for the varying fractionations. The experimental efforts so far have concentrated largely on the latter, the factors affecting the size of fractionation. Recently, however, the direct assessment of intracellular processes emerges as a promising means for the quantitative analysis of microbial sulfur isotope fractionation as a function of environmental or physiological variables. Here, we experimentally determined for the first time the sulfur isotope fractionation during APS reduction, the first reductive step in the dissimilatory sulfate reduction pathway, using the enzyme purified from Desulfovibrio vulgaris Miyazaki. APS reductase carried out the one-step, two-electron reduction of APS to sulfite, without the production of other metabolic intermediates. Nearly identical isotope effects were obtained at two different temperatures, while the rate of APS reduction more than quadrupled with a temperature increase from 20 to 32°C. When placed in context of the linear network model for microbial sulfur isotope fractionation, our finding could provide a new, semi-quantitative constraint on the sulfur cycle at levels from cellular to global.

  8. Results of chopper-controlled discharge life cycling studies on lead acid batteries

    NASA Technical Reports Server (NTRS)

    Ewashinka, J. G.; Sidik, S. M.

    1982-01-01

    A group of 108 state of the art nominally 6 volt lead acid batteries were tested in a program of one charge/discharge cycle per day for over two years or to ultimate battery failure. The primary objective was to determine battery cycle life as a function of depth of discharge (25 to 75 percent), chopper frequency (100 to 1000 Hz), duty cycle (25 to 87.5 percent), and average discharge current (20 to 260 A). The secondary objective was to determine the types of battery failure modes, if any, were due to the above parameters. The four parameters above were incorporated in a statistically designed test program.

  9. Anaerobic carbon metabolism by the tricarboxylic acid cycle

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vanlerberghe, G.C.; Horsey, A.K.; Weger, H.G.

    Nitrogen-limited cells of Selenastrum minutum (Naeg.) Collins are able to assimilate NH{sub 4}{sup +} in the dark under anaerobic conditions. Addition of NH{sub 4}{sup +} to anaerobic cells results in a threefold increase in tricarboxylic acid cycle (TCAC) CO{sub 2} efflux and an eightfold increase in the rate of anaplerotic carbon fixation via phosphoenspyruvate carboxylase. Both of these observations are consistent with increased TCAC carbon flow to supply intermediates for amino acid biosynthesis. Addition of H{sup 14}CO{sub 3}{sup {minus}} to anaerobic cells assimilating NH{sub 4}{sup +} results in the incorporation of radiolabel into the {alpha}-carboxyl carbon of glutamic acid. Incorporationmore » of radiolabel into glutamic acid is not simply a short-term phenomenon following NH{sub 4}{sup +} addition as the specific activity of glutamic acid increases over time. This indicates that this alga is able to maintain partial oxidative TCAC carbon flow while under anoxia to supply {alpha}ketoglutarate for glutamate production. During dark aerobic NH{sub 4}{sup +} assimilation, no radiolabel appears in fumarate or succinate and only a small amount occurs in malate. During anaerobic NH{sub 4}{sup +} assimilation, these metabolites contain a large proportion of the total radiolabel and radiolabel accumulates in succinate over time. Also, the ratio of dark carbon fixation to NH{sub 4}{sup +} assimilation is much higher under anaerobic than aerobic conditions. These observations suggest the operation of a partial reductive TCAC from oxaloacetic acid to malate, fumarate, and succinate. Such a pathway might contribute to redox balance in an anaerobic cell maintaining partial oxidative TCAC activity.« less

  10. Dietary long-chain unsaturated fatty acids acutely and differently reduce the activities of lipogenic enzymes and of citrate carrier in rat liver.

    PubMed

    Gnoni, Antonio; Giudetti, Anna M

    2016-09-01

    The activities of lipogenic enzymes appear to fluctuate with changes in the level and type of dietary fats. Polyunsaturated fatty acids (PUFAs) are known to induce on hepatic de novo lipogenesis (DNL) the highest inhibitory effect, which occurs through a long-term adaptation. Data on the acute effects of dietary fatty acids on DNL are lacking. In this study with rats, the acute 1-day effect of high-fat (15 % w/w) diets (HFDs) enriched in saturated fatty acids (SFAs) or unsaturated fatty acids (UFAs), i.e., monounsaturated (MUFA) and PUFA, of the ω-6 and ω-3 series on DNL and plasma lipid level was investigated; a comparison with a longer time feeding (21 days) was routinely carried out. After 1-day HFD administration UFA, when compared to SFA, reduced plasma triacylglycerol (TAG) level and the activities of the lipogenic enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), a decreased activity of the citrate carrier (CIC), a mitochondrial protein linked to lipogenesis, was also detected. In this respect, ω-3 PUFA was the most effective. On the other hand, PUFA maintained the effects at longer times, and the acute inhibition induced by MUFA feeding on DNL enzyme and CIC activities was almost nullified at 21 days. Mitochondrial fatty acid composition was slightly but significantly changed both at short- and long-term treatment, whereas the early changes in mitochondrial phospholipid composition vanished in long-term experiments. Our results suggest that in the early phase of administration, UFA coordinately reduced both the activities of de novo lipogenic enzymes and of CIC. ω-3 PUFA showed the greatest effect.

  11. Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes.

    PubMed

    Fujishima, Kosuke; Wang, Kendrick M; Palmer, Jesse A; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J

    2018-01-29

    Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/CysM). To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids. Using a cysteine-dependent auxotrophic E. coli strain, CysE function was rescued by cysteine-free and methionine-deficient enzymes, and CysM function was rescued by cysteine-free enzymes. CysK function, however, was not rescued in either case. Enzymatic assays showed that the enzymes responsible for rescuing the function in CysE and CysM also retained their activities in vitro. Additionally, substitution of the two highly conserved methionines in CysM decreased but did not eliminate overall activity. Engineering amino acid biosynthetic enzymes to lack the so-produced amino acids can provide insights into, and perhaps eventually fully recapitulate via a synthetic approach, the biogenesis of biotic amino acids.

  12. Alternative Oxidase Isoforms Are Differentially Activated by Tricarboxylic Acid Cycle Intermediates.

    PubMed

    Selinski, Jennifer; Hartmann, Andreas; Deckers-Hebestreit, Gabriele; Day, David A; Whelan, James; Scheibe, Renate

    2018-02-01

    The cyanide-insensitive alternative oxidase (AOX) is a non-proton-pumping ubiquinol oxidase that catalyzes the reduction of oxygen to water and is posttranslationally regulated by redox mechanisms and 2-oxo acids. Arabidopsis ( Arabidopsis thaliana ) possesses five AOX isoforms (AOX1A-AOX1D and AOX2). AOX1D expression is increased in aox1a knockout mutants from Arabidopsis (especially after restriction of the cytochrome c pathway) but cannot compensate for the lack of AOX1A, suggesting a difference in the regulation of these isoforms. Therefore, we analyzed the different AOX isoenzymes with the aim to identify differences in their posttranslational regulation. Seven tricarboxylic acid cycle intermediates (citrate, isocitrate, 2-oxoglutarate, succinate, fumarate, malate, and oxaloacetate) were tested for their influence on AOX1A, AOX1C, and AOX1D wild-type protein activity using a refined in vitro system. AOX1C is insensitive to all seven organic acids, AOX1A and AOX1D are both activated by 2-oxoglutarate, but only AOX1A is additionally activated by oxaloacetate. Furthermore, AOX isoforms cannot be transformed to mimic one another by substituting the variable cysteine residues at position III in the protein. In summary, we show that AOX isoforms from Arabidopsis are differentially fine-regulated by tricarboxylic acid cycle metabolites (most likely depending on the amino-terminal region around the highly conserved cysteine residues known to be involved in regulation by the 2-oxo acids pyruvate and glyoxylate) and propose that this is the main reason why they cannot functionally compensate for each other. © 2018 American Society of Plant Biologists. All Rights Reserved.

  13. Neuropsychiatric manifestations in late-onset urea cycle disorder patients.

    PubMed

    Serrano, Mercedes; Martins, Cecilia; Pérez-Dueñas, Belén; Gómez-López, Lilian; Murgui, Empar; Fons, Carmen; García-Cazorla, Angels; Artuch, Rafael; Jara, Fernando; Arranz, José A; Häberle, Johannes; Briones, Paz; Campistol, Jaume; Pineda, Mercedes; Vilaseca, Maria A

    2010-03-01

    Inherited urea cycle disorders represent one of the most common groups of inborn errors of metabolism. Late-onset urea cycle disorders caused by partial enzyme deficiencies may present with unexpected clinical phenotypes. We report 9 patients followed up in our hospital presenting late-onset urea cycle disorders who initially manifested neuropsychiatric/neurodevelopmental symptoms (the most prevalent neuropsychiatric/neurodevelopmental diagnoses were mental retardation, attention-deficit hyperactivity disorder [ADHD], language disorder, and delirium). Generally, these clinical pictures did not benefit from pharmacological treatment. Conversely, dietary treatment improved the symptoms. Regarding biochemical data, 2 patients showed normal ammonium but high glutamine levels. This study highlights the fact that neuropsychiatric/neurodevelopmental findings are common among the initial symptomatology of late-onset urea cycle disorders. The authors recommend that unexplained or nonresponsive neuropsychiatric/neurodevelopmental symptoms appearing during childhood or adolescence be followed by a study of ammonia and amino acid plasmatic levels to rule out a urea cycle disorder.

  14. Enzyme-enhanced fluorescence detection of DNA on etched optical fibers.

    PubMed

    Niu, Shu-yan; Li, Quan-yi; Ren, Rui; Zhang, Shu-sheng

    2009-05-15

    A novel DNA biosensor based on enzyme-enhanced fluorescence detection on etched optical fibers was developed. The hybridization complex of DNA probe and biotinylated target was formed on the etched optical fiber, and was then bound with streptavidin labeled horseradish peroxidase (streptavidin-HRP). The target DNA was quantified through the fluorescent detection of bi-p,p'-4-hydroxyphenylacetic acid (DBDA) generated from the substrate 4-hydroxyphenylacetic acid (p-HPA) under the catalysis of HRP, with a detection limit of 1 pM and a linear range from 1.69 pM to 169 pM. It is facile to regenerate this sensor through surface treatment with concentrated urea solution. It was discovered that the sensor can retain 70% of its original activity after three detection-regeneration cycles.

  15. Stoichiometry of Reducing Equivalents and Splitting of Water in the Citric Acid Cycle.

    ERIC Educational Resources Information Center

    Madeira, Vitor M. C.

    1988-01-01

    Presents a solution to the problem of finding the source of extra reducing equivalents, and accomplishing the stoichiometry of glucose oxidation reactions. Discusses the citric acid cycle and glycolysis. (CW)

  16. Effects of enzyme supplementation on the nutrient, amino acid, and energy utilization efficiency of citrus pulp and hawthorn pulp in Linwu ducks.

    PubMed

    Zhang, Xu; Li, Haobang; Jiang, Guitao; Wang, Xiangrong; Huang, Xuan; Li, Chuang; Wu, Duanqin; Dai, Qiuzhong

    2018-04-11

    The objective of this study was to evaluate the effects of enzyme supplementation on the nutrient, amino acid, and energy utilization efficiency of citrus pulp and hawthorn pulp as unusual feedstuffs in Linwu ducks. Forty ducks were assigned to each treatment group and fed diets with or without complex enzyme supplementation. All birds received the same quantity of raw material (60 g) via the force-feeding procedure. With the exception of leucine and phenylalanine, amino acid concentrations in hawthorn pulp were twice those in citrus pulp. Enzyme supplementation significantly increased apparent dry matter digestibility (ADM) of citrus pulp (P < 0.05), but had no significant effects (P > 0.05) on the apparent and true utilization rates of other nutrients, apparent metabolizable energy (AME), or true metabolizable energy (TME), from citrus pulp and hawthorn pulp by Linwu ducks. However, enzyme supplementation significantly increased (P < 0.05) apparent gross energy, true gross energy, AME, and TME of hawthorn pulp for Linwu ducks. There were no differences in the apparent and true utilization rates of amino acids from citrus pulp (P > 0.56) between the groups, with the exception of arginine (P < 0.05). There was an increasing trend in the apparent and true utilization rates of alanine (P = 0.06) and tyrosine (P = 0.074) from citrus pulp with enzyme supplementation. The apparent and true utilization rates of threonine in hawthorn pulp were increased significantly (P < 0.05) following enzyme supplementation. The addition of exogenous enzymes improved the forage quality of citrus pulp and hawthorn pulp, which represent potential feed resources for husbandry production.

  17. L-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea.

    PubMed

    Deutch, Charles E

    2013-11-01

    The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. L-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP(+)-dependent enzymes from chloroplasts and was separated from the NAD(+)-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis-Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K m for oxaloacetate was 20 μM; the K m for NADH was 22 μM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD(+) increased with pH but there was very little activity with NADP(+). At pH 7.0, the K m for L-malate was 5 mM and the K m for NAD(+) was 24 μM. The reductive activity was quite insensitive to inhibition by L-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55 °C. The reductive activity was consistently 10-20 times higher than the oxidative activity. These results indicate that the L-malate dehydrogenase in N. europaea is similar to other NAD(+)-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.

  18. Effects of dietary fats (fish, olive and high-oleic-acid sunflower oils) on lipid composition and antioxidant enzymes in rat liver.

    PubMed

    Ruiz-Gutiérrez, V; Pérez-Espinosa, A; Vázquez, C M; Santa-María, C

    1999-09-01

    The effects of two oleic-acid-rich diets (containing olive oil, OO, and high-oleic-acid sunflower oil, HOSO) on plasma and liver lipid composition detoxification enzyme activities, were compared with those of a fish-oil (FO) diet and a control diet. Compared with the control diet, plasma and hepatic total triacylglycerol concentrations were increased in the animals fed on the HOSO and OO diets and decreased in those fed on the FO diet. The animals fed on FO showed the highest level of cholesterol in the liver and had lower plasma cholesterol concentrations when compared with those fed on the two oleic-acid-rich diets. In comparison with the animals fed on the diets enriched in oleic acid, the FO group showed higher hepatic levels of polyunsaturated fatty acids of the n-3 series and lower levels of fatty acids of the n-6 series. Livers of FO-fed rats, compared with those of OO- and HOSO-fed rats showed: (1) significantly higher activities of catalase (EC 1.11.1.6) glutathione peroxidase (EC 1.11.1.9) and Cu/Zn superoxide dismutase (EC 1.15.1.1); (2) no differences in the NADPH-cytochrome c reductase (EC 1.6.99.3) activity. The HOSO diet had a similar effect on liver antioxidant enzyme activities as the OO diet. In conclusion, it appears that changes in the liver fatty acid composition due mainly to n-3 lipids may enhance the efficiency of the antioxidant defence system. The two monounsaturated fatty acids oils studied (OO and HOSO), with the same high content of oleic acid but different contents of natural antioxidants, had similar effects on the antioxidant enzyme activities measured.

  19. Phosphatidic acid and neurotransmission

    PubMed Central

    Raben, Daniel M.; Barber, Casey N.

    2016-01-01

    Lipids play a vital role in the health and functioning of neurons and interest in the physiological role of neuronal lipids is certainly increasing. One neuronal function in which neuronal lipids appears to play key roles in neurotransmission. Our understanding of the role of lipids in the synaptic vesicle cycle and neurotransmitter release is becoming increasingly more important. Much of the initial research in this area has highlighted the major roles played by the phosphoinositides (PtdIns), diacylglycerol (DAG), and phosphatidic acid (PtdOH). Of these, PtdOH has not received as much attention as the other lipids although its role and metabolism appears to be extremely important. This lipid has been shown to play a role in modulating both exocytosis and endocytosis although its precise role in either process is not well defined. The currently evidence suggest this lipid likely participates in key processes by altering membrane architecture necessary for membrane fusion, mediating the penetration of membrane proteins, serving as a precursor for other important SV cycling lipids, or activating essential enzymes. In this review, we address the sources of PtdOH, the enzymes involved in its production, the regulation of these enzymes, and its potential roles in neurotransmission in the central nervous system. PMID:27671966

  20. Phosphatidic acid and neurotransmission.

    PubMed

    Raben, Daniel M; Barber, Casey N

    2017-01-01

    Lipids play a vital role in the health and functioning of neurons and interest in the physiological role of neuronal lipids is certainly increasing. One neuronal function in which neuronal lipids appears to play key roles in neurotransmission. Our understanding of the role of lipids in the synaptic vesicle cycle and neurotransmitter release is becoming increasingly more important. Much of the initial research in this area has highlighted the major roles played by the phosphoinositides (PtdIns), diacylglycerol (DAG), and phosphatidic acid (PtdOH). Of these, PtdOH has not received as much attention as the other lipids although its role and metabolism appears to be extremely important. This lipid has been shown to play a role in modulating both exocytosis and endocytosis although its precise role in either process is not well defined. The currently evidence suggest this lipid likely participates in key processes by altering membrane architecture necessary for membrane fusion, mediating the penetration of membrane proteins, serving as a precursor for other important SV cycling lipids, or activating essential enzymes. In this review, we address the sources of PtdOH, the enzymes involved in its production, the regulation of these enzymes, and its potential roles in neurotransmission in the central nervous system. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Deficiency of cellulase activity measurements for enzyme evaluation.

    PubMed

    Pryor, Scott W; Nahar, Nurun

    2010-11-01

    Switchgrass was used as a model feedstock to determine the influence of pretreatment conditions and biomass quality on enzymatic hydrolysis using different enzyme products. Dilute sulfuric acid and soaking in aqueous ammonia pretreatments were used to produce biomass with varied levels of hemicellulose and lignin sheathing. Pretreated switchgrass solids were tested with simple enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) with three commercial enzyme products: Accellerase 1000 (Genencor), Spezyme CP (Genencor)/Novozyme 188 (Novozymes), and Celluclast/Novozyme 188 (Novozymes). Enzymes were loaded on a common activity basis (FPU/g cellulose and CBU/g cellulose). Despite identical enzyme loadings, glucose yields were significantly different for both acid and alkaline pretreatments but differences diminished as hydrolysis progressed for acid-pretreated biomass. Cellobiose concentrations in Accellerase treatments indicated an initial beta-glucosidase limitation that became less significant over time. SSF experiments showed that differences in glucose and ethanol yields could not be attributed to enzyme product inhibition. Yield discrepancies of glucose or ethanol in acid pretreatment, alkaline pretreatment, and acid pretreatment/SSF were as much as 15%, 19%, and 5%. These results indicate that standardized protocols for measuring enzyme activity may not be adequate for assessing activity using pretreated biomass substrates.

  2. DIBROMOACETIC ACID-INDUCED ELEVATIONS OF ESTRADIOL IN THE CYCLING AND OVARIECTOMOZED/ESTRADIOL-IMPLANTED FEMALE RAT

    EPA Science Inventory

    Goldman, JM and Murr, AS. Dibromoacetic Acid-induced Elevations of Estradiol in Both Cycling and Ovariectomized / Estradiol-implanted Female Rats

    ABSTRACT
    Haloacetic acids are one of the principal classes of disinfection by-products generated by the chlorination of mun...

  3. Structural aspects of denitrifying enzymes.

    PubMed

    Moura, I; Moura, J J

    2001-04-01

    The reduction of nitrate to nitrogen gas via nitrite, nitric oxide and nitrous oxide is the metabolic pathway usually known as denitrification, a key step in the nitrogen cycle. As observed for other elemental cycles, a battery of enzymes are utilized, namely the reductases for nitrate, nitrite, nitric oxide and nitrous oxide, as well as multiple electron donors that interact with these enzymes, in order to carry out the stepwise reactions that involve key intermediates. Because of the importance of this pathway (of parallel importance to the nitrogen-fixation pathway), efforts are underway to understand the structures of the participating enzymes and to uncover mechanistic aspects. Three-dimensional structures have been solved for the majority of these enzymes in the past few years, revealing the architecture of the active metal sites as well as global structural aspects, and possible mechanistic aspects. In addition, the recognition of specific electron-transfer partners raises important questions regarding specific electron-transfer pathways, partner recognition and control of metabolism.

  4. Extending food deprivation reverses the short-term lipolytic response to fasting: role of the triacylglycerol/fatty acid cycle.

    PubMed

    Weber, Jean-Michel; Reidy, Shannon P

    2012-05-01

    The effects of short-term food deprivation on lipid metabolism are well documented, but little is known about prolonged fasting. This study monitored the kinetics of glycerol (rate of appearance, R(a) glycerol) and non-esterified fatty acids (R(a) NEFA) in fasting rabbits. Our goals were to determine whether lipolysis is stimulated beyond values seen for short-term fasting, and to characterize the roles of primary (intracellular) and secondary (with transit through the circulation) triacylglycerol/fatty acid cycling (TAG/FA cycling) in regulating fatty acid allocation to oxidation or re-esterification. R(a) glycerol (9.62±0.72 to 15.29±0.96 μmol kg(-1) min(-1)) and R(a) NEFA (18.05±2.55 to 31.25±1.93 μmol kg(-1) min(-1)) were stimulated during the first 2 days of fasting, but returned to baseline after 4 days. An initial increase in TAG/FA cycling was followed by a reduction below baseline after 6 days without food, with primary and secondary cycling contributing to these responses. We conclude that the classic activation of lipolysis caused by short-term fasting is abolished when food deprivation is prolonged. High rates of re-esterification may become impossible to sustain, and TAG/FA cycling could decrease to reduce its cost to 3% of total energy expenditure. Throughout prolonged fasting, fatty acid metabolism gradually shifts towards increased oxidation and reduced re-esterification. Survival is achieved by pressing fuel selection towards the fatty acid dominance of energy metabolism and by slowing substrate cycles to assist metabolic suppression. However, TAG/FA cycling remains active even after prolonged fasting, suggesting that re-esterification is a crucial mechanism that cannot be stopped without harmful consequences.

  5. Effect of saturated fatty acid-rich dietary vegetable oils on lipid profile, antioxidant enzymes and glucose tolerance in diabetic rats

    PubMed Central

    Kochikuzhyil, Benson Mathai; Devi, Kshama; Fattepur, Santosh Raghunandan

    2010-01-01

    Objective: To study the effect of saturated fatty acid (SFA)-rich dietary vegetable oils on the lipid profile, endogenous antioxidant enzymes and glucose tolerance in type 2 diabetic rats. Materials and Methods: Type 2 diabetes was induced by administering streptozotocin (90 mg/kg, i.p.) in neonatal rats. Twenty-eight-day-old normal (N) and diabetic (D) male Wistar rats were fed for 45 days with a fat-enriched special diet (10%) prepared with coconut oil (CO) – lauric acid-rich SFA, palm oil (PO) – palmitic acid-rich SFA and groundnut oil (GNO) – control (N and D). Lipid profile, endogenous antioxidant enzymes and oral glucose tolerance tests were monitored. Results: D rats fed with CO (D + CO) exhibited a significant decrease in the total cholesterol and non-high-density lipoprotein cholesterol. Besides, they also showed a trend toward improving antioxidant enzymes and glucose tolerance as compared to the D + GNO group, whereas D + PO treatment aggravated the dyslipidemic condition while causing a significant decrease in the superoxide dismutase levels when compared to N rats fed with GNO (N + GNO). D + PO treatment also impaired the glucose tolerance when compared to N + GNO and D + GNO. Conclusion: The type of FA in the dietary oil determines its deleterious or beneficial effects. Lauric acid present in CO may protect against diabetes-induced dyslipidemia. PMID:20871763

  6. Modelling urea-cycle disorder citrullinemia type 1 with disease-specific iPSCs.

    PubMed

    Yoshitoshi-Uebayashi, Elena Yukie; Toyoda, Taro; Yasuda, Katsutaro; Kotaka, Maki; Nomoto, Keiko; Okita, Keisuke; Yasuchika, Kentaro; Okamoto, Shinya; Takubo, Noriyuki; Nishikubo, Toshiya; Soga, Tomoyoshi; Uemoto, Shinji; Osafune, Kenji

    2017-05-06

    Citrullinemia type 1 (CTLN1) is a urea cycle disorder (UCD) caused by mutations of the ASS1 gene, which is responsible for production of the enzyme argininosuccinate synthetase (ASS), and classically presented as life-threatening hyperammonemia in newborns. Therapeutic options are limited, and neurological sequelae may persist. To understand the pathophysiology and find novel treatments, induced pluripotent stem cells (iPSCs) were generated from a CTLN1 patient and differentiated into hepatocyte-like cells (HLCs). CTLN1-HLCs have lower ureagenesis, recapitulating part of the patient's phenotype. l-arginine, an amino acid clinically used for UCD treatment, improved this phenotype in vitro. Metabolome analysis revealed an increase in tricarboxylic acid (TCA) cycle metabolites in CTLN1, suggesting a connection between CTLN1 and the TCA cycle. This CTLN1-iPSC model improves the understanding of CTLN1 pathophysiology and can be used to pursue new therapeutic approaches. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Circadian oscillation of starch branching enzyme gene expression in the sorghum endosperm

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mutisya, J.; Sun, C.; Jansson, C.

    2009-08-31

    Expression of the three SBE genes, encoding starch branching enzymes, in the sorghum endosperm exhibited a diurnal rhythm during a 24-h cycle. Remarkably, the oscillation in SBE expression was maintained in cultured spikes after a 48-h dark treatment, also when fed a continuous solution of sucrose or abscisic acid. Our findings suggest that the rhythmicity in SBE expression in the endosperm is independent of cues from the photosynthetic source and that the oscillator resides within the endosperm itself.

  8. Remarkable Reproducibility of Enzyme Activity Profiles in Tomato Fruits Grown under Contrasting Environments Provides a Roadmap for Studies of Fruit Metabolism1[W][OPEN

    PubMed Central

    Biais, Benoît; Bénard, Camille; Beauvoit, Bertrand; Colombié, Sophie; Prodhomme, Duyên; Ménard, Guillaume; Bernillon, Stéphane; Gehl, Bernadette; Gautier, Hélène; Ballias, Patricia; Mazat, Jean-Pierre; Sweetlove, Lee; Génard, Michel; Gibon, Yves

    2014-01-01

    To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum ‘Moneymaker’) plants were grown under contrasting conditions (optimal for commercial, water limited, or shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids. The most remarkable result was the high reproducibility of enzyme activities throughout development, irrespective of conditions or location. Hierarchical clustering of enzyme activities also revealed tight relationships between metabolic pathways and phases of development. Thus, cell division was characterized by high activities of fructokinase, glucokinase, pyruvate kinase, and tricarboxylic acid cycle enzymes, indicating ATP production as a priority, whereas cell expansion was characterized by enzymes involved in the lower part of glycolysis, suggesting a metabolic reprogramming to anaplerosis. As expected, enzymes involved in the accumulation of sugars, citrate, and glutamate were strongly increased during ripening. However, a group of enzymes involved in ATP production, which is probably fueled by starch degradation, was also increased. Metabolites levels seemed more sensitive than enzymes to the environment, although such differences tended to decrease at ripening. The integration of enzyme and metabolite data obtained under contrasting growth conditions using principal component analysis suggests that, with the exceptions of alanine amino transferase and glutamate and malate dehydrogenase and malate, there are no links between single enzyme activities and metabolite time courses or levels. PMID:24474652

  9. Copper Efflux Is Induced during Anaerobic Amino Acid Limitation in Escherichia coli To Protect Iron-Sulfur Cluster Enzymes and Biogenesis

    PubMed Central

    Fung, Danny Ka Chun; Lau, Wai Yin; Chan, Wing Tat

    2013-01-01

    Adaptation to changing environments is essential to bacterial physiology. Here we report a unique role of the copper homeostasis system in adapting Escherichia coli to its host-relevant environment of anaerobiosis coupled with amino acid limitation. We found that expression of the copper/silver efflux pump CusCFBA was significantly upregulated during anaerobic amino acid limitation in E. coli without the supplement of exogenous copper. Inductively coupled plasma mass spectrometry analysis of the total intracellular copper content combined with transcriptional assay of the PcusC-lacZ reporter in the presence of specific Cu(I) chelators indicated that anaerobic amino acid limitation led to the accumulation of free Cu(I) in the periplasmic space of E. coli, resulting in Cu(I) toxicity. Cells lacking cusCFBA and another copper transporter, copA, under this condition displayed growth defects and reduced ATP production during fumarate respiration. Ectopic expression of the Fe-S cluster enzyme fumarate reductase (Frd), or supplementation with amino acids whose biosynthesis involves Fe-S cluster enzymes, rescued the poor growth of ΔcusC cells. Yet, Cu(I) treatment did not impair the Frd activity in vitro. Further studies revealed that the alternative Fe-S cluster biogenesis system Suf was induced during the anaerobic amino acid limitation, and ΔcusC enhanced this upregulation, indicating the impairment of the Fe-S cluster assembly machinery and the increased Fe-S cluster demands under this condition. Taken together, we conclude that the copper efflux system CusCFBA is induced during anaerobic amino acid limitation to protect Fe-S cluster enzymes and biogenesis from the endogenously originated Cu(I) toxicity, thus facilitating the physiological adaptation of E. coli. PMID:23893112

  10. Role of AMACR (α-methylacyl-CoA racemase) and MFE-1 (peroxisomal multifunctional enzyme-1) in bile acid synthesis in mice.

    PubMed

    Autio, Kaija J; Schmitz, Werner; Nair, Remya R; Selkälä, Eija M; Sormunen, Raija T; Miinalainen, Ilkka J; Crick, Peter J; Wang, Yuqin; Griffiths, William J; Reddy, Janardan K; Baes, Myriam; Hiltunen, J Kalervo

    2014-07-01

    Cholesterol is catabolized to bile acids by peroxisomal β-oxidation in which the side chain of C27-bile acid intermediates is shortened by three carbon atoms to form mature C24-bile acids. Knockout mouse models deficient in AMACR (α-methylacyl-CoA racemase) or MFE-2 (peroxisomal multifunctional enzyme type 2), in which this β-oxidation pathway is prevented, display a residual C24-bile acid pool which, although greatly reduced, implies the existence of alternative pathways of bile acid synthesis. One alternative pathway could involve Mfe-1 (peroxisomal multifunctional enzyme type 1) either with or without Amacr. To test this hypothesis, we generated a double knockout mouse model lacking both Amacr and Mfe-1 activities and studied the bile acid profiles in wild-type, Mfe-1 and Amacr single knockout mouse line and Mfe-1 and Amacr double knockout mouse lines. The total bile acid pool was decreased in Mfe-1-/- mice compared with wild-type and the levels of mature C24-bile acids were reduced in the double knockout mice when compared with Amacr-deficient mice. These results indicate that Mfe-1 can contribute to the synthesis of mature bile acids in both Amacr-dependent and Amacr-independent pathways.

  11. Serum aminotransferases in nonalcoholic fatty liver disease are a signature of liver metabolic perturbations at the amino acid and Krebs cycle level.

    PubMed

    Sookoian, Silvia; Castaño, Gustavo O; Scian, Romina; Fernández Gianotti, Tomas; Dopazo, Hernán; Rohr, Cristian; Gaj, Graciela; San Martino, Julio; Sevic, Ina; Flichman, Diego; Pirola, Carlos J

    2016-02-01

    Extensive epidemiologic studies have shown that cardiovascular disease and the metabolic syndrome (MetS) are associated with serum concentrations of liver enzymes; however, fundamental characteristics of this relation are currently unknown. We aimed to explore the role of liver aminotransferases in nonalcoholic fatty liver disease (NAFLD) and MetS. Liver gene- and protein-expression changes of aminotransferases, including their corresponding isoforms, were evaluated in a case-control study of patients with NAFLD (n = 42), which was proven through a biopsy (control subjects: n = 10). We also carried out a serum targeted metabolite profiling to the glycolysis, gluconeogenesis, and Krebs cycle (n = 48) and an exploration by the next-generation sequencing of aminotransferase genes (n = 96). An in vitro study to provide a biological explanation of changes in the transcriptional level and enzymatic activity of aminotransferases was included. Fatty liver was associated with a deregulated liver expression of aminotransferases, which was unrelated to the disease severity. Metabolite profiling showed that serum aminotransferase concentrations are a signature of liver metabolic perturbations, particularly at the amino acid metabolism and Krebs cycle level. A significant and positive association between systolic hypertension and liver expression levels of glutamic-oxaloacetic transaminase 2 (GOT2) messenger RNA (Spearman R = 0.42, P = 0.03) was observed. The rs6993 located in the 3' untranslated region of the GOT2 locus was significantly associated with features of the MetS, including arterial hypertension [P = 0.028; OR: 2.285 (95% CI: 1.024, 5.09); adjusted by NAFLD severity] and plasma lipid concentrations. In the context of an abnormal hepatic triglyceride accumulation, circulating aminotransferases rise as a consequence of the need for increased reactions of transamination to cope with the liver metabolic derangement that is associated with greater gluconeogenesis and

  12. The Multiple DSF-family QS Signals are Synthesized from Carbohydrate and Branched-chain Amino Acids via the FAS Elongation Cycle

    PubMed Central

    Zhou, Lian; Yu, Yonghong; Chen, Xiping; Diab, Abdelgader Abdeen; Ruan, Lifang; He, Jin; Wang, Haihong; He, Ya-Wen

    2015-01-01

    Members of the diffusible signal factor (DSF) family are a novel class of quorum sensing (QS) signals in diverse Gram-negative bacteria. Although previous studies have identified RpfF as a key enzyme for the biosynthesis of DSF family signals, many questions in their biosynthesis remain to be addressed. In this study with the phytopathogen Xanthomonas campestris pv. campestris (Xcc), we show that Xcc produces four DSF-family signals (DSF, BDSF, CDSF and IDSF) during cell culture, and that IDSF is a new functional signal characterized as cis-10-methyl-2-dodecenoic acid. Using a range of defined media, we further demonstrate that Xcc mainly produces BDSF in the presence of carbohydrates; leucine and valine are the primary precursor for DSF biosynthesis; isoleucine is the primary precursor for IDSF biosynthesis. Furthermore, our biochemical analyses show that the key DSF synthase RpfF has both thioesterase and dehydratase activities, and uses 3-hydroxydedecanoyl-ACP as a substrate to produce BDSF. Finally, our results show that the classic fatty acid synthesis elongation cycle is required for the biosynthesis of DSF-family signals. Taken all together, these findings establish a general biosynthetic pathway for the DSF-family quorum sensing signals. PMID:26289160

  13. A six-year longitudinal study of phosphorus enrichment on soil enzymes in acidic forest soils.

    NASA Astrophysics Data System (ADS)

    Deforest, J. L.; Freedman, Z.

    2017-12-01

    Acidic nitrogen (N) deposition may be shifting the nutrient economies of forest soils from one dominated by N more towards phosphorus (P) limitation. While the short-term responses of nutrient enrichment experiments are reported, there is a lack of information on the longer-term response mediating ecosystem nutrient dynamics, especially for P. We hypothesized that long-term soil P amendments should result in the persistent suppression of P-acquiring extracellular enzymes when compared with ambient soils. Alternatively, vegetation and/or the microbial community may have acclimated to require more P (i.e., communities more suitable to the altered nutrient economy) resulting in an increase in the activity of P-acquiring enzymes relative to carbon (C) and N-acquiring enzyme activity. To test the hypothesis, P availability was indirectly and/or directly increased by raising soil pH and/or the addition of phosphate fertilizer and maintained for six years. Study sites were in two North American eastern deciduous forest regions on glaciated soils with modest P availability and unglaciated with low P availability. For the glaciated sites, C:N acquiring enzyme activity remained stable and was insensitive to 6 years of elevated pH and/or P in the, but there was modest increases in the unglaciated site. Phosphorus-acquiring enzyme activity was insensitive to the treatments in the glaciated sites. For unglaciated sites, P-acquiring enzyme activity was suppressed under P addition in year one, rebounded in the second year, and was suppressed in the subsequent years. These results suggest that the basal nutrient resources of an ecosystem will have a very strong influence on its response to nutrient enrichment. Likewise, the second-year recovery of P-acquiring enzyme activity might be evidence of acclimation, but the gradual yearly suppression of these enzymes suggests the system has not reach a steady state.

  14. Release of enzymes from lysosomes by irradiation and the relation of lipid peroxide formation to enzyme release

    PubMed Central

    Wills, E. D.; Wilkinson, A. E.

    1966-01-01

    1. Acid phosphatase, cathepsin and β-glucuronidase are released from rat-liver lysosomes by irradiation in vitro. Enzyme release is detectable after a dose of 1krad and increases with dose up to 100krads. 2. Maximum radiation effects were observed when the lysosomes were kept for 20hr. at 4° or 20° after irradiation. 3. An atmosphere of nitrogen considerably decreases enzyme release from lysosomes. 4. Enzyme release is enhanced by ascorbic acid and decreased by vitamin E. 5. Irradiation causes formation of lipid peroxides in lysosomes, and enzyme release increases with lipid peroxide formation. 6. It is suggested that lipid peroxide formation leads to rupture of the lysosome membrane and allows release of the contained hydrolytic enzymes. PMID:5964962

  15. Methylcitrate cycle defines the bactericidal essentiality of isocitrate lyase for survival of Mycobacterium tuberculosis on fatty acids

    PubMed Central

    Eoh, Hyungjin; Rhee, Kyu Y.

    2014-01-01

    Few mutations attenuate Mycobacterium tuberculosis (Mtb) more profoundly than deletion of its isocitrate lyases (ICLs). However, the basis for this attenuation remains incompletely defined. Mtb’s ICLs are catalytically bifunctional isocitrate and methylisocitrate lyases required for growth on even and odd chain fatty acids. Here, we report that Mtb’s ICLs are essential for survival on both acetate and propionate because of its methylisocitrate lyase (MCL) activity. Lack of MCL activity converts Mtb’s methylcitrate cycle into a “dead end” pathway that sequesters tricarboxylic acid (TCA) cycle intermediates into methylcitrate cycle intermediates, depletes gluconeogenic precursors, and results in defects of membrane potential and intrabacterial pH. Activation of an alternative vitamin B12-dependent pathway of propionate metabolism led to selective corrections of TCA cycle activity, membrane potential, and intrabacterial pH that specifically restored survival, but not growth, of ICL-deficient Mtb metabolizing acetate or propionate. These results thus resolve the biochemical basis of essentiality for Mtb’s ICLs and survival on fatty acids. PMID:24639517

  16. A structural basis for the pH-dependent xanthophyll cycle in Arabidopsis thaliana.

    PubMed

    Arnoux, Pascal; Morosinotto, Tomas; Saga, Giorgia; Bassi, Roberto; Pignol, David

    2009-07-01

    Plants adjust their photosynthetic activity to changing light conditions. A central regulation of photosynthesis depends on the xanthophyll cycle, in which the carotenoid violaxanthin is converted into zeaxanthin in strong light, thus activating the dissipation of the excess absorbed energy as heat and the scavenging of reactive oxygen species. Violaxanthin deepoxidase (VDE), the enzyme responsible for zeaxanthin synthesis, is activated by the acidification of the thylakoid lumen when photosynthetic electron transport exceeds the capacity of assimilatory reactions: at neutral pH, VDE is a soluble and inactive enzyme, whereas at acidic pH, it attaches to the thylakoid membrane where it binds its violaxanthin substrate. VDE also uses ascorbate as a cosubstrate with a pH-dependent Km that may reflect a preference for ascorbic acid. We determined the structures of the central lipocalin domain of VDE (VDEcd) at acidic and neutral pH. At neutral pH, VDEcd is monomeric with its active site occluded within a lipocalin barrel. Upon acidification, the barrel opens up and the enzyme appears as a dimer. A channel linking the two active sites of the dimer can harbor the entire carotenoid substrate and thus may permit the parallel deepoxidation of the two violaxanthin beta-ionone rings, making VDE an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate.

  17. Let the substrate flow, not the enzyme: Practical immobilization of d-amino acid oxidase in a glass microreactor for effective biocatalytic conversions.

    PubMed

    Bolivar, Juan M; Tribulato, Marco A; Petrasek, Zdenek; Nidetzky, Bernd

    2016-11-01

    Exploiting enzymes for chemical synthesis in flow microreactors necessitates their reuse for multiple rounds of conversion. To achieve this goal, immobilizing the enzymes on microchannel walls is a promising approach, but practical methods for it are lacking. Using fusion to a silica-binding module to engineer enzyme adsorption to glass surfaces, we show convenient immobilization of d-amino acid oxidase on borosilicate microchannel plates. In confocal laser scanning microscopy, channel walls appeared uniformly coated with target protein. The immobilized enzyme activity was in the range expected for monolayer coverage of the plain surface with oxidase (2.37 × 10(-5)  nmol/mm(2) ). Surface attachment of the enzyme was completely stable under flow. The operational half-life of the immobilized oxidase (25°C, pH 8.0; soluble catalase added) was 40 h. Enzymatic oxidation of d-Met into α-keto-γ-(methylthio)butyric acid was characterized in single-pass and recycle reactor configurations, employing in-line measurement of dissolved O2 , and off-line determination of the keto-acid product. Reaction-diffusion time-scale analysis for different flow conditions showed that the heterogeneously catalyzed reaction was always slower than diffusion of O2 to the solid surface (DaII  ≤ 0.3). Potential of the microreactor for intensifying O2 -dependent biotransformations restricted by mass transfer in conventional reactors is thus revealed. Biotechnol. Bioeng. 2016;113: 2342-2349. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. Temperature effects on protein depolymerization and amino acid immobilization rates in soils.

    NASA Astrophysics Data System (ADS)

    Noll, Lisa; Hu, Yuntao; Zhang, Shasha; Zheng, Qing; Wanek, Wolfgang

    2017-04-01

    Increasing N deposition, land use change, elevated atmospheric CO2 concentrations and global warming have altered soil nitrogen (N) cycling during the last decades. Those changes affected ecosystem services, such as C and N sequestration in soils, which calls for a better understanding of soil N transformation processes. The cleavage of macromolecular organic N by extracellular enzymes maintains an ongoing flow of new bioavailable organic N into biotic systems and is considered to be the bottle neck of terrestrial N cycling in litter and soils. Recent studies showed that protein depolymerization is susceptible to changes in C and N availabilities. Based on general biological observations the temperature sensitivity of soil organic N processes is expected to depend on whether they are rather enzyme limited (i.e. Q10=2) or diffusion limited (i.e. Q10= 1.0 - 1.3). However, temperature sensitivities of protein depolymerization and amino acid immobilization are still unknown. We therefore here report short-term temperature effects on organic N transformation rates in soils differing in physicochemical parameters but not in climate. Soil samples were collected from two geologically distinct sites close to the LFZ Raumberg-Gumpenstein, Styria, Austria, each from three different management types (arable land, grassland, forest). Four replicates of mineral soil were taken from every site and management type. The area provides a unique opportunity to study geological and management controls in soils without confounding effects of climate and elevation. The soils differ in several soil chemical parameters, such as soil pH, base saturation, soil C: N ratio and SOM content as well as in soil physical parameters, such as soil texture, bulk density and water holding capacity. Soils were pre-incubated at 5, 15 and 25˚ C for one day. Protein depolymerization rates and amino acid immobilization rates were assessed by an isotope pool dilution assay with 15N labeled amino acids at

  19. Anaerobic Carbon Metabolism by the Tricarboxylic Acid Cycle 1

    PubMed Central

    Vanlerberghe, Greg C.; Horsey, Anne K.; Weger, Harold G.; Turpin, David H.

    1989-01-01

    Nitrogen-limited cells of Selenastrum minutum (Naeg.) Collins are able to assimilate NH4+ in the dark under anaerobic conditions. Addition of NH4+ to anaerobic cells results in a threefold increase in tricarboxylic acid cycle (TCAC) CO2 efflux and an eightfold increase in the rate of anaplerotic carbon fixation via phosphoenolpyruvate carboxylase. Both of these observations are consistent with increased TCAC carbon flow to supply intermediates for amino acid biosynthesis. Addition of H14CO3− to anaerobic cells assimilating NH4+ results in the incorporation of radiolabel into the α-carboxyl carbon of glutamic acid. Incorporation of radiolabel into glutamic acid is not simply a short-term phenomenon following NH4+ addition as the specific activity of glutamic acid increases over time. This indicates that this alga is able to maintain partial oxidative TCAC carbon flow while under anoxia to supply α-ketoglutarate for glutamate production. During dark aerobic NH4+ assimilation, no radiolabel appears in fumarate or succinate and only a small amount occurs in malate. During anaerobic NH4+ assimilation, these metabolites contain a large proportion of the total radiolabel and radiolabel accumulates in succinate over time. Also, the ratio of dark carbon fixation to NH4+ assimilation is much higher under anaerobic than aerobic conditions. These observations suggest the operation of a partial reductive TCAC from oxaloacetic acid to malate, fumarate, and succinate. Such a pathway might contribute to redox balance in an anaerobic cell maintaining partial oxidative TCAC activity. PMID:16667215

  20. Chronic fluoxetine treatment directs energy metabolism towards the citric acid cycle and oxidative phosphorylation in rat hippocampal nonsynaptic mitochondria.

    PubMed

    Filipović, Dragana; Costina, Victor; Perić, Ivana; Stanisavljević, Andrijana; Findeisen, Peter

    2017-03-15

    Fluoxetine (Flx) is the principal treatment for depression; however, the precise mechanisms of its actions remain elusive. Our aim was to identify protein expression changes within rat hippocampus regulated by chronic Flx treatment versus vehicle-controls using proteomics. Fluoxetine-hydrohloride (15mg/kg) was administered daily to adult male Wistar rats for 3weeks, and cytosolic and nonsynaptic mitochondrial hippocampal proteomes were analyzed. All differentially expressed proteins were functionally annotated according to biological process and molecular function using Uniprot and Blast2GO. Our comparative study revealed that in cytosolic and nonsynaptic mitochondrial fractions, 60 and 3 proteins respectively, were down-regulated, and 23 and 60 proteins, respectively, were up-regulated. Proteins differentially regulated in cytosolic and nonsynaptic mitochondrial fractions were primarily related to cellular and metabolic processes. Of the identified proteins, the expressions of calretinin and parvalbumine were confirmed. The predominant molecular functions of differentially expressed proteins in both cell hippocampal fractions were binding and catalytic activity. Most differentially expressed proteins in nonsynaptic mitochondria were catalytic enzymes involved in the pyruvate metabolism, citric acid cycle, oxidative phosphorylation, ATP synthesis, ATP transduction and glutamate metabolism. Results indicate that chronic Flx treatment may influence proteins involved in calcium signaling, cytoskeletal structure, chaperone system and stimulates energy metabolism via the upregulation of GAPDH expression in cytoplasm, as well as directing energy metabolism toward the citric acid cycle and oxidative phosphorylation in nonsynaptic mitochondria. This approach provides new insight into the chronic effects of Flx treatment on protein expression in a key brain region associated with stress response and memory. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. The Impact of Enzyme Characteristics on Corn Stover Fiber Degradation and Acid Production During Ensiled Storage

    NASA Astrophysics Data System (ADS)

    Ren, Haiyu; Richard, Tom L.; Moore, Kenneth J.

    Ensilage can be used to store lignocellulosic biomass before industrial bioprocessing. This study investigated the impacts of seven commerical enzyme mixtures derived from Aspergillus niger, Trichoderma reesei, and T. longibrachiatum. Treatments included three size grades of corn stover, two enzyme levels (1.67 and 5 IU/g dry matter based on hemicellulase), and various ratios of cellulase to hemicellulase (C ∶ H). The highest C ∶ H ratio tested, 2.38, derived from T. reesei, resulted in the most effective fermentation, with lactic acid as the dominant product. Enzymatic activity during storage may complement industrial pretreatment; creating synergies that could reduce total bioconversion costs.

  2. A Diel Flux Balance Model Captures Interactions between Light and Dark Metabolism during Day-Night Cycles in C3 and Crassulacean Acid Metabolism Leaves.

    PubMed

    Cheung, C Y Maurice; Poolman, Mark G; Fell, David A; Ratcliffe, R George; Sweetlove, Lee J

    2014-06-01

    Although leaves have to accommodate markedly different metabolic flux patterns in the light and the dark, models of leaf metabolism based on flux-balance analysis (FBA) have so far been confined to consideration of the network under continuous light. An FBA framework is presented that solves the two phases of the diel cycle as a single optimization problem and, thus, provides a more representative model of leaf metabolism. The requirement to support continued export of sugar and amino acids from the leaf during the night and to meet overnight cellular maintenance costs forces the model to set aside stores of both carbon and nitrogen during the day. With only minimal constraints, the model successfully captures many of the known features of C 3 leaf metabolism, including the recently discovered role of citrate synthesis and accumulation in the night as a precursor for the provision of carbon skeletons for amino acid synthesis during the day. The diel FBA model can be applied to other temporal separations, such as that which occurs in Crassulacean acid metabolism (CAM) photosynthesis, allowing a system-level analysis of the energetics of CAM. The diel model predicts that there is no overall energetic advantage to CAM, despite the potential for suppression of photorespiration through CO 2 concentration. Moreover, any savings in enzyme machinery costs through suppression of photorespiration are likely to be offset by the higher flux demand of the CAM cycle. It is concluded that energetic or nitrogen use considerations are unlikely to be evolutionary drivers for CAM photosynthesis. © 2014 American Society of Plant Biologists. All Rights Reserved.

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

    PubMed Central

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

    2016-01-01

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

  4. 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

  5. Cinnamic acid derivatives induce cell cycle arrest in carcinoma cell lines.

    PubMed

    Sova, Matej; Žižak, Željko; Stanković, Jelena A Antic; Prijatelj, Matevž; Turk, Samo; Juranić, Zorica D; Mlinarič-Raščan, Irena; Gobec, Stanislav

    2013-08-01

    Cinnamic acid derivatives can be found in plant material, and they possess a remarkable variety of biological effects. In the present study, we have investigated the cytotoxic effects of representative cinnamic acid esters and amides. The cytotoxicity was determined by MTT test on human cervix adenocarcinoma (HeLa), myelogenous leukemia (K562), malignant melanoma (Fem-x), and estrogen-receptor-positive breast cancer (MCF-7) cells, versus peripheral blood mononuclear cells (PBMCs) without or with the addition of the plant lectin phytohemaglutinin (PHA). The compounds tested showed significant cytotoxicity (IC50s between 42 and 166 µM) and furthermore selectivity of these cytotoxic effects on the malignant cell lines versus the PBMCs was also seen, especially when electron-withdrawing groups, such as a cyano group (compound 5), were present on the aromatic rings of the alcohol or amine parts of the cinnamic acid derivatives. The additional study on cell cycle phase distribution indicated that novel cinnamic acid derivatives inhibit cell growth by induction of cell death. Thus, cinnamic acids derivatives represent important lead compounds for further development of antineoplastic agents.

  6. Contribution of the tricarboxylic acid (TCA) cycle and the glyoxylate shunt in Saccharomyces cerevisiae to succinic acid production during dough fermentation.

    PubMed

    Rezaei, Mohammad N; Aslankoohi, Elham; Verstrepen, Kevin J; Courtin, Christophe M

    2015-07-02

    Succinic acid produced by yeast during bread dough fermentation can significantly affect the rheological properties of the dough. By introducing mutations in the model S288C yeast strain, we show that the oxidative pathway of the TCA cycle and the glyoxylate shunt contribute significantly to succinic acid production during dough fermentation. More specifically, deletion of ACO1 and double deletion of ACO1 and ICL1 resulted in a 36 and 77% decrease in succinic acid levels in fermented dough, respectively. Similarly, double deletion of IDH1 and IDP1 decreased succinic acid production by 85%, while also affecting the fermentation rate. By contrast, double deletion of SDH1 and SDH2 resulted in a two-fold higher succinic acid accumulation compared to the wild-type. Deletion of fumarate reductase activity (FRD1 and OSM1) in the reductive pathway of the TCA cycle did not affect the fermentation rate and succinic acid production. The changes in the levels of succinic acid produced by mutants Δidh1Δidp1 (low level) and Δsdh1Δsdh2 (high level) in fermented dough only resulted in small pH differences, reflecting the buffering capacity of dough at a pH of around 5.1. Moreover, Rheofermentometer analysis using these mutants revealed no difference in maximum dough height and gas retention capacity with the dough prepared with S288C. The impact of the changed succinic acid profile on the organoleptic or antimicrobial properties of bread remains to be demonstrated. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. SULPHUR-CONTAINING AMINO ACIDS METABOLISM IN EXPERIMENTAL HYPER- AND HYPOTHYROIDISM IN RATS.

    PubMed

    Nechiporuk, V; Zaichko, N; Korda, М; Melnyk, A; Koloshko, O

    2017-10-01

    Hyper- and hypothyroidism are some of the most common endocrinopathies that cause many metabolic disorders including amino acids metabolism. However, a specific molecular mechanism of thyroid hormones influence on sulphur-containing amino acids metabolism has not been established. The aim of our research was to investigate experimentally the influence of thyroid gland functional state on the main enzymatic systems of sulphur-containing amino acids metabolism in liver and kidneys, the content of homocysteine, cysteine and H2S in blood. The rats were administered with L-thyroxine and mercazolil to simulate the states of hyper- and hypothyroidism, which were confirmed by the content of fT3, fT4 and TSH in the blood. In liver and kidneys of the animals with hypothyroidism we observed the decrease in the activity of enzymes of remethylation cycle of S-adenosylmethioninsyntase, S-adenosylhomocysteinhyhdrolase, betaine-homocysteine methyltransferase. Suppression of transsulfuration transformation of homocysteine to cysteine in hypothyroidism was mainly due to the inhibition of cystathionine synthase activity of cystathionine-β-synthase, wherein cystathionase activity of cystathionine-γ-lyase was not changed. In animals with hypothyroidism we also noticed the inhibition of cysteine desulfunation reactions: the activity of enzymes of cystathionine-β-synthase, cystathionine-γ-lyase and cysteine aminotransferase significantly decreased in liver and kidneys. Experimental hyperthyroidism was accompanied by increase in activity of remethylation cycle enzymes, increase in cystationine synthase activity of cystathionine-β-synthase in liver and activity of these enzymes in kidneys. The simulation of hyperthyroidism led to the decrease of homocysteine concentration, and of hypothyroidism - to the increase of homocysteine and cysteine concentrations and reduced H2S content in blood of the animals. Thus, the significant risk factors for the development of atherosclerosis

  8. Problem-based test: replication of mitochondrial DNA during the cell cycle.

    PubMed

    Sétáló, György

    2013-01-01

    Terms to be familiar with before you start to solve the test: cell cycle, generation time, S-phase, cell culture synchronization, isotopic pulse-chase labeling, density labeling, equilibrium density-gradient centrifugation, buoyant density, rate-zonal centrifugation, nucleoside, nucleotide, kinase enzymes, polymerization of nucleic acids, re-replication block, cell fractionation, Svedberg (sedimentation constant = [ S]), nuclear DNA, mitochondrial DNA, heavy and light mitochondrial DNA chains, heteroplasmy, mitochondrial diseases Copyright © 2013 Wiley Periodicals, Inc.

  9. Catalytic and mechanical cycles in F-ATP synthases. Fourth in the Cycles Review Series.

    PubMed

    Dimroth, Peter; von Ballmoos, Christoph; Meier, Thomas

    2006-03-01

    Cycles have a profound role in cellular life at all levels of organization. Well-known cycles in cell metabolism include the tricarboxylic acid and the urea cycle, in which a specific carrier substrate undergoes a sequence of chemical transformations and is regenerated at the end. Other examples include the interconversions of cofactors, such as NADH or ATP, which are present in the cell in limiting amounts and have to be recycled effectively for metabolism to continue. Every living cell performs a rapid turnover of ATP to ADP to fulfil various energetic demands and effectively regenerates the ATP from ADP in an energy-consuming process. The turnover of the ATP cycle is impressive; a human uses about its body weight in ATP per day. Enzymes perform catalytic reaction cycles in which they undergo several chemical and physical transformations before they are converted back to their original states. The ubiquitous F1F(o) ATP synthase is of particular interest not only because of its biological importance, but also owing to its unique rotational mechanism. Here, we give an overview of the membrane-embedded F(o) sector, particularly with respect to the recent crystal structure of the c ring from Ilyobacter tartaricus, and summarize current hypotheses for the mechanism by which rotation of the c ring is generated.

  10. Amylolytic Enzymes Acquired from L-Lactic Acid Producing Enterococcus faecium K-1 and Improvement of Direct Lactic Acid Production from Cassava Starch.

    PubMed

    Unban, Kridsada; Kanpiengjai, Apinun; Takata, Goro; Uechi, Keiko; Lee, Wen-Chien; Khanongnuch, Chartchai

    2017-09-01

    An amylolytic lactic acid bacterium isolate K-1 was isolated from the wastewater of a cassava starch manufacturing factory and identified as Entercoccus faecium based on 16S rRNA gene sequence analysis. An extracellular α-amylase was purified to homogeneity and the molecular weight of the purified enzyme was approximately 112 kDa with optimal pH value and temperature measured of 7.0 and 40 °C, respectively. It was stable at a pH range of 6.0-7.0, but was markedly sensitive to high temperatures and low pH conditions, even at a pH value of 5. Ba 2+ , Al 3+ , and Co 2+ activated enzyme activity. This bacterium was capable of producing 99.2% high optically pure L-lactic acid of 4.3 and 8.2 g/L under uncontrolled and controlled pH at 6.5 conditions, respectively, in the MRS broth containing 10 g/L cassava starch as the sole carbon source when cultivated at 37 °C for 48 h. A control pH condition of 6.5 improved and stabilized the yield of L-lactic acid production directly from starch even at a high concentration of starch at up to 150 g/L. This paper is the first report describing the properties of purified α-amylase from E. faecium. Additionally, pullulanase and cyclodextrinase activities were also firstly recorded from E. faecium K-1.

  11. Magnetically responsive enzyme powders

    NASA Astrophysics Data System (ADS)

    Pospiskova, Kristyna; Safarik, Ivo

    2015-04-01

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

  12. 13-cis Retinoic acid induces apoptosis and cell cycle arrest in human SEB-1 sebocytes.

    PubMed

    Nelson, Amanda M; Gilliland, Kathryn L; Cong, Zhaoyuan; Thiboutot, Diane M

    2006-10-01

    Isotretinoin (13-cis retinoic acid (13-cis RA)) is the most potent inhibitor of sebum production, a key component in the pathophysiology of acne, yet its mechanism of action remains largely unknown. The effects of 13-cis RA, 9-cis retinoic acid (9-cis RA), and all-trans retinoic acid (ATRA) on cell proliferation, apoptosis, and cell cycle proteins were examined in SEB-1 sebocytes and keratinocytes. 13-cis RA causes significant dose-dependent and time-dependent decreases in viable SEB-1 sebocytes. A portion of this decrease can be attributed to cell cycle arrest as evidenced by decreased DNA synthesis, increased p21 protein expression, and decreased cyclin D1. Although not previously demonstrated in sebocytes, we report that 13-cis RA induces apoptosis in SEB-1 sebocytes as shown by increased Annexin V-FITC staining, increased TUNEL staining, and increased cleaved caspase 3 protein. Furthermore, the ability of 13-cis RA to induce apoptosis cannot be recapitulated by 9-cis RA or ATRA, and it is not inhibited by the presence of a retinoid acid receptor (RAR) pan-antagonist AGN 193109. Taken together these data indicate that 13-cis RA causes cell cycle arrest and induces apoptosis in SEB-1 sebocytes by a RAR-independent mechanism, which contributes to its sebosuppressive effect and the resolution of acne.

  13. Modeling and simulation of enzymatic gluconic acid production using immobilized enzyme and CSTR-PFTR circulation reaction system.

    PubMed

    Li, Can; Lin, Jianqun; Gao, Ling; Lin, Huibin; Lin, Jianqiang

    2018-04-01

    Production of gluconic acid by using immobilized enzyme and continuous stirred tank reactor-plug flow tubular reactor (CSTR-PFTR) circulation reaction system. A production system is constructed for gluconic acid production, which consists of a continuous stirred tank reactor (CSTR) for pH control and liquid storage and a plug flow tubular reactor (PFTR) filled with immobilized glucose oxidase (GOD) for gluconic acid production. Mathematical model is developed for this production system and simulation is made for the enzymatic reaction process. The pH inhibition effect on GOD is modeled by using a bell-type curve. Gluconic acid can be efficiently produced by using the reaction system and the mathematical model developed for this system can simulate and predict the process well.

  14. Microbial degradation of 2,4-dichlorophenoxyacetic acid: Insight into the enzymes and catabolic genes involved, their regulation and biotechnological implications.

    PubMed

    Kumar, Ajit; Trefault, Nicole; Olaniran, Ademola Olufolahan

    2016-01-01

    A considerable progress has been made to understand the mechanisms of biodegradation of 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D biodegradation pathway has been elucidated in many microorganisms including Cupriavidus necator JMP134 (previously known as Wautersia eutropha, Ralstonia eutropha and Alcaligenes eutrophus) and Pseudomonas strains. It generally involves the side chain removal of 2,4-D by α-ketoglutarate-dependent 2,4-D dioxygenase (tfdA) to form 2,4-dichlorophenol (2,4-DCP); hydroxylation of 2,4-DCP by 2,4-DCP hydroxylase (tfdB) to form dichlorocatechol; ortho or meta cleavage of dichlorocatechol by chlorocatechol 1,2-dioxygenase (tfdC) to form 2,4-dichloro-cis,cis-muconate; conversion of 2,4-dichloro-cis,cis-muconate to 2-chlorodienelactone by chloromuconate cycloisomerase (tfdD); conversion of 2-chlorodienelactone to 2-chloromaleylacetate by chlorodienelactone hydrolase (tfdE) and, finally, conversion of 2-chloromaleylacetate to 3-oxoadepate via maleylacetate by chloromaleylacetate reductase and maleylacetate reductase (tfdF), respectively, which is funnelled to the tricarboxylic acid cycle. The latest review on microbial breakdown of 2,4-D, other halogenated aromatic pesticides, and related compounds was compiled by Haggblom, however, a considerable progress has been made in this area of research since then. Thus, this review focuses on the recent advancement on 2,4-D biodegradation, the enzymes, and genes involved and their biotechlogical implications.

  15. Impact of unusual fatty acid synthesis on futile cycling through beta-oxidation and on gene expression in transgenic plants.

    PubMed

    Moire, Laurence; Rezzonico, Enea; Goepfert, Simon; Poirier, Yves

    2004-01-01

    Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal beta-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward beta-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through beta-oxidation than the expression profile of genes involved in lipid metabolism.

  16. Identification of PSD-95 Depalmitoylating Enzymes.

    PubMed

    Yokoi, Norihiko; Fukata, Yuko; Sekiya, Atsushi; Murakami, Tatsuro; Kobayashi, Kenta; Fukata, Masaki

    2016-06-15

    Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. α/β-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, Gαq, and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function. Protein palmitoylation, the most common lipid modification, dynamically

  17. Identification of PSD-95 Depalmitoylating Enzymes

    PubMed Central

    Yokoi, Norihiko; Sekiya, Atsushi; Murakami, Tatsuro; Kobayashi, Kenta

    2016-01-01

    Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. α/β-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, Gαq, and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function. SIGNIFICANCE STATEMENT Protein palmitoylation, the most common lipid

  18. Glutamate and GABA-metabolizing enzymes in post-mortem cerebellum in Alzheimer's disease: phosphate-activated glutaminase and glutamic acid decarboxylase.

    PubMed

    Burbaeva, G Sh; Boksha, I S; Tereshkina, E B; Savushkina, O K; Prokhorova, T A; Vorobyeva, E A

    2014-10-01

    Enzymes of glutamate and GABA metabolism in postmortem cerebellum from patients with Alzheimer's disease (AD) have not been comprehensively studied. The present work reports results of original comparative study on levels of phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase isoenzymes (GAD65/67) in autopsied cerebellum samples from AD patients and matched controls (13 cases in each group) as well as summarizes published evidence for altered levels of PAG and GAD65/67 in AD brain. Altered (decreased) levels of these enzymes and changes in links between amounts of these enzymes and other glutamate-metabolizing enzymes (such as glutamate dehydrogenase and glutamine synthetase-like protein) in AD cerebella suggest significantly impaired glutamate and GABA metabolism in this brain region, which was previously regarded as not substantially involved in AD pathogenesis.

  19. Engineered Production of Short Chain Fatty Acid in Escherichia coli Using Fatty Acid Synthesis Pathway

    PubMed Central

    Jawed, Kamran; Mattam, Anu Jose; Fatma, Zia; Wajid, Saima; Abdin, Malik Z.; Yazdani, Syed Shams

    2016-01-01

    Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron, for production of SCFAs in Escherichia coli utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. E. coli strain expressing tesBT gene yielded maximum butyric acid titer at 1.46 g L-1, followed by tesBF at 0.85 g L-1 and tesAT at 0.12 g L-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the fadD and fadE that initiates the fatty acid degradation cycle and overexpression of fadR that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. In vitro enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L-1 butyric acid and 17.5 g L-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in E. coli through FASII pathway and highlights role of genetic and process optimization to enhance the desired product. PMID:27466817

  20. Biochar affects soil organic matter cycling and microbial functions but does not alter microbial community structure in a paddy soil.

    PubMed

    Tian, Jing; Wang, Jingyuan; Dippold, Michaela; Gao, Yang; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2016-06-15

    The application of biochar (BC) in conjunction with mineral fertilizers is one of the most promising management practices recommended to improve soil quality. However, the interactive mechanisms of BC and mineral fertilizer addition affecting microbial communities and functions associated with soil organic matter (SOM) cycling are poorly understood. We investigated the SOM in physical and chemical fractions, microbial community structure (using phospholipid fatty acid analysis, PLFA) and functions (by analyzing enzymes involved in C and N cycling and Biolog) in a 6-year field experiment with BC and NPK amendment. BC application increased total soil C and particulate organic C for 47.4-50.4% and 63.7-74.6%, respectively. The effects of BC on the microbial community and C-cycling enzymes were dependent on fertilization. Addition of BC alone did not change the microbial community compared with the control, but altered the microbial community structure in conjunction with NPK fertilization. SOM fractions accounted for 55% of the variance in the PLFA-related microbial community structure. The particulate organic N explained the largest variation in the microbial community structure. Microbial metabolic activity strongly increased after BC addition, particularly the utilization of amino acids and amines due to an increase in the activity of proteolytic (l-leucine aminopeptidase) enzymes. These results indicate that microorganisms start to mine N from the SOM to compensate for high C:N ratios after BC application, which consequently accelerate cycling of stable N. Concluding, BC in combination with NPK fertilizer application strongly affected microbial community composition and functions, which consequently influenced SOM cycling. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Crassulacean acid metabolism-cycling in Euphorbia milii.

    PubMed

    Herrera, Ana

    2013-01-01

    Crassulacean acid metabolism (CAM) occurs in many Euphorbiaceae, particularly Euphorbia, a genus with C3 and C4 species as well. With the aim of contributing to our knowledge of the evolution of CAM in this genus, this study examined the possible occurrence of CAM in Euphorbia milii, a species with leaf succulence and drought tolerance suggestive of this carbon fixation pathway. Leaf anatomy consisted of a palisade parenchyma, a spongy parenchyma and a bundle sheath with chloroplasts, which indicates the possible functioning of C2 photosynthesis. No evidence of nocturnal CO2 fixation was found in plants of E. milii either watered or under drought; watered plants had a low nocturnal respiration rate (R). After 12 days without watering, the photosynthetic rate (P N) decreased 85 % and nocturnal R was nearly zero. Nocturnal H(+) accumulation (ΔH(+)) in watered plants was 18 ± 2 (corresponding to malate) and 18 ± 4 (citrate) μmol H(+) (g fresh mass)(-1). Respiratory CO2 recycling through acid synthesis contributed to a night-time water saving of 2 and 86 % in watered plants and plants under drought, respectively. Carbon isotopic composition (δ(13)C) was -25.2 ± 0.7 ‰ in leaves and -24.7 ± 0.1 ‰ in stems. Evidence was found for the operation of weak CAM in E. milii, with statistically significant ΔH(+), no nocturnal CO2 uptake and values of δ(13)C intermediate between C3 and constitutive CAM plants; ΔH(+) was apparently attributable to both malate and citrate. The results suggest that daily malate accumulation results from recycling of part of the nocturnal respiratory CO2, which helps explain the occurrence of an intermediate value of leaf δ(13)C. Euphorbia milii can be considered as a CAM-cycling species. The significance of the operation of CAM-cycling in E. milii lies in water conservation, rather than carbon acquisition. The possible occurrence of C2 photosynthesis merits research.

  2. The early ontogeny of digestive and metabolic enzyme activities in two commercial strains of arctic charr (Salvelinus alpinus L.).

    PubMed

    Lemieux, Hélène; Le François, Nathalie R; Blier, Pierre U

    2003-10-01

    The extent to which growth performance is linked to digestive or energetic capacities in the early life stages of a salmonid species was investigated. We compared two strains of Arctic charr known to have different growth potentials during their early development (Fraser and Yukon gold). Trypsin, lipase, and amylase activities of whole alevins were measured at regular intervals from hatching through 65 days of development. To assess catabolic ability, we also measured five enzymes representing the following metabolic pathways: amino acid oxidation (amino aspartate transferase), fatty acid oxidation (beta-hydroxy acyl CoA-dehydrogenase), tricarboxylic acid cycle (citrate synthase), glycolysis (pyruvate kinase), and anaerobic glycolysis (lactate dehydrogenase). The measurement of these enzyme activities in individual fish allowed a clear evaluation of digestive capacity in relation to energetic demand. We also compared triploid and diploid individuals within the Yukon gold strain. For the whole experimental period, diploid Yukon gold fish exhibited the highest growth rate (1.08+/-0.18% length/day) followed by triploid Yukon gold fish (1.00+/-0.28% length/day) and finally Fraser strain fish (0.84+/-0.28% length/day). When differences in enzyme activities were observed, the Fraser strain showed higher enzyme activities at a given length than the Yukon gold strain (diploid and triploid). Higher growth performance appears to be linked to lower metabolic capacity. Our results suggest that fish may have to reach an important increase in the ratio of digestive to catabolic enzyme activities or a leveling off of metabolic enzyme activities before the onset of large increases in mass. Copyright 2003 Wiley-Liss, Inc.

  3. Intracellular synthesis of glutamic acid in Bacillus methylotrophicus SK19.001, a glutamate-independent poly(γ-glutamic acid)-producing strain.

    PubMed

    Peng, Yingyun; Zhang, Tao; Mu, Wanmeng; Miao, Ming; Jiang, Bo

    2016-01-15

    Bacillus methylotrophicus SK19.001 is a glutamate-independent strain that produces poly(γ-glutamic acid) (γ-PGA), a polymer of D- and L-glutamic acids that possesses applications in food, the environment, agriculture, etc. This study was undertaken to explore the synthetic pathway of intracellular L- and D-glutamic acid in SK19.001 by investigating the effects of tricarboxylic acid cycle intermediates and different amino acids as metabolic precursors on the production of γ-PGA and analyzing the activities of the enzymes involved in the synthesis of L- and D-glutamate. Tricarboxylic acid cycle intermediates and amino acids could participate in the synthesis of γ-PGA via independent pathways in SK19.001. L-Aspartate aminotransferase, L-glutaminase and L-glutamate synthase were the enzymatic sources of L-glutamate. Glutamate racemase was responsible for the formation of D-glutamate for the synthesis of γ-PGA, and the synthetase had stereoselectivity for glutamate substrate. The enzymatic sources of L-glutamate were investigated for the first time in the glutamate-independent γ-PGA-producing strain, and multiple enzymatic sources of L-glutamate were verified in SK19.001, which will benefit efforts to improve production of γ-PGA with metabolic engineering strategies. © 2015 Society of Chemical Industry.

  4. Comparative study of toxicological and cell cycle effects of okadaic acid and dinophysistoxin-2 in primary rat hepatocytes.

    PubMed

    Rubiolo, J A; López-Alonso, H; Vega, F V; Vieytes, M R; Botana, L M

    2012-03-10

    To determine the relative toxicity and effects on the cell cycle of okadaic acid and dinophysistoxin-2 in primary hepatocyte cultures. Cytotoxicity was determined by the MTT method, caspase-3 activity and lactate dehydrogenase release to the medium. The cell cycle analysis was performed by imaging flow cytometry and the effect of the toxins on cell proliferation was studied by quantitative PCR and confocal microscopy. We show that dinophysistoxin-2 is less toxic than okadaic acid for primary hepatocytes with a similar difference in potency as that observed in vivo in mice after intraperitoneal injection. Both toxins induced apoptosis with caspase-3 increase. They also inhibited the hepatocytes cell cycle in G1 affecting diploid cells and diploid bi-nucleated cells. In proliferating hepatocytes exposed to the toxins, a decrease of p53 gene expression as well as a lower protein level was detected. Studies of the tubulin cytoskeleton in toxin treated cells, showed nuclear localization of this molecule and a granulated tubulin pattern in the cytoplasm. The results presented in this work show that the difference in toxicity between dinophysistoxin-2 and okadaic acid in cultured primary hepatocytes is the same as that observed in vivo after intraperitoneal injection. Okadaic acid and dinophysistoxin-2 arrest the cell cycle of hepatocytes at G1 even in diploid bi-nucleated cells. p53 and tubulin could be involved in the cell cycle inhibitory effect. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Legacy effects overwhelm the short-term effects of exotic plant invasion and restoration on soil microbial community structure, enzyme activities, and nitrogen cycling.

    PubMed

    Elgersma, Kenneth J; Ehrenfeld, Joan G; Yu, Shen; Vor, Torsten

    2011-11-01

    Plant invasions can have substantial consequences for the soil ecosystem, altering microbial community structure and nutrient cycling. However, relatively little is known about what drives these changes, making it difficult to predict the effects of future invasions. In addition, because most studies compare soils from uninvaded areas to long-established dense invasions, little is known about the temporal dependence of invasion impacts. We experimentally manipulated forest understory vegetation in replicated sites dominated either by exotic Japanese barberry (Berberis thunbergii), native Viburnums, or native Vacciniums, so that each vegetation type was present in each site-type. We compared the short-term effect of vegetation changes to the lingering legacy effects of the previous vegetation type by measuring soil microbial community structure (phospholipid fatty acids) and function (extracellular enzymes and nitrogen mineralization). We also replaced the aboveground litter in half of each plot with an inert substitute to determine if changes in the soil microbial community were driven by aboveground or belowground plant inputs. We found that after 2 years, the microbial community structure and function was largely determined by the legacy effect of the previous vegetation type, and was not affected by the current vegetation. Aboveground litter removal had only weak effects, suggesting that changes in the soil microbial community and nutrient cycling were driven largely by belowground processes. These results suggest that changes in the soil following either invasion or restoration do not occur quickly, but rather exhibit long-lasting legacy effects from previous belowground plant inputs.

  6. Effect of simulated acid rain on the litter decomposition of Quercus acutissima and Pinus massoniana in forest soil microcosms and the relationship with soil enzyme activities.

    PubMed

    Wang, Congyan; Guo, Peng; Han, Guomin; Feng, Xiaoguang; Zhang, Peng; Tian, Xingjun

    2010-06-01

    With the continuing increase in human activities, ecologists are increasingly interested in understanding the effects of acid rain on litter decomposition. Two dominant litters were chosen from Zijin Mountain in China: Quercus acutissima from a broad-leaved forest and Pinus massoniana from a coniferous forest. The litters were incubated in microcosms and treated with simulated acid rain (gradient pH levels). During a six-month incubation, changes in chemical composition (i.e., lignin, total carbohydrate, and nitrogen), litter mass losses, soil pH values, and activities of degradative enzymes were determined. Results showed that litter mass losses were depressed after exposure to acid rain and the effects of acid rain on the litter decomposition rates of needles were higher than on those of leaves. Results also revealed that simulated acid rain restrained the activities of cellulase, invertase, nitrate reductase, acid phosphatase, alkaline phosphatase, polyphenol oxidase, and urease, while it enhanced the activities of catalase in most cases during the six-month decomposition process. Catalase and polyphenol oxidase were primarily responsible for litter decomposition in the broad-leaved forest, while invertase, nitrate reductase, and urease were primarily responsible for litter decomposition in the coniferous forest. The results suggest acid rain-restrained litter decomposition may be due to the depressed enzymatic activities. According to the results of this study, soil carbon in subtropical forests would accumulate as a long-term consequence of continued acid rain. This may presumably alter the balance of ecosystem carbon flux, nutrient cycling, and humus formation, which may, in turn, have multiple effects on forest ecosystems. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  7. Nucleic acids digestion by enzymes in the stomach of snakehead (Channa argus) and banded grouper (Epinephelus awoara).

    PubMed

    Liu, Yu; Zhang, Yanfang; Jiang, Wei; Wang, Jing; Pan, Xiaoming; Wu, Wei; Cao, Minjie; Dong, Ping; Liang, Xingguo

    2017-02-01

    Dietary nucleic acids (NAs) were important nutrients. However, the digestion of NAs in stomach has not been studied. In this study, the digestion of NAs by enzymes from fish stomach was investigated. The snakehead pepsins (SP) which were the main enzymes in stomach were extracted and purified. The purity of SP was evaluated by SDS-PAGE and HPLC. The snakehead pepsin 2 (SP2) which was the main component in the extracts was used for investigating the protein and NAs digestion activity. SP2 could digest NAs, including λ DNA and salmon sperm DNA. Interestingly, the digestion could be inhibited by treatment of alkaline solution at pH 8.0 and pepstatin A, and the digestion could happen either in the presence or absence of hemoglobin (Hb) and BSA as the protein substrates. Similarly, the stomach enzymes of banded grouper also showed the NAs digestion activity. NAs could be digested by the stomach enzymes of snakehead and banded grouper. It may be helpful for understanding both animal nutrition and NAs metabolic pathway.

  8. Variant Amino Acid Residues Alter the Enzyme Activity of Peanut Type 2 Diacylglycerol Acyltransferases

    PubMed Central

    Zheng, Ling; Shockey, Jay; Bian, Fei; Chen, Gao; Shan, Lei; Li, Xinguo; Wan, Shubo; Peng, Zhenying

    2017-01-01

    Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triacylglycerol (TAG) biosynthesis via the acyl-CoA-dependent acylation of diacylglycerol. This reaction is a major control point in the Kennedy pathway for biosynthesis of TAG, which is the most important form of stored metabolic energy in most oil-producing plants. In this study, Arachis hypogaea type 2 DGAT (AhDGAT2) genes were cloned from the peanut cultivar ‘Luhua 14.’ Sequence analysis of 11 different peanut cultivars revealed a gene family of 8 peanut DGAT2 genes (designated AhDGAT2a-h). Sequence alignments revealed 21 nucleotide differences between the eight ORFs, but only six differences result in changes to the predicted amino acid (AA) sequences. A representative full-length cDNA clone (AhDGAT2a) was characterized in detail. The biochemical effects of altering the AhDGAT2a sequence to include single variable AA residues were tested by mutagenesis and functional complementation assays in transgenic yeast systems. All six mutant variants retained enzyme activity and produced lipid droplets in vivo. The N6D and A26P mutants also displayed increased enzyme activity and/or total cellular fatty acid (FA) content. N6D mutant mainly increased the content of palmitoleic acid, and A26P mutant mainly increased the content of palmitic acid. The A26P mutant grew well both in the presence of oleic and C18:2, but the other mutants grew better in the presence of C18:2. AhDGAT2 is expressed in all peanut organs analyzed, with high transcript levels in leaves and flowers. These levels are comparable to that found in immature seeds, where DGAT2 expression is most abundant in other plants. Over-expression of AhDGAT2a in tobacco substantially increased the FA content of transformed tobacco seeds. Expression of AhDGAT2a also altered transcription levels of endogenous tobacco lipid metabolic genes in transgenic tobacco, apparently creating a larger carbon ‘sink’ that supports increased FA levels. PMID

  9. Magnesium Induced Nucleophile Activation in the Guanylyltransferase mRNA Capping Enzyme

    PubMed Central

    Swift, Robert V.; Ong, Chau D.; Amaro, Rommie E.

    2012-01-01

    The messenger RNA guanylyltransferase, or mRNA capping enzyme, co-transcriptionally caps the 5′-end of nascent mRNA with GMP during the second in a set of three enzymatic reactions that result in the formation of an N7-methyl guanosine cap during mRNA maturation. The mRNA capping enzyme is characterized, in part, by a conserved lysine nucleophile that attacks the alpha-phosphorous atom of GTP, forming a lysine-GMP intermediate. Experiments have firmly established that magnesium is required for efficient intermediate formation, but have provided little insight into the requirement’s molecular origins. Using empirical and thermodynamic integration pKa estimates, along with conventional MD simulations, we show that magnesium binding likely activates the lysine nucleophile by increasing its acidity and by biasing the deprotonated nucleophile into conformations conducive to intermediate formation. These results provide additional functional understanding of an important enzyme in the mRNA transcript life cycle and allow functional analogies to be drawn that affect our understanding of the metal dependence of related superfamily members. PMID:23205906

  10. Using amphiphilic pseudo amino acid composition to predict enzyme subfamily classes.

    PubMed

    Chou, Kuo-Chen

    2005-01-01

    With protein sequences entering into databanks at an explosive pace, the early determination of the family or subfamily class for a newly found enzyme molecule becomes important because this is directly related to the detailed information about which specific target it acts on, as well as to its catalytic process and biological function. Unfortunately, it is both time-consuming and costly to do so by experiments alone. In a previous study, the covariant-discriminant algorithm was introduced to identify the 16 subfamily classes of oxidoreductases. Although the results were quite encouraging, the entire prediction process was based on the amino acid composition alone without including any sequence-order information. Therefore, it is worthy of further investigation. To incorporate the sequence-order effects into the predictor, the 'amphiphilic pseudo amino acid composition' is introduced to represent the statistical sample of a protein. The novel representation contains 20 + 2lambda discrete numbers: the first 20 numbers are the components of the conventional amino acid composition; the next 2lambda numbers are a set of correlation factors that reflect different hydrophobicity and hydrophilicity distribution patterns along a protein chain. Based on such a concept and formulation scheme, a new predictor is developed. It is shown by the self-consistency test, jackknife test and independent dataset tests that the success rates obtained by the new predictor are all significantly higher than those by the previous predictors. The significant enhancement in success rates also implies that the distribution of hydrophobicity and hydrophilicity of the amino acid residues along a protein chain plays a very important role to its structure and function.

  11. Arachidonic acid induces macrophage cell cycle arrest through the JNK signaling pathway.

    PubMed

    Shen, Ziying; Ma, Yunqing; Ji, Zhonghao; Hao, Yang; Yan, Xuan; Zhong, Yuan; Tang, Xiaochun; Ren, Wenzhi

    2018-02-09

    Arachidonic acid (AA) has potent pro-apoptotic effects on cancer cells at a low concentration and on macrophages at a very high concentration. However, the effects of AA on the macrophage cell cycle and related signaling pathways have not been fully investigated. Herein we aim to observe the effect of AA on macrophages cell cycle. AA exposure reduced the viability and number of macrophages in a dose- and time-dependent manner. The reduction in RAW264.7 cell viability was not caused by apoptosis, as indicated by caspase-3 and activated caspase-3 detection. Further research illustrated that AA exposure induced RAW264.7 cell cycle arrested at S phase, and some cell cycle-regulated proteins were altered accordingly. Moreover, JNK signaling was stimulated by AA, and the stimulation was partially reversed by a JNK signaling inhibitor in accordance with cell cycle-related factors. In addition, nuclear and total Foxo1/3a and phosphorylated Foxo1/3a were elevated by AA in a dose- and time-dependent manner, and this elevation was suppressed by the JNK signaling inhibitor. Our study demonstrated that AA inhibits macrophage viability by inducing S phase cell cycle arrest. The JNK signaling pathway and the downstream FoxO transcription factors are involved in AA-induced RAW264.7 cell cycle arrest.

  12. The antidiabetic drug metformin decreases mitochondrial respiration and tricarboxylic acid cycle activity in cultured primary rat astrocytes.

    PubMed

    Hohnholt, Michaela C; Blumrich, Eva-Maria; Waagepetersen, Helle S; Dringen, Ralf

    2017-11-01

    Metformin is an antidiabetic drug that is used daily by millions of patients worldwide. Metformin is able to cross the blood-brain barrier and has recently been shown to increase glucose consumption and lactate release in cultured astrocytes. However, potential effects of metformin on mitochondrial tricarboxylic acid (TCA) cycle metabolism in astrocytes are unknown. We investigated this by mapping 13 C labeling in TCA cycle intermediates and corresponding amino acids after incubation of primary rat astrocytes with [U- 13 C]glucose. The presence of metformin did not compromise the viability of cultured astrocytes during 4 hr of incubation, but almost doubled cellular glucose consumption and lactate release. Compared with control cells, the presence of metformin dramatically lowered the molecular 13 C carbon labeling (MCL) of the cellular TCA cycle intermediates citrate, α-ketoglutarate, succinate, fumarate, and malate, as well as the MCL of the TCA cycle intermediate-derived amino acids glutamate, glutamine, and aspartate. In addition to the total molecular 13 C labeling, analysis of the individual isotopomers of TCA cycle intermediates confirmed a severe decline in labeling and a significant lowering in TCA cycling ratio in metformin-treated astrocytes. Finally, the oxygen consumption of mitochondria isolated from metformin-treated astrocytes was drastically reduced in the presence of complex I substrates, but not of complex II substrates. These data demonstrate that exposure to metformin strongly impairs complex I-mediated mitochondrial respiration in astrocytes, which is likely to cause the observed decrease in labeling of mitochondrial TCA cycle intermediates and the stimulation of glycolytic lactate production. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  13. Flexible regulation of DNA displacement reaction through nucleic acid-recognition enzyme and its application in keypad lock system and biosensing.

    PubMed

    Li, Chao; Shi, Liu; Tao, Yaqin; Mao, Xiaoxia; Xiang, Yang; Li, Genxi

    2017-08-30

    Toehold-mediated DNA strand displacement reaction (SDR) plays pivotal roles for the construction of diverse dynamic DNA nanodevices. To date, many elements have been introduced into SDR system to achieve controllable activation and fine regulation. However, as the most relevant stimuli for nucleic acid involved reaction, nucleic acid-recognizing enzymes (NAEs) have received nearly no attention so far despite SDR often takes place in NAEs-enriched environment (i.e., biological fluids). Herein, we report a set of NAEs-controlled SDR strategies, which take full advantage of NAEs' properties. In this study, three different kinds of enzymes belonging to several classes (i.e., exonuclease, endonuclease and polymerase) have been used to activate or inhibit SDR, and more importantly, some mechanisms behind these strategies on how NAEs affect SDR have also been revealed. The exploration to use NAEs as possible cues to operate SDR will expand the available toolbox to build novel stimuli-fueled DNA nanodevices and could open the door to many applications including enzyme-triggered biocomputing and biosensing.

  14. DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets.

    PubMed

    Pinto-Fernandez, Adan; Kessler, Benedikt M

    2016-01-01

    Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors.

  15. DUBbing Cancer: Deubiquitylating Enzymes Involved in Epigenetics, DNA Damage and the Cell Cycle As Therapeutic Targets

    PubMed Central

    Pinto-Fernandez, Adan; Kessler, Benedikt M.

    2016-01-01

    Controlling cell proliferation is one of the hallmarks of cancer. A number of critical checkpoints ascertain progression through the different stages of the cell cycle, which can be aborted when perturbed, for instance by errors in DNA replication and repair. These molecular checkpoints are regulated by a number of proteins that need to be present at the right time and quantity. The ubiquitin system has emerged as a central player controlling the fate and function of such molecules such as cyclins, oncogenes and components of the DNA repair machinery. In particular, proteases that cleave ubiquitin chains, referred to as deubiquitylating enzymes (DUBs), have attracted recent attention due to their accessibility to modulation by small molecules. In this review, we describe recent evidence of the critical role of DUBs in aspects of cell cycle checkpoint control, associated DNA repair mechanisms and regulation of transcription, representing pathways altered in cancer. Therefore, DUBs involved in these processes emerge as potentially critical targets for the treatment of not only hematological, but potentially also solid tumors. PMID:27516771

  16. Sulfa drugs inhibit sepiapterin reduction and chemical redox cycling by sepiapterin reductase.

    PubMed

    Yang, Shaojun; Jan, Yi-Hua; Mishin, Vladimir; Richardson, Jason R; Hossain, Muhammad M; Heindel, Ned D; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2015-03-01

    Sepiapterin reductase (SPR) catalyzes the reduction of sepiapterin to dihydrobiopterin (BH2), the precursor for tetrahydrobiopterin (BH4), a cofactor critical for nitric oxide biosynthesis and alkylglycerol and aromatic amino acid metabolism. SPR also mediates chemical redox cycling, catalyzing one-electron reduction of redox-active chemicals, including quinones and bipyridinium herbicides (e.g., menadione, 9,10-phenanthrenequinone, and diquat); rapid reaction of the reduced radicals with molecular oxygen generates reactive oxygen species (ROS). Using recombinant human SPR, sulfonamide- and sulfonylurea-based sulfa drugs were found to be potent noncompetitive inhibitors of both sepiapterin reduction and redox cycling. The most potent inhibitors of sepiapterin reduction (IC50s = 31-180 nM) were sulfasalazine, sulfathiazole, sulfapyridine, sulfamethoxazole, and chlorpropamide. Higher concentrations of the sulfa drugs (IC50s = 0.37-19.4 μM) were required to inhibit redox cycling, presumably because of distinct mechanisms of sepiapterin reduction and redox cycling. In PC12 cells, which generate catecholamine and monoamine neurotransmitters via BH4-dependent amino acid hydroxylases, sulfa drugs inhibited both BH2/BH4 biosynthesis and redox cycling mediated by SPR. Inhibition of BH2/BH4 resulted in decreased production of dopamine and dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, and 5-hydroxytryptamine. Sulfathiazole (200 μM) markedly suppressed neurotransmitter production, an effect reversed by BH4. These data suggest that SPR and BH4-dependent enzymes, are "off-targets" of sulfa drugs, which may underlie their untoward effects. The ability of the sulfa drugs to inhibit redox cycling may ameliorate ROS-mediated toxicity generated by redox active drugs and chemicals, contributing to their anti-inflammatory activity. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  17. Metabolomics approach to assessing plasma 13- and 9-hydroxy-octadecadienoic acid and linoleic acid metabolite responses to 75-km cycling.

    PubMed

    Nieman, David C; Shanely, R Andrew; Luo, Beibei; Meaney, Mary Pat; Dew, Dustin A; Pappan, Kirk L

    2014-07-01

    Bioactive oxidized linoleic acid metabolites (OXLAMs) include 13- and 9-hydroxy-octadecadienoic acid (13-HODE + 9-HODE) and have been linked to oxidative stress, inflammation, and numerous pathological and physiological states. The purpose of this study was to measure changes in plasma 13-HODE + 9-HODE following a 75-km cycling bout and identify potential linkages to linoleate metabolism and established biomarkers of oxidative stress (F2-isoprostanes) and inflammation (cytokines) using a metabolomics approach. Trained male cyclists (N = 19, age 38.0 ± 1.6 yr, wattsmax 304 ± 10.5) engaged in a 75-km cycling time trial on their own bicycles using electromagnetically braked cycling ergometers (2.71 ± 0.07 h). Blood samples were collected preexercise, immediately post-, 1.5 h post-, and 21 h postexercise, and analyzed for plasma cytokines (IL-6, IL-8, IL-10, tumor necrosis factor-α, monocyte chemoattractant protein-1, granulocyte colony-stimulating factor), F2-isoprostanes, and shifts in metabolites using global metabolomics procedures with gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS). 13-HODE + 9-HODE increased 3.1-fold and 1.7-fold immediately post- and 1.5 h postexercise (both P < 0.001) and returned to preexercise levels by 21-h postexercise. Post-75-km cycling plasma levels of 13-HODE + 9-HODE were not significantly correlated with increases in plasma cytokines but were positively correlated with postexercise F2-isoprostanes (r = 0.75, P < 0.001), linoleate (r = 0.54, P = 0.016), arachidate (r = 0.77, P < 0.001), 12,13-dihydroxy-9Z-octadecenoate (12,13-DiHOME) (r = 0.60, P = 0.006), dihomo-linolenate (r = 0.57, P = 0.011), and adrenate (r = 0.56, P = 0.013). These findings indicate that prolonged and intensive exercise caused a transient, 3.1-fold increase in the stable linoleic acid oxidation product 13-HODE + 9-HODE and was related to increases in F2-isoprostanes, linoleate, and fatty acids in the linoleate

  18. Dominant mycorrhizal association of trees alters carbon and nutrient cycling by selecting for microbial groups with distinct enzyme function.

    PubMed

    Cheeke, Tanya E; Phillips, Richard P; Brzostek, Edward R; Rosling, Anna; Bever, James D; Fransson, Petra

    2017-04-01

    While it is well established that plants associating with arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) fungi cycle carbon (C) and nutrients in distinct ways, we have a limited understanding of whether varying abundance of ECM and AM plants in a stand can provide integrative proxies for key biogeochemical processes. We explored linkages between the relative abundance of AM and ECM trees and microbial functioning in three hardwood forests in southern Indiana, USA. Across each site's 'mycorrhizal gradient', we measured fungal biomass, fungal : bacterial (F : B) ratios, extracellular enzyme activities, soil carbon : nitrogen ratio, and soil pH over a growing season. We show that the percentage of AM or ECM trees in a plot promotes microbial communities that both reflect and determine the C to nutrient balance in soil. Soils dominated by ECM trees had higher F : B ratios and more standing fungal biomass than AM stands. Enzyme stoichiometry in ECM soils shifted to higher investment in extracellular enzymes needed for nitrogen and phosphorus acquisition than in C-acquisition enzymes, relative to AM soils. Our results suggest that knowledge of mycorrhizal dominance at the stand or landscape scale may provide a unifying framework for linking plant and microbial community dynamics, and predicting their effects on ecological function. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  19. Effect of calcium and salicylic acid on quality retention in relation to antioxidative enzymes in radish stored under refrigerated conditions.

    PubMed

    Devi, Jomika; Bhatia, Surekha; Alam, M S; Dhillon, Tarsem Singh

    2018-03-01

    Effect of post harvest treatments with calcium chloride (CaCl 2 ) and salicylic acid (SA) on physiological and biochemical parameters in relation to activities of antioxidative enzymes were investigated in radish. Radish of variety Punjab Safed Mooli 2 was harvested, washed and treated with CaCl 2 (1, 1.5 and 2%) or SA (1, 1.5 and 2 mM). Treated as well as untreated radish were placed in open trays and stored under refrigerated (5 ± 1 °C, 90% RH) conditions for 42 days. Treatment of radish with CaCl 2 and SA slowed down changes in physiological weight, colour, total soluble solids, ascorbic acid, titrable acidity, total phenolics and antioxidant activity. Treated samples exhibited higher enhancement in activities of antioxidant enzymes viz. catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), peroxidase (POD), dehydroascorbate reductase (DHAR) and monodehydro-ascorbate reductase (MDHAR) than untreated samples. However SA was found to be more effective in slowing down the metabolic activities of radish as compared to CaCl 2 treatment. Among all the treatments, 1.5 mM SA maintained the quality parameters to greater extent probably by reducing the oxidative stress to larger extent due to highest activities of antioxidative enzymes and can be used to enhance the shelf life of radish during refrigerated storage.

  20. The Randle cycle revisited: a new head for an old hat

    PubMed Central

    Hue, Louis; Taegtmeyer, Heinrich

    2009-01-01

    In 1963, Lancet published a paper by Randle et al. that proposed a “glucose-fatty acid cycle” to describe fuel flux between and fuel selection by tissues. The original biochemical mechanism explained the inhibition of glucose oxidation by fatty acids. Since then, the principle has been confirmed by many investigators. At the same time, many new mechanisms controlling the utilization of glucose and fatty acids have been discovered. Here, we review the known short- and long-term mechanisms involved in the control of glucose and fatty acid utilization at the cytoplasmic and mitochondrial level in mammalian muscle and liver under normal and pathophysiological conditions. They include allosteric control, reversible phosphorylation, and the expression of key enzymes. However, the complexity is formidable. We suggest that not all chapters of the Randle cycle have been written. PMID:19531645

  1. Enzyme electrochemical sensor electrode and method of making it

    DOEpatents

    Rishpon, Judith; Zawodzinski, Thomas A.; Gottesfeld, Shimshon

    1992-01-01

    An electrochemical sensor electrode is formed from an electronic conductor coated with a casting solution containing a perfluorosulfonic acid ionomer and a selected enzyme. The selected enzyme catalyzes a reaction between a predetermined substance in a solution and oxygen to form an electrochemically active compound that is detected at the electronic conductor. The resulting perfluorosulfonic acid polymer provides a stable matrix for the enzyme for long lived enzyme activity, wherein only thin coatings are required on the metal conductor. The polymer also advantageously repels interfering substances from contacting the enzyme and contains quantities of oxygen to maintain a sensing capability during conditions of oxygen depletion in the sample. In one particular embodiment, glucose oxidase is mixed with the perfluorosulfonic acid ionomer to form an electrode for glucose detection.

  2. A Structural Basis for the pH-Dependent Xanthophyll Cycle in Arabidopsis thaliana[C][W

    PubMed Central

    Arnoux, Pascal; Morosinotto, Tomas; Saga, Giorgia; Bassi, Roberto; Pignol, David

    2009-01-01

    Plants adjust their photosynthetic activity to changing light conditions. A central regulation of photosynthesis depends on the xanthophyll cycle, in which the carotenoid violaxanthin is converted into zeaxanthin in strong light, thus activating the dissipation of the excess absorbed energy as heat and the scavenging of reactive oxygen species. Violaxanthin deepoxidase (VDE), the enzyme responsible for zeaxanthin synthesis, is activated by the acidification of the thylakoid lumen when photosynthetic electron transport exceeds the capacity of assimilatory reactions: at neutral pH, VDE is a soluble and inactive enzyme, whereas at acidic pH, it attaches to the thylakoid membrane where it binds its violaxanthin substrate. VDE also uses ascorbate as a cosubstrate with a pH-dependent Km that may reflect a preference for ascorbic acid. We determined the structures of the central lipocalin domain of VDE (VDEcd) at acidic and neutral pH. At neutral pH, VDEcd is monomeric with its active site occluded within a lipocalin barrel. Upon acidification, the barrel opens up and the enzyme appears as a dimer. A channel linking the two active sites of the dimer can harbor the entire carotenoid substrate and thus may permit the parallel deepoxidation of the two violaxanthin β-ionone rings, making VDE an elegant example of the adaptation of an asymmetric enzyme to its symmetric substrate. PMID:19638474

  3. Hepatic and extrahepatic distribution of ornithine urea cycle enzymes in holocephalan elephant fish (Callorhinchus milii).

    PubMed

    Takagi, Wataru; Kajimura, Makiko; Bell, Justin D; Toop, Tes; Donald, John A; Hyodo, Susumu

    2012-04-01

    Cartilaginous fish comprise two subclasses, the Holocephali (chimaeras) and Elasmobranchii (sharks, skates and rays). Little is known about osmoregulatory mechanisms in holocephalan fishes except that they conduct urea-based osmoregulation, as in elasmobranchs. In the present study, we examined the ornithine urea cycle (OUC) enzymes that play a role in urea biosynthesis in the holocephalan elephant fish, Callorhinchus milii (cm). We obtained a single mRNA encoding carbamoyl phosphate synthetase III (cmCPSIII) and ornithine transcarbamylase (cmOTC), and two mRNAs encoding glutamine synthetases (cmGSs) and two arginases (cmARGs), respectively. The two cmGSs were structurally and functionally separated into two types: brain/liver/kidney-type cmGS1 and muscle-type cmGS2. Furthermore, two alternatively spliced transcripts with different sizes were found for cmgs1 gene. The longer transcript has a putative mitochondrial targeting signal (MTS) and was predominantly expressed in the liver and kidney. MTS was not found in the short form of cmGS1 and cmGS2. A high mRNA expression and enzyme activities were found in the liver and muscle. Furthermore, in various tissues examined, mRNA levels of all the enzymes except cmCPSIII were significantly increased after hatching. The data show that the liver is the important organ for urea biosynthesis in elephant fish, but, extrahepatic tissues such as the kidney and muscle may also contribute to the urea production. In addition to the role of the extrahepatic tissues and nitrogen metabolism, the molecular and functional characteristics of multiple isoforms of GSs and ARGs are discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Enzymes involved in plastid-targeted phosphatidic acid synthesis are essential for Plasmodium yoelii liver stage development

    PubMed Central

    Lindner, Scott E.; Sartain, Mark J.; Hayes, Kiera; Harupa, Anke; Moritz, Robert L.; Kappe, Stefan H. I.; Vaughan, Ashley M.

    2014-01-01

    SUMMARY Malaria parasites scavenge nutrients from their host but also harbor enzymatic pathways for de novo macromolecule synthesis. One such pathway is apicoplast-targeted type II fatty acid synthesis, which is essential for late liver stage development in rodent malaria. It is likely that fatty acids synthesized in the apicoplast are ultimately incorporated into membrane phospholipids necessary for exoerythrocytic merozoite formation. We hypothesized that these synthesized fatty acids are being utilized for apicoplast-targeted phosphatidic acid synthesis, the phospholipid precursor. Phosphatidic acid is typically synthesized in a three-step reaction utilizing three enzymes: glycerol 3-phosphate dehydrogenase, glycerol 3-phosphate acyltransferase and lysophosphatidic acid acyltransferase. The Plasmodium genome is predicted to harbor genes for both apicoplast- and cytosol/endoplasmic reticulum-targeted phosphatidic synthesis. Our research shows that apicoplast-targeted P. yoelii glycerol 3-phosphate dehydrogenase and glycerol 3-phosphate acyltransferase are expressed only during liver stage development and deletion of the encoding genes resulted in late liver stage growth arrest and lack of merozoite differentiation. However, the predicted apicoplast-targeted lysophosphatidic acid acyltransferase gene was refractory to deletion and was expressed solely in the endoplasmic reticulum throughout the parasite lifecycle. Our results suggest that P. yoelii has an incomplete apicoplast-targeted phosphatidic acid synthesis pathway that is essential for liver stage maturation. PMID:24330260

  5. Proteomic analysis reveals large amounts of decomposition enzymes and major metabolic pathways involved in algicidal process of Trametes versicolor F21a.

    PubMed

    Gao, Xueyan; Wang, Congyan; Dai, Wei; Ren, Shenrong; Tao, Fang; He, Xingbing; Han, Guomin; Wang, Wei

    2017-06-20

    A recent algicidal mode indicates that fungal mycelia can wrap and eliminate almost all co-cultivated algal cells within a short time span. However, the underlying molecular mechanism is rarely understood. We applied proteomic analysis to investigate the algicidal process of Trametes versicolor F21a and identified 3,754 fungal proteins. Of these, 30 fungal enzymes with endo- or exoglycosidase activities such as β-1,3-glucanase, α-galactosidase, α-glucosidase, alginate lyase and chondroitin lyase were significantly up-regulated. These proteins belong to Glycoside Hydrolases, Auxiliary Activities, Carbohydrate Esterases and Polysaccharide Lyases, suggesting that these enzymes may degrade lipopolysaccharides, peptidoglycans and alginic acid of algal cells. Additionally, peptidase, exonuclease, manganese peroxidase and cytochrome c peroxidase, which decompose proteins and DNA or convert other small molecules of algal cells, could be other major decomposition enzymes. Gene Ontology and KEGG pathway enrichment analysis demonstrated that pyruvate metabolism and tricarboxylic acid cycle pathways play a critical role in response to adverse environment via increasing energy production to synthesize lytic enzymes or uptake molecules. Carbon metabolism, selenocompound metabolism, sulfur assimilation and metabolism, as well as several amino acid biosynthesis pathways could play vital roles in the synthesis of nutrients required by fungal mycelia.

  6. Yeast Acid Phosphatases and Phytases: Production, Characterization and Commercial Prospects

    NASA Astrophysics Data System (ADS)

    Kaur, Parvinder; Satyanarayana, T.

    The element phosphorus is critical to all life forms as it forms the basic component of nucleic acids and ATP and has a number of indispensable biochemical roles. Unlike C or N, the biogeochemical cycling of phosphorus is very slow, and thus making it the growth-limiting element in most soils and aquatic systems. Phosphohydrolases (e.g. acid phosphatases and phytases) are enzymes that break the C-O-P ester bonds and provide available inorganic phosphorus from various inassimilable organic forms of phosphorus like phytates. These enzymes are of significant value in effectively combating phosphorus pollution. Although phytases and acid phosphatases are produced by various plants, animals and micro organisms, microbial sources are more promising for the production on a commercial scale. Yeasts being the simplest eukaryotes are ideal candidates for phytase and phos-phatase research due to their mostly non-pathogenic and GRAS status. They have not, however, been utilized to their full potential. This chapter focuses attention on the present state of knowledge on the production, characterization and potential commercial prospects of yeast phytases and acid phosphatases.

  7. Expression of a major surface protein of Trypanosoma brucei insect forms is controlled by the activity of mitochondrial enzymes.

    PubMed

    Vassella, Erik; Probst, Matthias; Schneider, André; Studer, Erwin; Renggli, Christina Kunz; Roditi, Isabel

    2004-09-01

    In cycling between the mammalian host and the tsetse fly vector, trypanosomes undergo major changes in energy metabolism and surface coat composition. Early procyclic (insect) forms in the tsetse fly midgut are coated by glycoproteins known as EP and GPEET procyclins. EP expression continues in late procyclic forms, whereas GPEET is down-regulated. In culture, expression of GPEET is modulated by glycerol or glucose. Here, we demonstrate that a glycerol-responsive element of 25 nucleotides within the 3' untranslated region of GPEET mRNA also controls expression by glucose and during development in the fly. In trypanosomes, mitochondrial ATP is produced mainly by the acetate: succinate-CoA transferase/succinyl-CoA synthetase (ASCT) cycle, the citric acid cycle, and the cytochromes. Silencing of the pyruvate dehydrogenase or succinyl-CoA synthetase from the ASCT cycle by RNA interference induces reexpression of GPEET in late procyclic forms, whereas inhibition of the citric acid cycle or the cytochromes has no effect. In contrast, inhibition of the alternative oxidase, the second branch of the electron transport chain, with salicylhydroxamic acid overrides the effect of glucose or glycerol and causes a reduction in the level of GPEET mRNA. Our results reveal a new mechanism by which expression of a surface glycoprotein is controlled by the activity of mitochondrial enzymes.

  8. Activation Energies for an Enzyme-Catalyzed and Acid-Catalyzed Hydrolysis: An Introductory Interdisciplinary Experiment for Chemists and Biochemists.

    ERIC Educational Resources Information Center

    Adams, K. R.; Meyers, M. B.

    1985-01-01

    Background information, procedures used, and typical results obtained are provided for an experiment in which students determine and compare the Arrhenius activation energies (Ea) for the hydrolysis of salicin. This reaction is subject to catalysis both by acid and by the enzyme emulsin (beta-d-glucoside glycohydrolase). (JN)

  9. Duty cycle dependent chemical structure and wettability of RF pulsed plasma copolymers of acrylic acid and octafluorocyclobutane

    NASA Astrophysics Data System (ADS)

    Muzammil, I.; Li, Y. P.; Li, X. Y.; Lei, M. K.

    2018-04-01

    Octafluorocyclobutane and acrylic acid (C4F8-co-AA) plasma copolymer coatings are deposited using a pulsed wave (PW) radio frequency (RF) plasma on low density polyethylene (LDPE). The influence of duty cycle in pulsed process with the monomer feed rate on the surface chemistry and wettability of C4F8-co-AA plasma polymer coatings is studied. The concentration of the carboxylic acid (hydrophilic) groups increase, and that of fluorocarbon (hydrophobic) groups decrease by lowering the duty cycle. The combined effect of surface chemistry and surface morphology of the RF pulsed plasma copolymer coatings causes tunable surface wettability and surface adhesion. The gradual emergence of hydrophilic contents leads to surface heterogeneity by lowering duty cycle causing an increased surface adhesion in hydrophobic coatings. The C4F8-co-AA plasma polymer coatings on the nanotextured surfaces are tuned from repulsive superhydrophobicity to adhesive superhydrophobicity, and further to superhydrophilicity by adjusting the duty cycles with the monomer feed rates.

  10. Oriented and selective enzyme immobilization on functionalized silica carrier using the cationic binding module Z basic2: design of a heterogeneous D-amino acid oxidase catalyst on porous glass.

    PubMed

    Bolivar, Juan M; Nidetzky, Bernd

    2012-06-01

    D-amino acid oxidase from Trigonopsis variabilis (TvDAO) is applied in industry for the synthesis of pharmaceutical intermediates. Because free TvDAO is extremely sensitive to exposure to gas-liquid interfaces, biocatalytic processing is usually performed with enzyme immobilizates that offer enhanced stability under bubble aeration. We herein present an "Immobilization by Design" approach that exploits engineered charge complementarity between enzyme and carrier to optimize key features of the immobilization of TvDAO. A fusion protein between TvDAO and the positively charged module Z(basic2) was generated, and a corresponding oppositely charged carrier was obtained by derivatization of mesoporous glass with 3-(trihydroxysilyl)-1-propane-sulfonic acid. Using 250 mM NaCl for charge screening at pH 7.0, the Z(basic2) fusion of TvDAO was immobilized directly from E. coli cell extract with almost absolute selectivity and full retention of catalytic effectiveness of the isolated enzyme in solution. Attachment of the homodimeric enzyme to the carrier was quasi-permanent in low-salt buffer but fully reversible upon elution with 5 M NaCl. Immobilized TvDAO was not sensitive to bubble aeration and received substantial (≥ tenfold) stabilization of the activity at 45°C as compared to free enzyme, suggesting immobilization via multisubunit oriented interaction of enzyme with the insoluble carrier. The Z(basic2) enzyme immobilizate was demonstrated to serve as re-usable heterogeneous catalyst for D-amino acid oxidation. Z(basic2) -mediated binding on a sulfonic acid group-containing glass carrier constitutes a generally useful strategy of enzyme immobilization that supports transition from case-specific empirical development to rational design. Copyright © 2012 Wiley Periodicals, Inc.

  11. Respiratory enzymes of Thiobacillus ferrooxidans. Kinetic properties of an acid-stable iron:rusticyanin oxidoreductase.

    PubMed

    Blake, R C; Shute, E A

    1994-08-09

    Rusticyanin is an acid-stable, soluble blue copper protein found in abundance in the periplasmic space of Thiobacillus ferrooxidans, an acidophilic bacterium capable of growing autotrophically on soluble ferrous sulfate. An acid-stable iron:rusticyanin oxidoreductase activity was partially purified from cell-free extracts of T. ferrooxidans. The enzyme-catalyzed, iron-dependent reduction of the rusticyanin exhibited three kinetic properties characteristic of aerobic iron oxidation by whole cells. (i) A survey of 14 different anions indicated that catalysis by the oxidoreductase occurred only in the presence of sulfate or selenate, an anion specificity identical to that of whole cells. (ii) Saturation with both sulfatoiron(II) and the catalyst produced a concentration-independent rate constant of 3 s-1 for the reduction of the rusticyanin, which is an electron transfer reaction sufficiently rapid to account for the flux of electrons through the iron respiratory chain. (iii) Values for the enzyme-catalyzed pseudo-first-order rate constants for the reduction of the rusticyanin showed a hyperbolic dependence on the concentration of sulfatoiron(II) with a half-maximal effect at 300 microM, a value similar to the apparent KM for iron shown by whole cells. On the basis of these favorable comparisons between the behavior patterns of isolated biomolecules and those of whole cells, this iron:rusticyanin oxidoreductase is postulated to be the primary cellular oxidant of ferrous ions in the iron respiratory electron transport chain of T. ferrooxidans.

  12. Impact of L-FABP and glucose on polyunsaturated fatty acid induction of PPARα-regulated β-oxidative enzymes

    PubMed Central

    Petrescu, Anca D.; Huang, Huan; Martin, Gregory G.; McIntosh, Avery L.; Storey, Stephen M.; Landrock, Danilo; Kier, Ann B.

    2013-01-01

    Liver fatty acid binding protein (L-FABP) is the major soluble protein that binds very-long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs) in hepatocytes. However, nothing is known about L-FABP's role in n-3 PUFA-mediated peroxisome proliferator activated receptor-α (PPARα) transcription of proteins involved in long-chain fatty acid (LCFA) β-oxidation. This issue was addressed in cultured primary hepatocytes from wild-type, L-FABP-null, and PPARα-null mice with these major findings: 1) PUFA-mediated increase in the expression of PPARα-regulated LCFA β-oxidative enzymes, LCFA/LCFA-CoA binding proteins (L-FABP, ACBP), and PPARα itself was L-FABP dependent; 2) PPARα transcription, robustly potentiated by high glucose but not maltose, a sugar not taken up, correlated with higher protein levels of these LCFA β-oxidative enzymes and with increased LCFA β-oxidation; and 3) high glucose altered the potency of n-3 relative to n-6 PUFA. This was not due to a direct effect of glucose on PPARα transcriptional activity nor indirectly through de novo fatty acid synthesis from glucose. Synergism was also not due to glucose impacting other signaling pathways, since it was observed only in hepatocytes expressing both L-FABP and PPARα. Ablation of L-FABP or PPARα as well as treatment with MK886 (PPARα inhibitor) abolished/reduced PUFA-mediated PPARα transcription of these genes, especially at high glucose. Finally, the PUFA-enhanced L-FABP distribution into nuclei with high glucose augmentation of the L-FABP/PPARα interaction reveals not only the importance of L-FABP for PUFA induction of PPARα target genes in fatty acid β-oxidation but also the significance of a high glucose enhancement effect in diabetes. PMID:23238934

  13. Determination of asparagine, glutamine and pyrrolidonecarboxylic acid in total enzymic hydrolysates of peptides and glycopeptides by gas–liquid chromatography

    PubMed Central

    Hediger, Hedy; Stevens, Richard L.; Brandenberger, Hans; Schmid, Karl

    1973-01-01

    A new procedure for the qualitative and quantitative determination of asparagine, glutamine and pyrrolidonecarboxylic acid in total enzymic hydrolysates of peptides and glycopeptides based on g.l.c. has been developed. Under the conditions of esterification and trifluoroacetylation N-trifluoroacetylaspartic acid mono-n-butyl ester was formed from asparagine and N-trifluoroacetylglutamic acid mono-n-butyl ester from both glutamine and pyrrolidonecarboxylic acid. To distinguish between the latter two compounds, the esterification was carried out at room temperature yielding 30% of esterified pyrrolidonecarboxylic acid but less than 1% of esterified glutamine. In extending the g.l.c. of amino acids, the previously unknown positions in the g.l.c. elution pattern of the following amino acids could also be reproducibly determined: carboxymethylcysteine, homoserine, hydroxylysine and ∈-methyl-lysine. Further, certain glycopeptides were investigated and the artifacts due to their carbohydrate moieties were determined. PMID:4733240

  14. Enzymic Synthesis of Indole-3-Acetyl-1-O-β-d-Glucose 1

    PubMed Central

    Leznicki, Antoni J.; Bandurski, Robert S.

    1988-01-01

    The synthesis of indole-3-acetyl-1-O-β-d-glucose from indole-3-acetic acid (IAA) and uridine diphosphoglucose (UDPG) has been shown to be a reversible reaction with the equilibrium away from ester formation and toward formation of IAA. The enzyme occurs primarily in the liquid endosperm of the corn kernel but some activity occurs in the embryo. It is relatively specific showing no glucose ester formation with oxindole-3-acetic acid or 7-hydroxy-oxindole-3-acetic acid, and low activity with phenylpropene acids, such as ρ-coumaric acid. The enzyme is also specific for the nucleotide sugar showing no activity with UDPGalactose or UDPXylose. The enzyme is inhibited by inorganic pyrophosphate, by phosphate esters and by phospholipids, particularly phosphatidyl ethanolamine. The enzyme is inhibited by zeatin, by 2,4-dichlorophenoxy-acetic acid, by IAA-myo-inositol and IAA-glucan, but not by zeatin riboside, and only weakly by gibberellic acid, abscisic acid, and kinetin. The reaction is slightly stimulated by both calcium and calmodulin and, in some cases, by thiol compounds. The role of this enzyme in the homeostatic control of indole-3-acetic acid levels in Zea mays is discussed. PMID:11537439

  15. Activation and Repression of Epstein-Barr Virus and Kaposi's Sarcoma-Associated Herpesvirus Lytic Cycles by Short- and Medium-Chain Fatty Acids

    PubMed Central

    Gorres, Kelly L.; Daigle, Derek; Mohanram, Sudharshan

    2014-01-01

    ABSTRACT The lytic cycles of Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are induced in cell culture by sodium butyrate (NaB), a short-chain fatty acid (SCFA) histone deacetylase (HDAC) inhibitor. Valproic acid (VPA), another SCFA and an HDAC inhibitor, induces the lytic cycle of KSHV but blocks EBV lytic reactivation. To explore the hypothesis that structural differences between NaB and VPA account for their functional effects on the two related viruses, we investigated the capacity of 16 structurally related short- and medium-chain fatty acids to promote or prevent lytic cycle reactivation. SCFAs differentially affected EBV and KSHV reactivation. KSHV was reactivated by all SCFAs that are HDAC inhibitors, including phenylbutyrate. However, several fatty acid HDAC inhibitors, such as isobutyrate and phenylbutyrate, did not reactivate EBV. Reactivation of KSHV lytic transcripts could not be blocked completely by any fatty acid tested. In contrast, several medium-chain fatty acids inhibited lytic activation of EBV. Fatty acids that blocked EBV reactivation were more lipophilic than those that activated EBV. VPA blocked activation of the BZLF1 promoter by NaB but did not block the transcriptional function of ZEBRA. VPA also blocked activation of the DNA damage response that accompanies EBV lytic cycle activation. Properties of SCFAs in addition to their effects on chromatin are likely to explain activation or repression of EBV. We concluded that fatty acids stimulate the two related human gammaherpesviruses to enter the lytic cycle through different pathways. IMPORTANCE Lytic reactivation of EBV and KSHV is needed for persistence of these viruses and plays a role in carcinogenesis. Our direct comparison highlights the mechanistic differences in lytic reactivation between related human oncogenic gammaherpesviruses. Our findings have therapeutic implications, as fatty acids are found in the diet and produced by the human microbiota

  16. Antioxidative Peptides Derived from Enzyme Hydrolysis of Bone Collagen after Microwave Assisted Acid Pre-Treatment and Nitrogen Protection

    PubMed Central

    Lin, Yun-Jian; Le, Guo-Wei; Wang, Jie-Yun; Li, Ya-Xin; Shi, Yong-Hui; Sun, Jin

    2010-01-01

    This study focused on the preparation method of antioxidant peptides by enzymatic hydrolysis of bone collagen after microwave assisted acid pre-treatment and nitrogen protection. Phosphoric acid showed the highest ability of hydrolysis among the four other acids tested (hydrochloric acid, sulfuric acid and/or citric acid). The highest degree of hydrolysis (DH) was 9.5% using 4 mol/L phosphoric acid with a ratio of 1:6 under a microwave intensity of 510 W for 240 s. Neutral proteinase gave higher DH among the four protease tested (Acid protease, neutral protease, Alcalase and papain), with an optimum condition of: (1) ratio of enzyme and substrate, 4760 U/g; (2) concentration of substrate, 4%; (3) reaction temperature, 55 °C and (4) pH 7.0. At 4 h, DH increased significantly (P < 0.01) under nitrogen protection compared with normal microwave assisted acid pre-treatment hydrolysis conditions. The antioxidant ability of the hydrolysate increased and reached its maximum value at 3 h; however DH decreased dramatically after 3 h. Microwave assisted acid pre-treatment and nitrogen protection could be a quick preparatory method for hydrolyzing bone collagen. PMID:21151439

  17. Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis

    PubMed Central

    Guarnieri, Michael T.; Chou, Yat-Chen; Salvachúa, Davinia; Mohagheghi, Ali; St. John, Peter C.; Peterson, Darren J.; Bomble, Yannick J.

    2017-01-01

    ABSTRACT Actinobacillus succinogenes, a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes, enabling examination of SA flux determinants via knockout of the primary competing pathways—namely, acetate and formate production—and overexpression of the key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe. IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes. Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Overall, this work demonstrates genetic modifications that can lead to succinic

  18. Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis.

    PubMed

    Guarnieri, Michael T; Chou, Yat-Chen; Salvachúa, Davinia; Mohagheghi, Ali; St John, Peter C; Peterson, Darren J; Bomble, Yannick J; Beckham, Gregg T

    2017-09-01

    Actinobacillus succinogenes , a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO 2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO 2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes , enabling examination of SA flux determinants via knockout of the primary competing pathways-namely, acetate and formate production-and overexpression of the key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe. IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Overall, this work demonstrates genetic modifications that can lead to succinic acid

  19. Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Guarnieri, Michael T.; Chou, Yat -Chen; Salvachua, Davinia Rodriquez

    Actinobacillus succinogenes, a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO 2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO 2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes, enabling examination of SA flux determinants via knockout of the primary competing pathways—namely, acetate and formate production—and overexpression of themore » key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe. IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes. Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Altogether, this work demonstrates genetic modifications that can lead to succinic

  20. Metabolic Engineering of Actinobacillus succinogenes Provides Insights into Succinic Acid Biosynthesis

    DOE PAGES

    Guarnieri, Michael T.; Chou, Yat -Chen; Salvachua, Davinia Rodriquez; ...

    2017-06-16

    Actinobacillus succinogenes, a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO 2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO 2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes, enabling examination of SA flux determinants via knockout of the primary competing pathways—namely, acetate and formate production—and overexpression of themore » key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe. IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes. Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Altogether, this work demonstrates genetic modifications that can lead to succinic

  1. Beta-ketoacyl-acyl carrier protein synthase IV: a key enzyme for regulation of medium-chain fatty acid synthesis in Cuphea lanceolata seeds.

    PubMed

    Schütt, Burkhardt Siegfried; Abbadi, Amine; Loddenkötter, Brigitte; Brummel, Monika; Spener, Friedrich

    2002-09-01

    With the aim of elucidating the mechanisms involved in the biosynthesis of medium-chain fatty acids in Cuphea lanceolata Ait., a crop accumulating up to 90% decanoic acid in seed triacylglycerols, cDNA clones of a beta-ketoacyl-acyl carrier protein (ACP) synthase IV (clKAS IV, EC 2.3.1.41) were isolated from C. lanceolata seed embryos. The amino acid sequence deduced from clKAS IV cDNA showed 80% identity to other plant KAS II-type enzymes, 55% identity towards plant KAS I and over 90% towards other Cuphea KAS IV-type sequences. Recombinant clKAS IV was functionally overexpressed in Escherichia coli, and substrate specificity of purified enzyme showed strong preference for elongation of short-chain and medium-chain acyl-ACPs (C4- to C10-ACP) with nearly equal activity. Further elongation steps were catalysed with distinctly less activity. Moreover, short- and medium-chain acyl-ACPs exerted a chain-length-specific and concentration-dependent substrate inhibition of clKAS IV. Based on these findings a regulatory mechanism for medium-chain fatty acid synthesis in C. lanceolata is presented.

  2. The emerging role and targetability of the TCA cycle in cancer metabolism.

    PubMed

    Anderson, Nicole M; Mucka, Patrick; Kern, Joseph G; Feng, Hui

    2018-02-01

    The tricarboxylic acid (TCA) cycle is a central route for oxidative phosphorylation in cells, and fulfills their bioenergetic, biosynthetic, and redox balance requirements. Despite early dogma that cancer cells bypass the TCA cycle and primarily utilize aerobic glycolysis, emerging evidence demonstrates that certain cancer cells, especially those with deregulated oncogene and tumor suppressor expression, rely heavily on the TCA cycle for energy production and macromolecule synthesis. As the field progresses, the importance of aberrant TCA cycle function in tumorigenesis and the potentials of applying small molecule inhibitors to perturb the enhanced cycle function for cancer treatment start to evolve. In this review, we summarize current knowledge about the fuels feeding the cycle, effects of oncogenes and tumor suppressors on fuel and cycle usage, common genetic alterations and deregulation of cycle enzymes, and potential therapeutic opportunities for targeting the TCA cycle in cancer cells. With the application of advanced technology and in vivo model organism studies, it is our hope that studies of this previously overlooked biochemical hub will provide fresh insights into cancer metabolism and tumorigenesis, subsequently revealing vulnerabilities for therapeutic interventions in various cancer types.

  3. Effects of traditionally used anxiolytic botanicals on enzymes of the gamma-aminobutyric acid (GABA) system.

    PubMed

    Awad, R; Levac, D; Cybulska, P; Merali, Z; Trudeau, V L; Arnason, J T

    2007-09-01

    In Canada, the use of botanical natural health products (NHPs) for anxiety disorders is on the rise, and a critical evaluation of their safety and efficacy is required. The purpose of this study was to determine whether commercially available botanicals directly affect the primary brain enzymes responsible for gamma-aminobutyric acid (GABA) metabolism. Anxiolytic plants may interact with either glutamic acid decarboxylase (GAD) or GABA transaminase (GABA-T) and ultimately influence brain GABA levels and neurotransmission. Two in vitro rat brain homogenate assays were developed to determine the inhibitory concentrations (IC50) of aqueous and ethanolic plant extracts. Approximately 70% of all extracts that were tested showed little or no inhibitory effect (IC50 values greater than 1 mg/mL) and are therefore unlikely to affect GABA metabolism as tested. The aqueous extract of Melissa officinalis (lemon balm) exhibited the greatest inhibition of GABA-T activity (IC50 = 0.35 mg/mL). Extracts from Centella asiatica (gotu kola) and Valeriana officinalis (valerian) stimulated GAD activity by over 40% at a dose of 1 mg/mL. On the other hand, both Matricaria recutita (German chamomile) and Humulus lupulus (hops) showed significant inhibition of GAD activity (0.11-0.65 mg/mL). Several of these species may therefore warrant further pharmacological investigation. The relation between enzyme activity and possible in vivo mode of action is discussed.

  4. Superoxide Triggers an Acid Burst in Saccharomyces cerevisiae to Condition the Environment of Glucose-starved Cells*

    PubMed Central

    Baron, J. Allen; Laws, Kaitlin M.; Chen, Janice S.; Culotta, Valeria C.

    2013-01-01

    Although yeast cells grown in abundant glucose tend to acidify their extracellular environment, they raise the pH of the environment when starved for glucose or when grown strictly with non-fermentable carbon sources. Following prolonged periods in this alkaline phase, Saccharomyces cerevisiae cells will switch to producing acid. The mechanisms and rationale for this “acid burst” were unknown. Herein we provide strong evidence for the role of mitochondrial superoxide in initiating the acid burst. Yeast mutants lacking the mitochondrial matrix superoxide dismutase (SOD2) enzyme, but not the cytosolic Cu,Zn-SOD1 enzyme, exhibited marked acceleration in production of acid on non-fermentable carbon sources. Acid production is also dramatically enhanced by the superoxide-producing agent, paraquat. Conversely, the acid burst is eliminated by boosting cellular levels of Mn-antioxidant mimics of SOD. We demonstrate that the acid burst is dependent on the mitochondrial aldehyde dehydrogenase Ald4p. Our data are consistent with a model in which mitochondrial superoxide damage to Fe-S enzymes in the tricarboxylic acid (TCA) cycle leads to acetate buildup by Ald4p. The resultant expulsion of acetate into the extracellular environment can provide a new carbon source to glucose-starved cells and enhance growth of yeast. By triggering production of organic acids, mitochondrial superoxide has the potential to promote cell population growth under nutrient depravation stress. PMID:23281478

  5. Conformational diversity and computational enzyme design

    PubMed Central

    Lassila, Jonathan K.

    2010-01-01

    The application of computational protein design methods to the design of enzyme active sites offers potential routes to new catalysts and new reaction specificities. Computational design methods have typically treated the protein backbone as a rigid structure for the sake of computational tractability. However, this fixed-backbone approximation introduces its own special challenges for enzyme design and it contrasts with an emerging picture of natural enzymes as dynamic ensembles with multiple conformations and motions throughout a reaction cycle. This review considers the impact of conformational variation and dynamics on computational enzyme design and it highlights new approaches to addressing protein conformational diversity in enzyme design including recent advances in multistate design, backbone flexibility, and computational library design. PMID:20829099

  6. Inhibition of hydrolytic enzymes by gold compounds. I. beta-Glucuronidase and acid phosphatase by sodium tetrachloroaurate (III) and potassium tetrabromoaurate (III).

    PubMed

    Lee, M T; Ahmed, T; Friedman, M E

    1989-01-01

    Purified bovine liver beta-glucuronidase (beta-D-glucuronide glucuronohydrolase, EC 3.2.1.32) and wheat germ acid phosphatase (orthophosphoric monoesterphosphohydrolase, EC 3.1.3.2) were inhibited with freshly dissolved and 24 h aquated tetrahaloaurate (III) compounds. Rate and equilibrium inhibition constants were measured. From this data two acid phosphatases species were observed. Equilibrium inhibition constants ranged from 1 to 12.5 microM for the various gold compounds toward both enzymes. The first order rate constants ranged between 0.005 and 0.04 min.-1 for most reactions with the exception of the fast reacting acid phosphatase which had values as high as 2.6 and 2.8 min.-1. It is observed that the beta-glucuronidase is rapidly inhibited during the equilibrium phase before the more slower reaction covalent bond formation takes place. The acid phosphatases form the covalent bonds more rapidly, especially the faster reacting species suggesting a unique difference in the active site geometry to that of the more slowly reacting species. The tightly bonded gold (III)-enzyme complex is probably the reason for its toxicity and non-anti-inflammatory use as a drug.

  7. Impact of Unusual Fatty Acid Synthesis on Futile Cycling through β-Oxidation and on Gene Expression in Transgenic Plants1[w

    PubMed Central

    Moire, Laurence; Rezzonico, Enea; Goepfert, Simon; Poirier, Yves

    2004-01-01

    Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal β-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward β-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through β-oxidation than the expression profile of genes involved in lipid metabolism. PMID:14671017

  8. Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors.

    PubMed

    Iglesias, Jose; Lamontagne, Julien; Erb, Heidi; Gezzar, Sari; Zhao, Shangang; Joly, Erik; Truong, Vouy Linh; Skorey, Kathryn; Crane, Sheldon; Madiraju, S R Murthy; Prentki, Marc

    2016-01-01

    Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

  9. Simplified assays of lipolysis enzymes for drug discovery and specificity assessment of known inhibitors

    PubMed Central

    Iglesias, Jose; Lamontagne, Julien; Erb, Heidi; Gezzar, Sari; Zhao, Shangang; Joly, Erik; Truong, Vouy Linh; Skorey, Kathryn; Crane, Sheldon; Madiraju, S. R. Murthy; Prentki, Marc

    2016-01-01

    Lipids are used as cellular building blocks and condensed energy stores and also act as signaling molecules. The glycerolipid/ fatty acid cycle, encompassing lipolysis and lipogenesis, generates many lipid signals. Reliable procedures are not available for measuring activities of several lipolytic enzymes for the purposes of drug screening, and this resulted in questionable selectivity of various known lipase inhibitors. We now describe simple assays for lipolytic enzymes, including adipose triglyceride lipase (ATGL), hormone sensitive lipase (HSL), sn-1-diacylglycerol lipase (DAGL), monoacylglycerol lipase, α/β-hydrolase domain 6, and carboxylesterase 1 (CES1) using recombinant human and mouse enzymes either in cell extracts or using purified enzymes. We observed that many of the reported inhibitors lack specificity. Thus, Cay10499 (HSL inhibitor) and RHC20867 (DAGL inhibitor) also inhibit other lipases. Marked differences in the inhibitor sensitivities of human ATGL and HSL compared with the corresponding mouse enzymes was noticed. Thus, ATGListatin inhibited mouse ATGL but not human ATGL, and the HSL inhibitors WWL11 and Compound 13f were effective against mouse enzyme but much less potent against human enzyme. Many of these lipase inhibitors also inhibited human CES1. Results describe reliable assays for measuring lipase activities that are amenable for drug screening and also caution about the specificity of the many earlier described lipase inhibitors. PMID:26423520

  10. Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mueller, Edith E., E-mail: ed.mueller@salk.at; Mayr, Johannes A., E-mail: h.mayr@salk.at; Zimmermann, Franz A., E-mail: f.zimmermann@salk.at

    2012-01-20

    Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complexmore » II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.« less

  11. A biochemical and physicochemical comparison of two recombinant enzymes used for enzyme replacement therapies of hunter syndrome.

    PubMed

    Chung, Yo Kyung; Sohn, Young Bae; Sohn, Jong Mun; Lee, Jieun; Chang, Mi Sun; Kwun, Younghee; Kim, Chi Hwa; Lee, Jin Young; Yook, Yeon Joo; Ko, Ah-Ra; Jin, Dong-Kyu

    2014-05-01

    Mucopolysaccharidosis II (MPS II, Hunter syndrome; OMIM 309900) is an X-linked lysosomal storage disease caused by a deficiency in the enzyme iduronate-2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs). For enzyme replacement therapy (ERT) of Hunter syndrome, two recombinant enzymes, idursulfase (Elaprase(®), Shire Human Genetic Therapies, Lexington, MA) and idursulfase beta (Hunterase(®), Green Cross Corporation, Yongin, Korea), are currently available in Korea. To compare the biochemical and physicochemical differences between idursulfase and idursulfase beta, we examined the formylglycine (FGly) content, specific enzyme activity, mannose-6-phosphate (M6P) content, sialic acid content, and in vitro cell uptake activity of normal human fibroblasts of these two enzymes.The FGly content, which determines the enzyme activity, of idursulfase beta was significantly higher than that of idursulfase (79.4 ± 0.9 vs. 68.1 ± 2.2 %, P < 0.001). In accordance with the FGly content, the specific enzyme activity of idursulfase beta was significantly higher than that of idursulfase (42.6 ± 1.1 vs. 27.8 ± 0.9 nmol/min/μg protein, P < 0.001). The levels of M6P and sialic acid were not significantly different (2.4 ± 0.1 vs 2.4 ± 0.3 mol/mol protein for M6P and 12.3 ± 0.7 vs. 12.4 ± 0.4 mol/mol protein for sialic acid). However, the cellular uptake activity of the normal human fibroblasts in vitro showed a significant difference (Kuptake, 5.09 ± 0.96 vs. 6.50 ± 1.28 nM protein, P = 0.017).In conclusion, idursulfase beta exhibited significantly higher specific enzyme activity than idursulfase, resulting from higher FGly content. These biochemical differences may be partly attributed to clinical efficacy. However, long-term clinical evaluations of Hunter syndrome patients treated with these two enzymes will be needed to demonstrate the clinical implications of significant difference of the enzyme activity and the FGly content.

  12. AN ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) METHOD FOR THE URINARY BIOMONITORING OF 2,4-DICHLOROPHRENOCYACETIC ACID (2,4-D)

    EPA Science Inventory

    An enzyme-linked immunosorbent assay (ELISA) method was developed to quantitatively measure 2,4-dichlorophenoyacetic acid (2,4-D) in human urine. Samples were diluted (1:5) with phosphate-buffered saline, 0.05% Tween 20, with 0.02% sodium azide, and analyzed by a 96-microwekk pl...

  13. Comparative genomics of citric-acid producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Andersen, Mikael R.; Salazar, Margarita; Schaap, Peter

    2011-06-01

    The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compels additional exploration. We therefore undertook whole genome sequencing of the acidogenic A. niger wild type strain (ATCC 1015), and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence and half the telomeric regionsmore » have been elucidated. Moreover, sequence information from ATCC 1015 was utilized to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 megabase of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis revealed up-regulation of the electron transport chain, specifically the alternative oxidative pathway in ATCC 1015, while CBS 513.88 showed significant up regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases and protein transporters.« less

  14. Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase

    PubMed Central

    Lloyd, Julie C.; Raines, Christine A.

    2011-01-01

    In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein–protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the ‘non-regulatory’ A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed. PMID:21498632

  15. Inter-species variation in the oligomeric states of the higher plant Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase.

    PubMed

    Howard, Thomas P; Lloyd, Julie C; Raines, Christine A

    2011-07-01

    In darkened leaves the Calvin cycle enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) form a regulatory multi-enzyme complex with the small chloroplast protein CP12. GAPDH also forms a high molecular weight regulatory mono-enzyme complex. Given that there are different reports as to the number and subunit composition of these complexes and that enzyme regulatory mechanisms are known to vary between species, it was reasoned that protein-protein interactions may also vary between species. Here, this variation is investigated. This study shows that two different tetramers of GAPDH (an A2B2 heterotetramer and an A4 homotetramer) have the capacity to form part of the PRK/GAPDH/CP12 complex. The role of the PRK/GAPDH/CP12 complex is not simply to regulate the 'non-regulatory' A4 GAPDH tetramer. This study also demonstrates that the abundance and nature of PRK/GAPDH/CP12 interactions are not equal in all species and that whilst NAD enhances complex formation in some species, this is not sufficient for complex formation in others. Furthermore, it is shown that the GAPDH mono-enzyme complex is more abundant as a 2(A2B2) complex, rather than the larger 4(A2B2) complex. This smaller complex is sensitive to cellular metabolites indicating that it is an important regulatory isoform of GAPDH. This comparative study has highlighted considerable heterogeneity in PRK and GAPDH protein interactions between closely related species and the possible underlying physiological basis for this is discussed.

  16. Cutinase-Like Enzyme from the Yeast Cryptococcus sp. Strain S-2 Hydrolyzes Polylactic Acid and Other Biodegradable Plastics

    PubMed Central

    Masaki, Kazuo; Kamini, Numbi Ramudu; Ikeda, Hiroko; Iefuji, Haruyuki

    2005-01-01

    A purified lipase from the yeast Cryptococcus sp. strain S-2 exhibited remote homology to proteins belonging to the cutinase family rather than to lipases. This enzyme could effectively degrade the high-molecular-weight compound polylactic acid, as well as other biodegradable plastics, including polybutylene succinate, poly (ɛ-caprolactone), and poly(3-hydroxybutyrate). PMID:16269800

  17. The Tricarboxylic Acid Cycle, an Ancient Metabolic Network with a Novel Twist

    PubMed Central

    Mailloux, Ryan J.; Bériault, Robin; Lemire, Joseph; Singh, Ranji; Chénier, Daniel R.; Hamel, Robert D.; Appanna, Vasu D.

    2007-01-01

    The tricarboxylic acid (TCA) cycle is an essential metabolic network in all oxidative organisms and provides precursors for anabolic processes and reducing factors (NADH and FADH2) that drive the generation of energy. Here, we show that this metabolic network is also an integral part of the oxidative defence machinery in living organisms and α-ketoglutarate (KG) is a key participant in the detoxification of reactive oxygen species (ROS). Its utilization as an anti-oxidant can effectively diminish ROS and curtail the formation of NADH, a situation that further impedes the release of ROS via oxidative phosphorylation. Thus, the increased production of KG mediated by NADP-dependent isocitrate dehydrogenase (NADP-ICDH) and its decreased utilization via the TCA cycle confer a unique strategy to modulate the cellular redox environment. Activities of α-ketoglutarate dehydrogenase (KGDH), NAD-dependent isocitrate dehydrogenase (NAD-ICDH), and succinate dehydrogenase (SDH) were sharply diminished in the cellular systems exposed to conditions conducive to oxidative stress. These findings uncover an intricate link between TCA cycle and ROS homeostasis and may help explain the ineffective TCA cycle that characterizes various pathological conditions and ageing. PMID:17668068

  18. [Effects of elevated ozone concentrations on enzyme activities and organic acids content in wheat rhizospheric soil.

    PubMed

    Yin, Wei Qin; Jing, Hao Qi; Wang, Ya Bo; Wei, Si Yu; Sun, Yue; Wang, Sheng Sen; Wang, Xuai Zhi

    2018-02-01

    The elevated concentration of tropospheric ozone (O 3 ) is an important global climate change driver, with adverse impacts on soil ecological environment and crop growth. In this study, a pot experiment was carried out in an open top chamber (OTC), to investigate the effects of elevated ozone concentration on soil enzyme activities (catalase, polyphenol oxidase, dehydrogenase and invertase), organic acids contents (oxalic acid, citric acid and malic acid) at different growth stages (tillering, jointing, heading and ripening stages) of wheat, and combined with the rhizospheric soil physicochemical properties and plant root characteristics to analyze the underlying reasons. The results showed that, elevated ozone concentration increased soil catalase, polyphenol oxidase, dehydrogenase and invertase activities at wheat ripening period to different degrees, with the effects on the activities of catalase and polyphenol oxidase being statistically significant. At the heading stage, activities of dehydrogenase and invertase were significantly increased by up to 76.7%. At the ripening stage, elevated ozone concentration significantly increased the content of citric acid and malic acid and redox potential (Eh) in rhizospheric soil, but reduced soil pH, electrical conductivity, total carbon and nitrogen. For root characteristics, elevated ozone concentrations significantly reduced the wheat root biomass, total root length and root surface area but increased the average root diameter.

  19. Condensing enzymes from Cuphea wrightii associated with medium chain fatty acid biosynthesis.

    PubMed

    Slabaugh, M B; Leonard, J M; Knapp, S J

    1998-03-01

    Seed oils of most Cuphea species contain > 90% medium chain (C8-C14) fatty acids. Thioesterases with specificity for these substrates are important determinants of the medium chain phenotype. The role of condensing enzymes, however, has not been investigated. cDNA clones encoding beta-ketoacyl-acyl carrier protein (ACP) synthase (KAS) were isolated from C. wrightii, a C10/C12-producing species. Deduced amino acid sequences of four unique clones were approximately 60% identical to plant KAS I sequences and approximately 75% identical to a distinct class of KAS sequences recently identified in castor and barley. A 46 kDa protein that was observed only in developing and mature seed was detected using antiserum directed against recombinant Cuphea KAS protein. The 46 kDa protein was abundant in developing seeds of six medium chain-producing Cuphea species but barely detected in one long chain-producing species. A 48 kDa protein identified immunologically as KAS I was expressed in both medium and long chain-producing Cuphea species and was detected in all tissues tested. In in vitro assays, extracts from C. wrightii and C. viscosissima developing embryos were unable to extend fatty acid chains beyond C10 following treatment with 10 microns cerulenin, a potent inhibitor of KAS I. However, a C. viscosissima mutant, cpr-1, whose seed oils are deficient in caprate relative to wild type, was impaired in extension of C8 to C10 in this assay and Western analysis revealed a specific deficiency in 46 kDa KAS in cpr-1 embryos. These results implicate cerulenin-resistant condensing activity in production of medium chain fatty acids in Cuphea.

  20. Neuroprotection of ebselen against ischemia/reperfusion injury involves GABA shunt enzymes.

    PubMed

    Seo, Jeong Yeol; Lee, Choong Hyun; Cho, Jun Hwi; Choi, Jung Hoon; Yoo, Ki-Yeon; Kim, Dae Won; Park, Ok Kyu; Li, Hua; Choi, Soo Young; Hwang, In Koo; Won, Moo-Ho

    2009-10-15

    Seleno-organic compound, ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), is a substrate with radical-scavenging activity. In this study, we observed the neuroprotective effects of ebselen against ischemic damage and on GABA shunt enzymes such as glutamic acid decarboxylase 67 (GAD67), GABA transaminse (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) in the hippocampal CA1 region after 5 min of transient forebrain ischemia in gerbils. For this, vehicle (physiological saline) or ebselen was administered 30 min before or after ischemia/reperfusion and sacrificed 4 days after ischemia/reperfusion. The administration of ebselen significantly reduced the neuronal death in the CA1 region induced by ischemia/reperfusion. In addition, treatment with ebselen markedly elevated GAD67, GABA-T and SSADH immunoreactivity and their protein levels compared to that in the vehicle-treated group, respectively. These results suggest that ebselen protects neurons from ischemic damage via control of the expressions of GABA shunt enzymes to enter the TCA cycle.

  1. Reciprocal regulation of p53 and malic enzymes modulates metabolism and senescence.

    PubMed

    Jiang, Peng; Du, Wenjing; Mancuso, Anthony; Wellen, Kathryn E; Yang, Xiaolu

    2013-01-31

    Cellular senescence both protects multicellular organisms from cancer and contributes to their ageing. The pre-eminent tumour suppressor p53 has an important role in the induction and maintenance of senescence, but how it carries out this function remains poorly understood. In addition, although increasing evidence supports the idea that metabolic changes underlie many cell-fate decisions and p53-mediated tumour suppression, few connections between metabolic enzymes and senescence have been established. Here we describe a new mechanism by which p53 links these functions. We show that p53 represses the expression of the tricarboxylic-acid-cycle-associated malic enzymes ME1 and ME2 in human and mouse cells. Both malic enzymes are important for NADPH production, lipogenesis and glutamine metabolism, but ME2 has a more profound effect. Through the inhibition of malic enzymes, p53 regulates cell metabolism and proliferation. Downregulation of ME1 and ME2 reciprocally activates p53 through distinct MDM2- and AMP-activated protein kinase-mediated mechanisms in a feed-forward manner, bolstering this pathway and enhancing p53 activation. Downregulation of ME1 and ME2 also modulates the outcome of p53 activation, leading to strong induction of senescence, but not apoptosis, whereas enforced expression of either malic enzyme suppresses senescence. Our findings define physiological functions of malic enzymes, demonstrate a positive-feedback mechanism that sustains p53 activation, and reveal a connection between metabolism and senescence mediated by p53.

  2. How Enzymes Work: A Look through the Perspective of Molecular Viscoelastic Properties

    NASA Astrophysics Data System (ADS)

    Qu, Hao; Zocchi, Giovanni

    2013-01-01

    We present nanorheology measurements on the folded state of an enzyme that show directly that the (ensemble-averaged) stress-strain relations are nonlinear and frequency dependent beyond 1-Å deformation. We argue that this frequency dependence allows for opening a nonequilibrium cycle in the force-deformation plane if the forward and backward conformational changes of the enzyme during catalysis happen at different speeds. Using a heuristic model for the experimentally established viscoelastic properties of the enzyme, we examine a number of general features of enzymatic action. We find that the proposed viscoelastic cycle is consistent with the linear decrease of the speed of motor proteins with load. We find a relation between the stall force and the maximum rate for enzymes (in general) and motors (in particular). We estimate the stall force of the motor protein kinesin from thermodynamic quantities and estimate the maximum rate of enzymes from purely mechanical quantities. We propose that the viscoelastic cycle provides a framework for considering mechanochemical coupling in enzymes on the basis of possibly universal materials properties of the folded state of proteins.

  3. Determining the N and O isotope effects of microbial nitrite reduction: the global N cycle implications of an enzyme-dependent isotope effect

    NASA Astrophysics Data System (ADS)

    Martin, T. S.; Casciotti, K. L.

    2014-12-01

    The marine nitrogen (N) cycle is a dynamic system of critical importance, since nitrogen is the limiting nutrient in over half of the world's oceans. Denitrification and anammox, the main N loss processes from the ocean, have different effects on carbon cycling and greenhouse gas emission. Understanding the balance between the two processes is vital to understanding the role of the N cycle in global climate change. One approach for investigating these processes is by using stable isotope analysis to estimate the relative magnitudes of N fluxes, particularly for biologically mediated processes. In order to make the most of the currently available isotope analysis techniques, it is necessary to know the isotope effects for each processes occurring in the environment. Nitrite reduction is an important step in denitrification. Previous work had begun to explore the N isotope effects for nitrite reduction, but no oxygen (O) isotope effect has been measured. Additionally, no consideration has been given to the type of nitrite reductase carrying out the reaction. There are two main types of respiratory nitrite reductase, one that is Cu-based and another that is Fe-based. We performed batch culture experiments with denitrifier strains possessing either a Cu-type or Fe-type nitrite reductase. Both N and O isotope effects for nitrite reduction were determined for each of these experiments by measuring the NO2- concentration, as well as the N and O isotopes of nitrite and applying a Rayleigh fractionation model. Both the N and O isotope effects were found to be significantly different between the two types of enzymes. This enzyme-linked difference in isotope effects emphasizes the importance of microbial community composition within the global N cycle.

  4. Lactic acid bacteria affect serum cholesterol levels, harmful fecal enzyme activity, and fecal water content.

    PubMed

    Lee, Do Kyung; Jang, Seok; Baek, Eun Hye; Kim, Mi Jin; Lee, Kyung Soon; Shin, Hea Soon; Chung, Myung Jun; Kim, Jin Eung; Lee, Kang Oh; Ha, Nam Joo

    2009-06-11

    Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as lower cholesterol. Although present in many foods, most trials have been in spreads or dairy products. Here we tested whether Bifidobacteria isolates could lower cholesterol, inhibit harmful enzyme activities, and control fecal water content. In vitro culture experiments were performed to evaluate the ability of Bifidobacterium spp. isolated from healthy Koreans (20 approximately 30 years old) to reduce cholesterol-levels in MRS broth containing polyoxyethanylcholesterol sebacate. Animal experiments were performed to investigate the effects on lowering cholesterol, inhibiting harmful enzyme activities, and controlling fecal water content. For animal studies, 0.2 ml of the selected strain cultures (108 approximately 109 CFU/ml) were orally administered to SD rats (fed a high-cholesterol diet) every day for 2 weeks. B. longum SPM1207 reduced serum total cholesterol and LDL levels significantly (p < 0.05), and slightly increased serum HDL. B. longum SPM1207 also increased fecal LAB levels and fecal water content, and reduced body weight and harmful intestinal enzyme activities. Daily consumption of B. longum SPM1207 can help in managing mild to moderate hypercholesterolemia, with potential to improve human health by helping to prevent colon cancer and constipation.

  5. Stabilization of enzymes in ionic liquids via modification of enzyme charge.

    PubMed

    Nordwald, Erik M; Kaar, Joel L

    2013-09-01

    Due to the propensity of ionic liquids (ILs) to inactivate enzymes, the development of strategies to improve enzyme utility in these solvents is critical to fully exploit ILs for biocatalysis. We have developed a strategy to broadly improve enzyme utility in ILs based on elucidating the effect of charge modifications on the function of enzymes in IL environments. Results of stability studies in aqueous-IL mixtures indicated a clear connection between the ratio of enzyme-containing positive-to-negative sites and enzyme stability in ILs. Stability studies of the effect of [BMIM][Cl] and [EMIM][EtSO4 ] on chymotrypsin specifically found an optimum ratio of positively-charged amine-to-negatively-charged acid groups (0.39). At this ratio, the half-life of chymotrypsin was increased 1.6- and 4.3-fold relative to wild-type chymotrypsin in [BMIM][Cl] and [EMIM][EtSO4 ], respectively. The half-lives of lipase and papain were similarly increased as much as 4.0 and 2.4-fold, respectively, in [BMIM][Cl] by modifying the ratio of positive-to-negative sites of each enzyme. More generally, the results of stability studies found that modifications that reduce the ratio of enzyme-containing positive-to-negative sites improve enzyme stability in ILs. Understanding the impact of charge modification on enzyme stability in ILs may ultimately be exploited to rationally engineer enzymes for improved function in IL environments. Copyright © 2013 Wiley Periodicals, Inc.

  6. Low-temperature Storage of Cucumbers Induces Changes in the Organic Acid Content and in Citrate Synthase Activity

    USDA-ARS?s Scientific Manuscript database

    To elucidate the cause of reported pyruvate accumulation in chilled stored cucumbers (Cucumis sativus L.) cv. ‘Toppugurin’, we have examined differences in the extent of incorporation of acetate-1,2-14C into the tricarboxylic acid (TCA) cycle and the specific activity of the enzyme citrate synthase ...

  7. A viscous solvent enables information transfer from gene-length nucleic acids in a model prebiotic replication cycle

    NASA Astrophysics Data System (ADS)

    He, Christine; Gállego, Isaac; Laughlin, Brandon; Grover, Martha A.; Hud, Nicholas V.

    2017-04-01

    Many hypotheses concerning the nature of early life assume that genetic information was once transferred through the template-directed synthesis of RNA, before the emergence of coded enzymes. However, attempts to demonstrate enzyme-free, template-directed synthesis of nucleic acids have been limited by 'strand inhibition', whereby transferring information from a template strand in the presence of its complementary strand is inhibited by the stability of the template duplex. Here, we use solvent viscosity to circumvent strand inhibition, demonstrating information transfer from a gene-length template (>300 nt) within a longer (545 bp or 3 kb) duplex. These results suggest that viscous environments on the prebiotic Earth, generated periodically by water evaporation, could have facilitated nucleic acid replication—particularly of long, structured sequences such as ribozymes. Our approach works with DNA and RNA, suggesting that viscosity-mediated replication is possible for a range of genetic polymers, perhaps even for informational polymers that may have preceded RNA.

  8. Reconstruction of Cysteine Biosynthesis Using Engineered Cysteine-Free and Methionine-Free Enzymes

    NASA Technical Reports Server (NTRS)

    Wang, Kendrick; Fujishima, Kosuke; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J.

    2016-01-01

    Ten of the proteinogenic amino acids can be generated abiotically while the remaining thirteen require biology for their synthesis. Paradoxically, the biosynthesis pathways observed in nature require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine. Here, we substituted alternate amino acids for cysteine and also methionine, which is biosynthesized from cysteine, in serine acetyl transferase (CysE) and O-acetylserine sulfhydrylase (CysM). CysE function was rescued by cysteine-and-methionine-free enzymes and CysM function was rescued by cysteine-free enzymes. Structural modeling suggests that methionine stabilizes CysM and is present in the active site of CysM. Cysteine is not conserved among CysE and CysM protein orthologs, suggesting that cysteine is not functionally important for its own synthesis. Engineering biosynthetic enzymes that lack the amino acids being synthesized provides insights into the evolution of amino acid biosynthesis and pathways for bioengineering.

  9. A Diel Flux Balance Model Captures Interactions between Light and Dark Metabolism during Day-Night Cycles in C3 and Crassulacean Acid Metabolism Leaves1[C][W][OPEN

    PubMed Central

    Cheung, C.Y. Maurice; Poolman, Mark G.; Fell, David. A.; Ratcliffe, R. George; Sweetlove, Lee J.

    2014-01-01

    Although leaves have to accommodate markedly different metabolic flux patterns in the light and the dark, models of leaf metabolism based on flux-balance analysis (FBA) have so far been confined to consideration of the network under continuous light. An FBA framework is presented that solves the two phases of the diel cycle as a single optimization problem and, thus, provides a more representative model of leaf metabolism. The requirement to support continued export of sugar and amino acids from the leaf during the night and to meet overnight cellular maintenance costs forces the model to set aside stores of both carbon and nitrogen during the day. With only minimal constraints, the model successfully captures many of the known features of C3 leaf metabolism, including the recently discovered role of citrate synthesis and accumulation in the night as a precursor for the provision of carbon skeletons for amino acid synthesis during the day. The diel FBA model can be applied to other temporal separations, such as that which occurs in Crassulacean acid metabolism (CAM) photosynthesis, allowing a system-level analysis of the energetics of CAM. The diel model predicts that there is no overall energetic advantage to CAM, despite the potential for suppression of photorespiration through CO2 concentration. Moreover, any savings in enzyme machinery costs through suppression of photorespiration are likely to be offset by the higher flux demand of the CAM cycle. It is concluded that energetic or nitrogen use considerations are unlikely to be evolutionary drivers for CAM photosynthesis. PMID:24596328

  10. Involvement of organic acids and amino acids in ameliorating Ni(II) toxicity induced cell cycle dysregulation in Caulobacter crescentus: a metabolomics analysis.

    PubMed

    Jain, Abhishek; Chen, Wei Ning

    2018-05-01

    Nickel (Ni(II)) toxicity is addressed by many different bacteria, but bacterial responses to nickel stress are still unclear. Therefore, we studied the effect of Ni(II) toxicity on cell proliferation of α-proteobacterium Caulobacter crescentus. Next, we showed the mechanism that allows C. crescentus to survive in Ni(II) stress condition. Our results revealed that the growth of C. crescentus is severely affected when the bacterium was exposed to different Ni(II) concentrations, 0.003 mM slightly affected the growth, 0.008 mM reduced the growth by 50%, and growth was completely inhibited at 0.015 mM. It was further shown that Ni(II) toxicity induced mislocalization of major regulatory proteins such as MipZ, FtsZ, ParB, and MreB, resulting in dysregulation of the cell cycle. GC-MS metabolomics analysis of Ni(II) stressed C. crescentus showed an increased level of nine important metabolites including TCA cycle intermediates and amino acids. This indicates that changes in central carbon metabolism and nitrogen metabolism are linked with the disruption of cell division process. Addition of malic acid, citric acid, alanine, proline, and glutamine to 0.015 mM Ni(II)-treated C. crescentus restored its growth. Thus, the present work shows a protective effect of these organic acids and amino acids on Ni(II) toxicity. Metabolic stimulation through the PutA/GlnA pathway, accelerated degradation of CtrA, and Ni-chelation by organic acids or amino acids are some of the possible mechanisms suggested to be involved in enhancing C. crescentus's tolerance. Our results shed light on the mechanism of increased Ni(II) tolerance in C. crescentus which may be useful in bioremediation strategies and synthetic biology applications such as the development of whole cell biosensor.

  11. Four distinct types of E.C. 1.2.1.30 enzymes can catalyze the reduction of carboxylic acids to aldehydes.

    PubMed

    Stolterfoht, Holly; Schwendenwein, Daniel; Sensen, Christoph W; Rudroff, Florian; Winkler, Margit

    2017-09-10

    Increasing demand for chemicals from renewable resources calls for the development of new biotechnological methods for the reduction of oxidized bio-based compounds. Enzymatic carboxylate reduction is highly selective, both in terms of chemo- and product selectivity, but not many carboxylate reductase enzymes (CARs) have been identified on the sequence level to date. Thus far, their phylogeny is unexplored and very little is known about their structure-function-relationship. CARs minimally contain an adenylation domain, a phosphopantetheinylation domain and a reductase domain. We have recently identified new enzymes of fungal origin, using similarity searches against genomic sequences from organisms in which aldehydes were detected upon incubation with carboxylic acids. Analysis of sequences with known CAR functionality and CAR enzymes recently identified in our laboratory suggests that the three-domain architecture mentioned above is modular. The construction of a distance tree with a subsequent 1000-replicate bootstrap analysis showed that the CAR sequences included in our study fall into four distinct subgroups (one of bacterial origin and three of fungal origin, respectively), each with a bootstrap value of 100%. The multiple sequence alignment of all experimentally confirmed CAR protein sequences revealed fingerprint sequences of residues which are likely to be involved in substrate and co-substrate binding and one of the three catalytic substeps, respectively. The fingerprint sequences broaden our understanding of the amino acids that might be essential for the reduction of organic acids to the corresponding aldehydes in CAR proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Cinnamic Acid, an Autoinducer of Its Own Biosynthesis, Is Processed via Hca Enzymes in Photorhabdus luminescens▿

    PubMed Central

    Chalabaev, Sabina; Turlin, Evelyne; Bay, Sylvie; Ganneau, Christelle; Brito-Fravallo, Emma; Charles, Jean-François; Danchin, Antoine; Biville, Francis

    2008-01-01

    Photorhabdus luminescens, an entomopathogenic bacterium and nematode symbiont, has homologues of the Hca and Mhp enzymes. In Escherichia coli, these enzymes catalyze the degradation of the aromatic compounds 3-phenylpropionate (3PP) and cinnamic acid (CA) and allow the use of 3PP as sole carbon source. P. luminescens is not able to use 3PP and CA as sole carbon sources but can degrade them. Hca dioxygenase is involved in this degradation pathway. P. luminescens synthesizes CA from phenylalanine via a phenylalanine ammonia-lyase (PAL) and degrades it via the not-yet-characterized biosynthetic pathway of 3,5-dihydroxy-4-isopropylstilbene (ST) antibiotic. CA induces its own synthesis by enhancing the expression of the stlA gene that codes for PAL. P. luminescens bacteria release endogenous CA into the medium at the end of exponential growth and then consume it. Hca dioxygenase is involved in the consumption of endogenous CA but is not required for ST production. This suggests that CA is consumed via at least two separate pathways in P. luminescens: the biosynthesis of ST and a pathway involving the Hca and Mhp enzymes. PMID:18245247

  13. Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase.

    PubMed

    Selak, Mary A; Armour, Sean M; MacKenzie, Elaine D; Boulahbel, Houda; Watson, David G; Mansfield, Kyle D; Pan, Yi; Simon, M Celeste; Thompson, Craig B; Gottlieb, Eyal

    2005-01-01

    Several mitochondrial proteins are tumor suppressors. These include succinate dehydrogenase (SDH) and fumarate hydratase, both enzymes of the tricarboxylic acid (TCA) cycle. However, to date, the mechanisms by which defects in the TCA cycle contribute to tumor formation have not been elucidated. Here we describe a mitochondrion-to-cytosol signaling pathway that links mitochondrial dysfunction to oncogenic events: succinate, which accumulates as a result of SDH inhibition, inhibits HIF-alpha prolyl hydroxylases in the cytosol, leading to stabilization and activation of HIF-1alpha. These results suggest a mechanistic link between SDH mutations and HIF-1alpha induction, providing an explanation for the highly vascular tumors that develop in the absence of VHL mutations.

  14. Characterization of a dual specificity aryl acid adenylation enzyme with dual function in nikkomycin biosynthesis.

    PubMed

    Moon, Mary; Van Lanen, Steven G

    2010-09-01

    Nikkomycin Z is a dipeptide antifungal antibiotic characterized by two nonproteinogenic amino acids, nikkomycin C(Z) and 4-(4'-hydroxy-2'-pyridinyl)-homothreonine (HPHT). The HPHT scaffold is assembled by an aldol reaction between 2-oxobutyrate and picolinaldehyde, the latter of which is derived from picolinic acid that is activated and loaded to coenzyme A by the aryl-activating adenylation enzyme, NikE. We now provide evidence that NikE is also involved in the activation and loading of the alpha-keto acid precursor, 4-(2'-pyridinyl)-2-oxo-4-hydroxyisovalerate (POHIV), to a phosphopantetheinyl group of an acyl carrier protein domain of NikT. POHIV was synthesized using Escherichia coli 2-dehydro-3-deoxy-phosphogluconate aldolase, and phenylalanine dehydrogenase from Bacillus sp. NRRL B-14911 was used to prepare the alpha-amino acid, 4-(2'-pyridinyl)-homothreonine (PHT). Using the carboxylic acid-dependent, ATP-[(32)P]PP(i) exchange assay, NikE is shown to activate both picolinic acid and POHIV but not PHT. Furthermore, NikE loads POHIV to holo-NikT to generate a new thioester-linked intermediate, which was not observed using a NikT(S33A) mutant. Thus, NikE activates two distinct carboxylic acids to form two new thioester intermediates, one of which is subsequently reduced to the aldehyde and the other that likely serves as a substrate for the aminotransferase domain of NikT prior to condensation with nikkomycin C(Z) to yield the dipeptide. Copyright 2010 Wiley Periodicals, Inc.

  15. [Combined effects of copper and simulated acid rain on copper accumulation, growth, and antioxidant enzyme activities of Rumex acetosa].

    PubMed

    He, Shan-Ying; Gao, Yong-Jie; Shentu, Jia-Li; Chen, Kun-Bai

    2011-02-01

    A pot experiment was conducted to study the combined effects of Cu (0-1500 mg x kg(-1)) and simulated acid rain (pH 2.5-5.6) on the copper accumulation, growth, and antioxidant enzyme activities of Rumex acetosa. With the increasing concentration of soil Cu, the Cu accumulation in R. acetosa increased, being higher in root than in stem and leaf. The exposure to low pH acid rain promoted the Cu uptake by R. acetosa. With the increase of soil Cu concentration and/or of acid rain acidity, the biomass of R. acetosa decreased, leaf and root MDA contents increased and had good correlation with soil Cu concentration, and the SOD and POD activities in leaf and root displayed a decreasing trend after an initial increase. This study showed that R. acetosa had a strong adaptive ability to Cu and acid rain stress, exhibiting a high application potential in the remediation of Cu-contaminated soil in acid rain areas.

  16. Increased saccharification yields from aspen biomass upon treatment with enzymatically generated peracetic acid.

    PubMed

    Duncan, Shona; Jing, Qing; Katona, Adrian; Kazlauskas, Romas J; Schilling, Jonathan; Tschirner, Ulrike; Aldajani, Waleed Wafa

    2010-03-01

    The recalcitrance of lignocellulosic biomass to enzymatic release of sugars (saccharification) currently limits its use as feedstock for biofuels. Enzymatic hydrolysis of untreated aspen wood releases only 21.8% of the available sugars due primarily to the lignin barrier. Nature uses oxidative enzymes to selectively degrade lignin in lignocellulosic biomass, but thus far, natural enzymes have been too slow for industrial use. In this study, oxidative pretreatment with commercial peracetic acid (470 mM) removed 40% of the lignin (from 19.9 to 12.0 wt.% lignin) from aspen and enhanced the sugar yields in subsequent enzymatic hydrolysis to about 90%. Increasing the amount of lignin removed correlated with increasing yields of sugar release. Unfortunately, peracetic acid is expensive, and concentrated forms can be hazardous. To reduce costs and hazards associated with using commercial peracetic acid, we used a hydrolase to catalyze the perhydrolysis of ethyl acetate generating 60-70 mM peracetic acid in situ as a pretreatment to remove lignin from aspen wood. A single pretreatment was insufficient, but multiple cycles (up to eight) removed up to 61.7% of the lignin enabling release of >90% of the sugars during saccharification. This value corresponds to a predicted 581 g of fermentable sugars from 1 kg of aspen wood. Improvements in the enzyme stability are needed before the enzymatically generated peracetic acid is a commercially viable alternative.

  17. Chlorogenic acid ameliorates isoproterenol-induced myocardial injury in rats by stabilizing mitochondrial and lysosomal enzymes.

    PubMed

    Akila, Palaniyandi; Asaikumar, Lourthurani; Vennila, Lakshmanan

    2017-01-01

    This study was deliberated to aspire the effects of chlorogenic acid (CGA) against myocardial infarction (MI) induced by Isoproterenol (ISO), in a rat model. In the pathology of MI, enzymes released due to the mitochondrial and lysosomal lipid peroxidation play an integral role. Induction of rats with ISO (85mg/kg BW) for 2 consecutive days resulted in a significant decrease in the activities of heart mitochondrial enzymes isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (α-KGDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH). The activities of lysosomal enzymes (β- glucosidase, β-glucuronidase, α-galactosidase, β-galactosidase, cathepsin-B and cathepsin-D) were increased significantly in the heart tissue. A prominent expression of LDH 1 and LDH 2 isoenzymes in the serum were observed and changes in the Electrocardiographic (ECG) patterns were also recorded in the ISO-induced rats. The prior administrations of CGA (40mg/kg BW) for 19days markedly ameliorated ISO induced alterations in ECG and significantly restored the activities of all the above enzymes in the heart of ISO-induced rats, which substantiates the stress stabilizing action of CGA. Oral administration of CGA (40mg/kg BW) to normal rats did not show any significant changes. These biochemical functional alterations were supported by the histology of heart (Massion's trichrome and Picrosirius red staining for collagen formation). Thereupon, this study shows that 40mg/kg BW of CGA gives protection against ISO-induced MI and demonstrates that CGA has a significant effect in the protection of heart. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  18. Effect of caffeine, caffeic acid and their various combinations on enzymes of cholinergic, monoaminergic and purinergic systems critical to neurodegeneration in rat brain-In vitro.

    PubMed

    Akomolafe, S F; Akinyemi, A J; Ogunsuyi, O B; Oyeleye, S I; Oboh, G; Adeoyo, O O; Allismith, Y R

    2017-09-01

    Caffeine and caffeic acid are two bioactive compounds that are present in plant foods and are major constituent of coffee, cocoa, tea, cola drinks and chocolate. Although not structurally related, caffeine and caffeic acid has been reported to elicit neuroprotective properties. However, their different proportional distribution in food sources and possible effect of such interactions are not often taken into consideration. Therefore, in this study, we investigated the effect of caffeine, caffeic acid and their various combinations on activities of some enzymes [acetylcholinesterase (AChE), monoamine oxidase (MAO) ecto-nucleoside triphosphate diphosphohydrolase (E-NTPase), ecto-5 1 -nucleotidase (E-NTDase) and Na + /K + ATPase relevant to neurodegeneration in vitro in rat brain. The stock concentration of caffeine and caffiec acid and their various proportional combinations were prepared and their interactions with the activities of these enzymes were assessed (in vitro) in different brain structures. The Fe 2+ and Cu 2+ chelating abilities of the samples were also investigated. The results revealed that caffeine, caffeic acid and their various combinations exhibited inhibitory effect on activities of AChE, MAO, E-NTPase and E-NTDase, but stimulatory effect on Na + /K + ATPase activity. The combinations also exhibited Fe 2+ and Cu 2+ chelating abilities. Considering the various combinations, a higher caffeine to caffeic acid ratio produced significantly highest enzyme modulatory effects; these were significantly lower to the effect of caffeine alone but significantly higher than the effect of caffeic acid alone. These findings may provide new insight into the effect of proportional combination of these bioactive compounds as obtained in many foods especially with respect to their neuroprotective effects. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. The inhibitory effect of convulsant agents on the enzyme in brain which inactivates nerveside.

    PubMed

    Toh, C C

    1969-07-01

    1. An enzyme which can be extracted from brain inactivates nerveside in the optimum pH range 5.8-7.0.2. The polybasic acids trypan blue and its analogue trypan red, bromphenol blue and its analogue bromthymol blue at concentrations of 0.22 mM and ethylenediaminetetra-acetic acid (EDTA) at a concentration of 1 mM are strong inhibitors of the enzyme.3. Penicillin which is a monobasic carboxylic acid also inhibits the enzyme but only if concentrations as high as 3.6 mM are used. The antibiotic streptomycin which is a basic substance does not inhibit the enzyme.4. Caffeine at a concentration of 7.2 mM only weakly inhibits the enzyme.5. Chymotrypsin and wheat germ acid phosphatase also inactivate nerveside at pH 5.9 and are inhibited by the acidic dyes and penicillin. EDTA inhibits wheat germ phosphatase but activates chymotrypsin.6. Inactivation of nerveside by the brain enzyme and by wheat germ phosphatase is different from the action of chymotrypsin. Nerveside solutions incubated with chymotrypsin completely lose all biological activity whereas if incubation is carried out with either the brain enzyme or wheat germ acid phosphatase a residual biological activity remains even when the concentration of these two enzymes is increased. This residual biological activity is due to a peptide as it is destroyed by chymotrypsin.7. The manner in which nerveside is inactivated by the brain enzyme is uncertain as the preparation of the latter contained phosphodiesterase and protease activities which were similarly inhibited by the acid dyes, penicillin and EDTA.8. Pentylenetetrazole, picrotoxin, strychnine and tetanus toxin do not inhibit the brain enzyme.9. The nerveside-inactivating enzyme is not identical with the Substance P-inactivating enzyme in brain as the former is inhibited by EDTA while the latter is not.

  20. Temperature sensitivity differences with depth and season between carbon, nitrogen, and phosphorus cycling enzyme activities in an ombrotrophic peatland system

    NASA Astrophysics Data System (ADS)

    Steinweg, J. M.; Kostka, J. E.; Hanson, P. J.; Schadt, C. W.

    2017-12-01

    Northern peatlands have large amounts of soil organic matter due to reduced decomposition. Breakdown of organic matter is initially mediated by extracellular enzymes, the activity of which may be controlled by temperature, moisture, and substrate availability, all of which vary seasonally throughout the year and with depth. In typical soils the majority of the microbial biomass and decomposition occurs within the top 30cm due to reduced organic matter inputs in the subsurface however peatlands by their very nature contain large amounts of organic matter throughout their depth profile. We hypothesized that potential enzyme activity would be greatest at the surface of the peat due to a larger microbial biomass compared to 40cm and 175cm below the surface and that temperature sensitivity would be greatest at the surface during winter but lowest during the summer due to high temperatures and enzyme efficiency. Peat samples were collected in February, July, and August 2012 from the DOE Spruce and Peatland Responses Under Climatic and Environmental Change project at Marcell Experimental Forest S1 bog. We measured potential activity of hydrolytic enzymes involved in three different nutrient cycles: beta-glucosidase (carbon), leucine amino peptidase (nitrogen), and phosphatase (phosphorus) at 15 temperature points ranging from 3°C to 65°C. Enzyme activity decreased with depth as expected but there was no concurrent change in activation energy (Ea). The reduction in enzyme activity with depth indicates a smaller pool which coincided with a decreased microbial biomass. Differences in enzyme activity with depth also mirrored the changes in peat composition from the acrotelm to the catotelm. Season did play a role in temperature sensitivity with Ea of β-glucosidase and phosphatase being the lowest in August as expected but leucine amino peptidase (a nitrogen acquiring enzyme) Ea was not influenced by season. As temperatures rise, especially in winter months, enzymatic

  1. Efficacy of azelaic acid on hepatic key enzymes of carbohydrate metabolism in high fat diet induced type 2 diabetic mice.

    PubMed

    Muthulakshmi, Shanmugam; Saravanan, Ramalingam

    2013-06-01

    Azelaic acid (AzA), a C9 linear α,ω-dicarboxylic acid, is found in whole grains namely wheat, rye, barley, oat seeds and sorghum. The study was performed to investigate whether AzA exerts beneficial effect on hepatic key enzymes of carbohydrate metabolism in high fat diet (HFD) induced type 2 diabetic C57BL/6J mice. C57BL/6J mice were fed high fat diet for 10 weeks and subjected to intragastric administration of various doses (20 mg, 40 mg and 80 mg/kg BW) of AzA daily for the subsequent 5 weeks. Rosiglitazone (RSG) was used as reference drug. Body weight, food intake, plasma glucose, plasma insulin, blood haemoglobin (Hb), blood glycosylated haemoglobin (HbA1c), liver glycolytic enzyme (hexokinase), hepatic shunt enzyme (glucose-6-phosphate dehydrogenase), gluconeogenic enzymes(glucose-6-phosphatase and fructose-1,6-bisphosphatase), liver glycogen, plasma and liver triglycerides were examined in mice fed with normal standard diet (NC), high fat diet (HFD), HFD with AzA (HFD + AzA) and HFD with rosiglitazone (HFD + RSG). Among the three doses, 80 mg/kg BW of AzA was able to positively regulate plasma glucose, insulin, blood HbA1c and haemoglobin levels by significantly increasing the activity of hexokinase and glucose-6-phosphate dehydrogenase and significantly decreasing the activity of glucose-6-phosphatase and fructose-1,6-bisphosphatase thereby increasing the glycogen content in the liver. From this study, we put forward that AzA could significantly restore the levels of plasma glucose, insulin, HbA1c, Hb, liver glycogen and carbohydrate metabolic key enzymes to near normal in diabetic mice and hence, AzA may be useful as a biomaterial in the development of therapeutic agents against high fat diet induced T2DM. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  2. Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88

    PubMed Central

    Andersen, Mikael R.; Salazar, Margarita P.; Schaap, Peter J.; van de Vondervoort, Peter J.I.; Culley, David; Thykaer, Jette; Frisvad, Jens C.; Nielsen, Kristian F.; Albang, Richard; Albermann, Kaj; Berka, Randy M.; Braus, Gerhard H.; Braus-Stromeyer, Susanna A.; Corrochano, Luis M.; Dai, Ziyu; van Dijck, Piet W.M.; Hofmann, Gerald; Lasure, Linda L.; Magnuson, Jon K.; Menke, Hildegard; Meijer, Martin; Meijer, Susan L.; Nielsen, Jakob B.; Nielsen, Michael L.; van Ooyen, Albert J.J.; Pel, Herman J.; Poulsen, Lars; Samson, Rob A.; Stam, Hein; Tsang, Adrian; van den Brink, Johannes M.; Atkins, Alex; Aerts, Andrea; Shapiro, Harris; Pangilinan, Jasmyn; Salamov, Asaf; Lou, Yigong; Lindquist, Erika; Lucas, Susan; Grimwood, Jane; Grigoriev, Igor V.; Kubicek, Christian P.; Martinez, Diego; van Peij, Noël N.M.E.; Roubos, Johannes A.; Nielsen, Jens; Baker, Scott E.

    2011-01-01

    The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compel additional exploration. We therefore undertook whole-genome sequencing of the acidogenic A. niger wild-type strain (ATCC 1015) and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence, and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was used to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 Mb of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis supported up-regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases, and protein transporters in the protein producing CBS 513.88 strain. Our results and data sets from this integrative systems biology analysis resulted in a snapshot of fungal evolution and will support further optimization of cell factories based on filamentous fungi. PMID:21543515

  3. Effect of high-intensity intermittent swimming training on fatty acid oxidation enzyme activity in rat skeletal muscle.

    PubMed

    Terada, Shin; Tabata, Izumi; Higuchi, Mitsuru

    2004-02-01

    We previously reported that high-intensity exercise training significantly increased citrate synthase (CS) activity, a marker of oxidative enzyme, in rat skeletal muscle to a level equaling that attained after low-intensity prolonged exercise training (Terada et al., J Appl Physiol 90: 2019-2024, 2001). Since mitochondrial oxidative enzymes and fatty acid oxidation (FAO) enzymes are often increased simultaneously, we assessed the effect of high-intensity intermittent swimming training on FAO enzyme activity in rat skeletal muscle. Male Sprague-Dawley rats (3 to 4 weeks old) were assigned to a 10-day period of high-intensity intermittent exercise training (HIT), low-intensity prolonged exercise training (LIT), or sedentary control conditions. In the HIT group, the rats repeated fourteen 20 s swimming sessions with a weight equivalent to 14-16% of their body weight. Between the exercise sessions, a 10 s pause was allowed. Rats in the LIT group swam 6 h/day in two 3 h sessions separated by 45 min of rest. CS activity in the triceps muscle of rats in the HIT and LIT groups was significantly higher than that in the control rats by 36 and 39%, respectively. Furthermore, 3-beta hydroxyacyl-CoA dehydrogenase (HAD) activity, an important enzyme in the FAO pathway in skeletal muscle, was higher in the two training groups than in the control rats (HIT: 100%, LIT: 88%). No significant difference in HAD activity was observed between the two training groups. In conclusion, the present investigation demonstrated that high-intensity intermittent swimming training elevated FAO enzyme activity in rat skeletal muscle to a level similar to that attained after 6 h of low-intensity prolonged swimming exercise training.

  4. Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.

    PubMed

    Pellegrini, Vanessa O A; Serpa, Viviane Isabel; Godoy, Andre S; Camilo, Cesar M; Bernardes, Amanda; Rezende, Camila A; Junior, Nei Pereira; Franco Cairo, João Paulo L; Squina, Fabio M; Polikarpov, Igor

    2015-11-01

    Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

  5. Interaction between chitosan and its related enzymes: A review.

    PubMed

    Shinya, Shoko; Fukamizo, Tamo

    2017-11-01

    Chitosan-related enzymes including chitosanases, exo-β-glucosaminidases, and enzymes having chitosan-binding modules recognize ligands through electrostatic interactions between the acidic amino acids in proteins and amino groups of chitosan polysaccharides. However, in GH8 chitosanases, several aromatic residues are also involved in substrate recognition through stacking interactions, and these enzymes consequently hydrolyze β-1,4-glucan as well as chitosan. The binding grooves of these chitosanases are extended and opened at both ends of the grooves, so that the enzymes can clamp a long chitosan polysaccharide. The association/dissociation of positively charged glucosamine residues to/from the binding pocket of a GH2 exo-β-glucosaminidase controls the p K a of the catalytic acid, thereby maintaining the high catalytic potency of the enzyme. In contrast to chitosanases, chitosan-binding modules only accommodate a couple of glucosamine residues, predominantly recognizing the non-reducing end glucosamine residue of chitosan by electrostatic interactions and a hydrogen-bonding network. These structural findings on chitosan-related enzymes may contribute to future applications for the efficient conversion of the chitin/chitosan biomass. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Non-enzymic beta-decarboxylation of aspartic acid.

    NASA Technical Reports Server (NTRS)

    Doctor, V. M.; Oro, J.

    1972-01-01

    Study of the mechanism of nonenzymic beta-decarboxylation of aspartic acid in the presence of metal ions and pyridoxal. The results suggest that aspartic acid is first converted to oxalacetic acid by transamination with pyridoxal which in turn is converted to pyridoxamine. This is followed by decarboxylation of oxalacetic acid to form pyruvic acid which transaminates with pyridoxamine to form alanine. The possible significance of these results to prebiotic molecular evolution is briefly discussed.

  7. Starch-Branching Enzyme IIa Is Required for Proper Diurnal Cycling of Starch in Leaves of Maize1[OA

    PubMed Central

    Yandeau-Nelson, Marna D.; Laurens, Lieve; Shi, Zi; Xia, Huan; Smith, Alison M.; Guiltinan, Mark J.

    2011-01-01

    Starch-branching enzyme (SBE), a glucosyl transferase, is required for the highly regular pattern of α-1,6 bonds in the amylopectin component of starch. In the absence of SBEIIa, as shown previously in the sbe2a mutant of maize (Zea mays), leaf starch has drastically reduced branching and the leaves exhibit a severe senescence-like phenotype. Detailed characterization of the maize sbe2a mutant revealed that SBEIIa is the primary active branching enzyme in the leaf and that in its absence plant growth is affected. Both seedling and mature sbe2a mutant leaves do not properly degrade starch during the night, resulting in hyperaccumulation. In mature sbe2a leaves, starch hyperaccumulation is greatest in visibly senescing regions but also observed in green tissue and is correlated to a drastic reduction in photosynthesis within the leaf. Starch granules from sbe2a leaves observed via scanning electron microscopy and transmission electron microscopy analyses are larger, irregular, and amorphous as compared with the highly regular, discoid starch granules observed in wild-type leaves. This appears to trigger premature senescence, as shown by an increased expression of genes encoding proteins known to be involved in senescence and programmed cell death processes. Together, these results indicate that SBEIIa is required for the proper diurnal cycling of transitory starch within the leaf and suggest that SBEIIa is necessary in producing an amylopectin structure amenable to degradation by starch metabolism enzymes. PMID:21508184

  8. Cations in component reactions of `malic' enzyme catalysis

    PubMed Central

    Tsai, C. S.; Tsai, Y. H.; Samad, R. A.

    1971-01-01

    The `malic' enzyme (EC 1.1.1.40) has been purified (300-fold) from wheat germ and its abilities to catalyse the decarboxylation and the hydrogenation of oxaloacetic acid and oxaloacetate esters was studied. The free 1-carboxyl group is essential for the interaction of oxaloacetates and substituted oxaloacetates with the enzyme via cations. The free 4-carboxyl group is required for the decarboxylation but is not indispensable for the hydrogenation. At high concentrations, cations inhibit the enzymic hydrogenation of oxaloacetic acid but not that of 4-ethyl oxaloacetate. A plausible inhibitory mechanism is proposed. PMID:4399519

  9. Enzymes in Fermented Fish.

    PubMed

    Giyatmi; Irianto, H E

    Fermented fish products are very popular particularly in Southeast Asian countries. These products have unique characteristics, especially in terms of aroma, flavor, and texture developing during fermentation process. Proteolytic enzymes have a main role in hydrolyzing protein into simpler compounds. Fermentation process of fish relies both on naturally occurring enzymes (in the muscle or the intestinal tract) as well as bacteria. Fermented fish products processed using the whole fish show a different characteristic compared to those prepared from headed and gutted fish. Endogenous enzymes like trypsin, chymotrypsin, elastase, and aminopeptidase are the most involved in the fermentation process. Muscle tissue enzymes like cathepsins, peptidases, transaminases, amidases, amino acid decarboxylases, glutamic dehydrogenases, and related enzymes may also play a role in fish fermentation. Due to the decreased bacterial number during fermentation, contribution of microbial enzymes to proteolysis may be expected prior to salting of fish. Commercial enzymes are supplemented during processing for specific purposes, such as quality improvement and process acceleration. In the case of fish sauce, efforts to accelerate fermentation process and to improve product quality have been studied by addition of enzymes such as papain, bromelain, trypsin, pepsin, and chymotrypsin. © 2017 Elsevier Inc. All rights reserved.

  10. Interaction of Gut Microbiota with Bile Acid Metabolism and its Influence on Disease States

    PubMed Central

    Staley, Christopher; Weingarden, Alexa R.

    2016-01-01

    Primary bile acids serve important roles in cholesterol metabolism, lipid digestion, host-microbe interactions, and regulatory pathways in the human host. While most bile acids are reabsorbed and recycled via enterohepatic cycling, ~5% serve as substrates for bacterial biotransformation in the colon. Enzymes involved in various transformations have been characterized from cultured gut bacteria and reveal taxa-specific distribution. More recently, bioinformatic approaches have revealed greater diversity in isoforms of these enzymes, and the microbial species in which they are found. Thus, the functional roles played by the bile acid-transforming gut microbiota and the distribution of resulting secondary bile acids, in the bile acid pool, may be profoundly affected by microbial community structure and function. Bile acids and the composition of the bile acid pool have historically been hypothesized to be associated with several disease states, including recurrent Clostridium difficile infection, inflammatory bowel diseases, metabolic syndrome, and several cancers. Recently, however, emphasis has been placed on how microbial communities in the dysbiotic gut may alter the bile acid pool to potentially cause or mitigate disease onset. This review highlights the current understanding of the interactions between the gut microbial community, bile acid biotransformation, and disease states, and addresses future directions to better understand these complex associations. PMID:27888332

  11. Design of Stomach Acid-Stable and Mucin-Binding Enzyme Polymer Conjugates.

    PubMed

    Cummings, Chad S; Campbell, Alan S; Baker, Stefanie L; Carmali, Sheiliza; Murata, Hironobu; Russell, Alan J

    2017-02-13

    The reduced immunogenicity and increased stability of protein-polymer conjugates has made their use in therapeutic applications particularly attractive. However, the physicochemical interactions between polymer and protein, as well as the effect of this interaction on protein activity and stability, are still not fully understood. In this work, polymer-based protein engineering was used to examine the role of polymer physicochemical properties on the activity and stability of the chymotrypsin-polymer conjugates and their degree of binding to intestinal mucin. Four different chymotrypsin-polymer conjugates, each with the same polymer density, were synthesized using "grafting-from" atom transfer radical polymerization. The influence of polymer charge on chymotrypsin-polymer conjugate mucin binding, bioactivity, and stability in stomach acid was determined. Cationic polymers covalently attached to chymotrypsin showed high mucin binding, while zwitterionic, uncharged, and anionic polymers showed no mucin binding. Cationic polymers also increased chymotrypsin activity from pH 6-8, while zwitterionic polymers had no effect, and uncharged and anionic polymers decreased enzyme activity. Lastly, cationic polymers decreased the tendency of chymotrypsin to structurally unfold at extremely low pH, while uncharged and anionic polymers induced unfolding more quickly. We hypothesized that when polymers are covalently attached to the surface of a protein, the degree to which those polymers interact with the protein surface is the predominant determinant of whether the polymer will stabilize or inactivate the protein. Preferential interactions between the polymer and the protein lead to removal of water from the surface of the protein, and this, we believe, inactivates the enzyme.

  12. Ontogenetic changes in digestive enzyme activities and the amino acid profile of starry flounder Platichthys stellatus

    NASA Astrophysics Data System (ADS)

    Song, Zhidong; Wang, Jiying; Qiao, Hongjin; Li, Peiyu; Zhang, Limin; Xia, Bin

    2016-09-01

    Ontogenetic changes in digestive enzyme activities and the amino acid (AA) profile of starry flounder, Platichthys stellatus, were investigated and limiting amino acids were estimated compared with the essential AA profile between larvae and live food to clarify starry flounder larval nutritional requirements. Larvae were collected at the egg stage and 0, 2, 4, 7, 12, 17, 24 days after hatching (DAH) for analysis. Larvae grew from 1.91 mm at hatching to 12.13 mm at 24 DAH. Trypsin and chymotrypsin activities changed slightly by 4 DAH and then increased significantly 4 DAH. Pepsin activity increased sharply beginning 17 DAH. Lipase activity increased significantly 4 DAH and increased progressively with larval growth. Amylase activity was also detected in newly hatched larvae and increased 7 DAH followed by a gradual decrease. High free amino acid (FAA) content was detected in starry flounder eggs (110.72 mg/g dry weight). Total FAA content dropped to 43.29 mg/g in 4-DAH larvae and then decreased gradually to 13.74 mg/g in 24-DAH larvae. Most FAAs (except lysine and methionine) decreased >50% in 4-DAH larvae compared with those in eggs and then decreased to the lowest values in 24-DAH larvae. Changes in the protein amino acid (PAA) profile were much milder than those observed for FAAs. Most PAAs increased gradually during larval development, except lysine and phenylalanine. The percentages of free threonine, valine, isoleucine, and leucine decreased until the end of the trial, whereas the protein forms of these four AAs followed the opposite trend. A comparison of the essential AA composition of live food (rotifers, Artemia nauplii, and Artemia metanauplii) and larvae suggested that methionine was potentially the first limiting AA. These results may help develop starry flounder larviculture methods by solving the AA imbalance in live food. Moreover, the increased digestive enzyme activities indicate the possibility of introducing artificial compound feed.

  13. Residual Tensile Property of Plain Woven Jute Fiber/Poly(Lactic Acid) Green Composites during Thermal Cycling

    PubMed Central

    Katogi, Hideaki; Takemura, Kenichi; Akiyama, Motoki

    2016-01-01

    This study investigated the residual tensile properties of plain woven jute fiber reinforced poly(lactic acid) (PLA) during thermal cycling. Temperature ranges of thermal cycling tests were 35–45 °C and 35–55 °C. The maximum number of cycles was 103 cycles. The quasi-static tensile tests of jute fiber, PLA, and composite were conducted after thermal cycling tests. Thermal mechanical analyses of jute fiber and PLA were conducted after thermal cycling tests. Results led to the following conclusions. For temperatures of 35–45 °C, tensile strength of composite at 103 cycles decreased 10% compared to that of composite at 0 cycles. For temperatures of 35–55 °C, tensile strength and Young’s modulus of composite at 103 cycles decreased 15% and 10%, respectively, compared to that of composite at 0 cycles. Tensile properties and the coefficient of linear expansion of PLA and jute fiber remained almost unchanged after thermal cycling tests. From observation of a fracture surface, the length of fiber pull out in the fracture surface of composite at 103 cycles was longer than that of composite at 0 cycles. Therefore, tensile properties of the composite during thermal cycling were decreased, probably because of the decrease of interfacial adhesion between the fiber and resin. PMID:28773694

  14. Adding tetrahydrofuran to dilute acid pretreatment provides new insights into substrate changes that greatly enhance biomass deconstruction by Clostridium thermocellum and fungal enzymes

    DOE PAGES

    Thomas, Vanessa A.; Donohoe, Bryon S.; Li, Mi; ...

    2017-11-30

    Consolidated bioprocessing (CBP) by anaerobes, such as Clostridium thermocellum, which combine enzyme production, hydrolysis, and fermentation are promising alternatives to historical economic challenges of using fungal enzymes for biological conversion of lignocellulosic biomass. However, limited research has integrated CBP with real pretreated biomass, and understanding how pretreatment impacts subsequent deconstruction by CBP vs. fungal enzymes can provide valuable insights into CBP and suggest other novel biomass deconstruction strategies. This study focused on determining the effect of pretreatment by dilute sulfuric acid alone (DA) and with tetrahydrofuran (THF) addition via co-solvent-enhanced lignocellulosic fractionation (CELF) on deconstruction of corn stover and Populusmore » with much different recalcitrance by C. thermocellum vs. fungal enzymes and changes in pretreated biomass related to these differences.« less

  15. Adding tetrahydrofuran to dilute acid pretreatment provides new insights into substrate changes that greatly enhance biomass deconstruction by Clostridium thermocellum and fungal enzymes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Thomas, Vanessa A.; Donohoe, Bryon S.; Li, Mi

    Consolidated bioprocessing (CBP) by anaerobes, such as Clostridium thermocellum, which combine enzyme production, hydrolysis, and fermentation are promising alternatives to historical economic challenges of using fungal enzymes for biological conversion of lignocellulosic biomass. However, limited research has integrated CBP with real pretreated biomass, and understanding how pretreatment impacts subsequent deconstruction by CBP vs. fungal enzymes can provide valuable insights into CBP and suggest other novel biomass deconstruction strategies. This study focused on determining the effect of pretreatment by dilute sulfuric acid alone (DA) and with tetrahydrofuran (THF) addition via co-solvent-enhanced lignocellulosic fractionation (CELF) on deconstruction of corn stover and Populusmore » with much different recalcitrance by C. thermocellum vs. fungal enzymes and changes in pretreated biomass related to these differences.« less

  16. Occurrence of enzymes involved in biosynthesis of indole-3-acetic acid from indole-3-acetonitrile in plant-associated bacteria, Agrobacterium and Rhizobium.

    PubMed Central

    Kobayashi, M; Suzuki, T; Fujita, T; Masuda, M; Shimizu, S

    1995-01-01

    The occurrence of a hitherto unknown pathway involving the action of two enzymes, a nitrile hydratase and an amidase for the biosynthesis of indole-3-acetic acid was discovered in phytopathogenic bacteria Agrobacterium tumefaciens and in leguminous bacteria Rhizobium. The nitrile hydratase acting on indole-3-acetonitrile was purified to homogeneity through only two steps from the cell-free extract of A. tumefaciens. The molecular mass of the purified enzyme estimated by HPLC was about 102 kDa, and the enzyme consisted of four subunits identical in molecular mass. The enzyme exhibited a broad absorption spectrum in the visible range with absorption maxima at 408 nm and 705 nm, and it contained cobalt and iron. The enzyme stoichiometrically catalyzed the hydration of indole-3-acetonitrile into indole-3-acetamide with a specific activity of 13.7 mol per min per mg and a Km of 7.9 microM. Images Fig. 1 PMID:11607511

  17. Purification and Characterization of the Crown Gall-specific Enzyme, Octopine Synthase 1

    PubMed Central

    Hack, Ethan; Kemp, John D.

    1980-01-01

    A single enzyme catalyzes the synthesis of all four N2-(1-carboxyethyl)-amino acid derivatives found in a crown gall tumor tissue induced by Agrobacterium tumefaciens (E. F. Sm. and Town.) Conn strain B6 on sunflower (Helianthus annuus L.). This enzyme, octopine synthase, has been purified by ammonium sulfate fractionation and chromatography on diethylaminoethylcellulose, blue agarose, and hydroxylapatite. The purified enzyme has all the N2-(1-carboxyethyl)-amino acid synthesizing activities found in crude preparations, and the relative activities with six amino acids remain nearly constant during purification. Although the maximum velocities (V) and Michaelis constants (Km) differ, the ratio V/Km is the same for all amino acid substrates. Thus an equimolar mixture of amino acids will give rise to an equimolar mixture of products. The kinetic properties of the enzyme are consistent with a partially ordered mechanism with arginine (NADPH, then arginine or pyruvate). Octopine synthase is a monomeric enzyme with a molecular weight of 39,000 by gel filtration and 38,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images PMID:16661312

  18. Response of soil physicochemical properties and enzyme activities to long-term reclamation of coastal saline soil, Eastern China.

    PubMed

    Xie, Xuefeng; Pu, Lijie; Wang, Qiqi; Zhu, Ming; Xu, Yan; Zhang, Meng

    2017-12-31

    Soil enzyme activity during different years of reclamation and land use patterns could indicate changes in soil quality. The objective of this research is to explore the dynamics of 5 soil enzyme activities (dehydrogenase, amylase, urease, acid phosphatase and alkaline phosphatase) involved in C, N, and P cycling and their responses to changes in soil physicochemical properties resulting from long-term reclamation of coastal saline soil. Soil samples from a total of 55 sites were collected from a coastal reclamation area with different years of reclamation (0, 7, 32, 40, 63a) in this study. The results showed that both long-term reclamation and land use patterns have significant effects on soil physicochemical properties and enzyme activities. Compared with the bare flat, soil water content, soil bulk density, pH and electrical conductivity showed a decreasing trend after reclamation, whereas soil organic carbon, total nitrogen and total phosphorus tended to increase. Dehydrogenase, amylase and acid phosphatase activities initially increased and then decreased with increasing years of reclamation, whereas urease and alkaline phosphatase activities were characterized by an increase-decrease-increase trend. Moreover, urease, acid phosphatase and alkaline phosphatase activities exhibited significant differences between coastal saline soil with 63years of reclamation and bare flat, whereas dehydrogenase and amylase activities remained unchanged. Aquaculture ponds showed higher soil water content, pH and EC but lower soil organic carbon, total nitrogen and total phosphorus than rapeseed, broad bean and wheat fields. Rapeseed, broad bean and wheat fields displayed higher urease and alkaline phosphatase activities and lower dehydrogenase, amylase and acid phosphatase activities compared with aquaculture ponds. Redundancy analysis revealed that the soil physicochemical properties explained 74.5% of the variation in soil enzyme activities and that an obvious relationship

  19. Glutathionylation of α-ketoglutarate dehydrogenase: The chemical nature and relative susceptibility of the cofactor lipoic acid to modification

    PubMed Central

    McLain, Aaron L.; Cormier, Peter J.; Kinter, Michael; Szweda, Luke I.

    2013-01-01

    α-Ketoglutarate dehydrogenase (KGDH) is reversibly inhibited when rat heart mitochondria are exposed to hydrogen peroxide (H2O2). H2O2-induced inhibition occurs through the formation of a mixed disulfide between a protein sulfhydryl and glutathione. Upon consumption of H2O2, glutaredoxin can rapidly remove glutathione, resulting in regeneration of enzyme activity. KGDH is a key regulatory site within the Krebs cycle. Glutathionylation of the enzyme may therefore represent an important means to control mitochondrial function in response to oxidative stress. We have previously provided indirect evidence that glutathionylation occurs on lipoic acid, a cofactor covalently bound to the E2 subunit of KGDH. However, lipoic acid contains two vicinal sulfhydryls and rapid disulfide exchange might be predicted to preclude stable glutathionylation. The current study sought conclusive identification of the site and chemistry of KGDH glutathionylation and factors that control the degree and rate of enzyme inhibition. We present evidence that, upon reaction of free lipoic acid with oxidized glutathione in solution, disulfide exchange occurs rapidly, producing oxidized lipoic acid and reduced glutathione. This prevents the stable formation of a glutathione–lipoic acid adduct. Nevertheless, 1:1 lipoic acid–glutathione adducts are formed on KGDH because the second sulfhydryl on lipoic acid is unable to participate in disulfide exchange in the enzyme’s native conformation. The maximum degree of KGDH inhibition that can be achieved by treatment of mitochondria with H2O2 is 50%. Results indicate that this is not due to glutathionylation of a subpopulation of the enzyme but, rather, the unique susceptibility of lipoic acid on a subset of E2 subunits within each enzyme complex. Calcium enhances the rate of glutathionylation by increasing the half-life of reduced lipoic acid during enzyme catalysis. This does not, however, alter the maximal level of inhibition, providing further

  20. Glutathionylation of α-ketoglutarate dehydrogenase: the chemical nature and relative susceptibility of the cofactor lipoic acid to modification.

    PubMed

    McLain, Aaron L; Cormier, Peter J; Kinter, Michael; Szweda, Luke I

    2013-08-01

    α-Ketoglutarate dehydrogenase (KGDH) is reversibly inhibited when rat heart mitochondria are exposed to hydrogen peroxide (H2O2). H2O2-induced inhibition occurs through the formation of a mixed disulfide between a protein sulfhydryl and glutathione. Upon consumption of H2O2, glutaredoxin can rapidly remove glutathione, resulting in regeneration of enzyme activity. KGDH is a key regulatory site within the Krebs cycle. Glutathionylation of the enzyme may therefore represent an important means to control mitochondrial function in response to oxidative stress. We have previously provided indirect evidence that glutathionylation occurs on lipoic acid, a cofactor covalently bound to the E2 subunit of KGDH. However, lipoic acid contains two vicinal sulfhydryls and rapid disulfide exchange might be predicted to preclude stable glutathionylation. The current study sought conclusive identification of the site and chemistry of KGDH glutathionylation and factors that control the degree and rate of enzyme inhibition. We present evidence that, upon reaction of free lipoic acid with oxidized glutathione in solution, disulfide exchange occurs rapidly, producing oxidized lipoic acid and reduced glutathione. This prevents the stable formation of a glutathione-lipoic acid adduct. Nevertheless, 1:1 lipoic acid-glutathione adducts are formed on KGDH because the second sulfhydryl on lipoic acid is unable to participate in disulfide exchange in the enzyme's native conformation. The maximum degree of KGDH inhibition that can be achieved by treatment of mitochondria with H2O2 is 50%. Results indicate that this is not due to glutathionylation of a subpopulation of the enzyme but, rather, the unique susceptibility of lipoic acid on a subset of E2 subunits within each enzyme complex. Calcium enhances the rate of glutathionylation by increasing the half-life of reduced lipoic acid during enzyme catalysis. This does not, however, alter the maximal level of inhibition, providing further

  1. Distinct Prominent Roles for Enzymes of Plasmodium berghei Heme Biosynthesis in Sporozoite and Liver Stage Maturation

    PubMed Central

    Matuschewski, Kai; Haussig, Joana M.

    2016-01-01

    Malarial parasites have evolved complex regulation of heme supply and disposal to adjust to heme-rich and -deprived host environments. In addition to its own pathway for heme biosynthesis, Plasmodium likely harbors mechanisms for heme scavenging from host erythrocytes. Elaborate compartmentalization of de novo heme synthesis into three subcellular locations, including the vestigial plastid organelle, indicates critical roles in life cycle progression. In this study, we systematically profile the essentiality of heme biosynthesis by targeted gene deletion of enzymes in early steps of this pathway. We show that disruption of endogenous heme biosynthesis leads to a first detectable defect in oocyst maturation and sporogony in the Anopheles vector, whereas blood stage propagation, colonization of mosquito midguts, or initiation of oocyst development occurs indistinguishably from that of wild-type parasites. Although sporozoites are produced by parasites lacking an intact pathway for heme biosynthesis, they are absent from mosquito salivary glands, indicative of a vital role for heme biosynthesis only in sporozoite maturation. Rescue of the first defect in sporogony permitted analysis of potential roles in liver stages. We show that liver stage parasites benefit from but do not strictly depend upon their own aminolevulinic acid synthase and that they can scavenge aminolevulinic acid from the host environment. Together, our experimental genetics analysis of Plasmodium enzymes for heme biosynthesis exemplifies remarkable shifts between the use of endogenous and host resources during life cycle progression. PMID:27600503

  2. Multiple turnovers of the nicotino-enzyme PdxB require α-keto acids as co-substrates

    PubMed Central

    Rudolph, Johannes; Kim, Juhan; Copley, Shelley D.

    2012-01-01

    PdxB catalyzes the second step in the biosynthesis of pyridoxal phosphate by oxidizing 4-phospho-D-erythronate (4PE) to 2-oxo-3-hydroxy-4-phospho-butanoate (OHPB) with concomitant reduction of NAD+ to NADH. PdxB is a nicotino-enzyme wherein the NAD(H) cofactor remains tightly bound to PdxB. It has been a mystery how PdxB performs multiple turnovers since addition of free NAD+ does not re-oxidize the enzyme-bound NADH following conversion of 4PE to OHPB. We have solved this mystery by demonstrating that a variety of physiologically available α-ketoacids serve as oxidants of PdxB to sustain multiple turnovers. In a coupled assay using the next two enzymes of the biosynthetic pathway for pyridoxal phosphate (SerC and PdxA), we have found that α-ketoglutarate, oxaloacetic acid, and pyruvate are equally good substrates for PdxB (kcat/Km values ~ 1 × 104 M-1s-1). The kinetic parameters for the substrate 4PE include a kcat of 1.4 s-1, a Km of 2.9 μM, and a kcat/Km of 6.7 × 106 M-1s-1. Additionally, we have characterized the stereochemistry of α-ketoglutarate reduction by showing that D-2-HGA, but not L-2-HGA, is a competitive inhibitor vs. 4PE and a noncompetitive inhibitor vs. α-ketoglutarate. PMID:20831184

  3. Effect of tricarboxylic acid cycle regulator on carbon retention and organic component transformation during food waste composting.

    PubMed

    Lu, Qian; Zhao, Yue; Gao, Xintong; Wu, Junqiu; Zhou, Haixuan; Tang, Pengfei; Wei, Qingbin; Wei, Zimin

    2018-05-01

    Composting is an environment friendly method to recycling organic waste. However, with the increasing concern about greenhouse gases generated in global atmosphere, it is significant to reduce the emission of carbon dioxide (CO 2 ). This study analyzes tricarboxylic acid (TCA) cycle regulators on the effect of reducing CO 2 emission, and the relationship among organic component (OC) degradation and transformation and microorganism during composting. The results showed that adding adenosine tri-phosphate (ATP) and nicotinamide adenine dinucleotide (NADH) could enhance the transformation of OC and increase the diversity of microorganism community. Malonic acid (MA) as a competitive inhibitor could decrease the emission of CO 2 by inhibiting the TCA cycle. A structural equation model was established to explore effects of different OC and microorganism on humic acid (HA) concentration during composting. Furthermore, added MA provided an environmental benefit in reducing the greenhouse gas emission for manufacture sustainable products. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. Effect of ω-3 and ω-9 fatty acid rich oils on lipoxygenases and cyclooxygenases enzymes and on the growth of a mammary adenocarcinoma model.

    PubMed

    Comba, Andrea; Maestri, Damian M; Berra, María A; Garcia, Carolina Paola; Das, Undurti N; Eynard, Aldo R; Pasqualini, María E

    2010-10-08

    Nutritional factors play a major role in cancer initiation and development. Dietary polyunsaturated fatty acids (PUFAs) have the ability to induce modifications in the activity of lipoxygenase (LOX) and cyclooxygenase (COX) enzymes that affect tumour growth. We studied the effect of two diets enriched in 6% Walnut and Peanut oils that are rich in ω-3 and ω9 PUFAs respectively on a murine mammary gland adenocarcinoma as compared with the control (C) that received commercial diet. Peanut oil enriched diet induced an increase in membrane arachidonic acid (AA) content and the cyclooxygenase enzyme derived 12-HHT (p < 0.05) and simultaneously showed decrease in 12-LOX, 15-LOX-2, 15-LOX-1 and PGE activities (p < 0.05) that corresponded to higher apoptosis and lower mitosis seen in this group (p < 0.05). Furthermore, Peanut oil group showed lower T-cell infiltration (p < 0.05), number of metastasis (p < 0.05) and tumour volume (p < 0.05) and longer survival rate compared to other groups. The results of the present study showed that Peanut oil-enriched diet protects against mammary cancer development by modulating tumour membrane fatty acids composition and LOX and COX enzyme activities.

  5. Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass.

    PubMed

    Kim, Youngmi; Ximenes, Eduardo; Mosier, Nathan S; Ladisch, Michael R

    2011-04-07

    Liquid hot water, steam explosion, and dilute acid pretreatments of lignocellulose generate soluble inhibitors which hamper enzymatic hydrolysis as well as fermentation of sugars to ethanol. Toxic and inhibitory compounds will vary with pretreatment and include soluble sugars, furan derivatives (hydroxymethyl fulfural, furfural), organic acids (acetic, formic and, levulinic acid), and phenolic compounds. Their effect is seen when an increase in the concentration of pretreated biomass in a hydrolysis slurry results in decreased cellulose conversion, even though the ratio of enzyme to cellulose is kept constant. We used lignin-free cellulose, Solka Floc, combined with mixtures of soluble components released during pretreatment of wood, to prove that the decrease in the rate and extent of cellulose hydrolysis is due to a combination of enzyme inhibition and deactivation. The causative agents were extracted from wood pretreatment liquid using PEG surfactant, activated charcoal or ethyl acetate and then desorbed, recovered, and added back to a mixture of enzyme and cellulose. At enzyme loadings of either 1 or 25mg protein/g glucan, the most inhibitory components, later identified as phenolics, decreased the rate and extent of cellulose hydrolysis by half due to both inhibition and precipitation of the enzymes. Full enzyme activity occurred when the phenols were removed. Hence detoxification of pretreated woods through phenol removal is expected to reduce enzyme loadings, and therefore reduce enzyme costs, for a given level of cellulose conversion. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Not all boronic acids with a five-membered cycle induce tremor, neuronal damage and decreased dopamine.

    PubMed

    Pérez-Rodríguez, Maribel; García-Mendoza, Esperanza; Farfán-García, Eunice D; Das, Bhaskar C; Ciprés-Flores, Fabiola J; Trujillo-Ferrara, José G; Tamay-Cach, Feliciano; Soriano-Ursúa, Marvin A

    2017-09-01

    Several striatal toxins can be used to induce motor disruption. One example is MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), whose toxicity is accepted as a murine model of parkinsonism. Recently, 3-Thienylboronic acid (3TB) was found to produce motor disruption and biased neuronal damage to basal ganglia in mice. The aim of this study was to examine the toxic effects of four boronic acids with a close structural relationship to 3TB (all having a five-membered cycle), as well as boric acid and 3TB. These boron-containing compounds were compared to MPTP regarding brain access, morphological disruption of the CNS, and behavioral manifestations of such disruption. Data was collected through acute toxicity evaluations, motor behavior tests, necropsies, determination of neuronal survival by immunohistochemistry, Raman spectroscopic analysis of brain tissue, and HPLC measurement of dopamine in substantia nigra and striatum tissue. Each compound showed a distinct profile for motor disruption. For example, motor activity was not disrupted by boric acid, but was decreased by two boronic acids (caused by a sedative effect). 3TB, 2-Thienyl and 2-furanyl boronic acid gave rise to shaking behavior. The various manifestations generated by these compounds can be linked, in part, to different levels of dopamine (measured by HPLC) and degrees of neuronal damage in the basal ganglia and cerebellum. Clearly, motor disruption is not induced by all boronic acids with a five-membered cycle as substituent. Possible explanations are given for the diverse chemico-morphological changes and degrees of disruption of the motor system, considering the role of boron and the structure-toxicity relationship. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. [Effect of cultivation conditions on the growth and activities of sulfur metabolism enzymes and carboxylases of Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41].

    PubMed

    Egorova, M A; Tsaplina, I A; Zakharchuk, L M; Bogdanova, T I; Krasil'nikova, E N

    2004-01-01

    The moderately thermophilic acidophilic bacterium Sulfobacillus thermosulfidooxidans subsp. asporogenes strain 41 is capable of utilizing sulfides of gold-arsenic concentrate and elemental sulfur as a source of energy. The growth in the presence of S0 under auto- or mixotrophic conditions was less stable compared with the media containing iron monoxide. The enzymes involved in oxidation of sulfur inorganic compounds--thiosulfate-oxidizing enzyme, tetrathionate hydrolase, rhodonase, adenylyl sulfate reductase, sulfite oxidase, and sulfur oxygenase--were discovered in the cells of Sulfobacillus grown in the mineral medium containing 0.02% yeast extract and either sulfur or iron monoxide and thiosulfate. Cell-free extracts of the cultures grown in the medium with sulfur under auto- or mixotrophic conditions displayed activity of the key enzyme of the Calvin cycle--ribulose bisphosphate carboxylase--and several other enzymes involved in heterotrophic fixation of carbonic acid. Activities of carboxylases depended on the composition of cultivation media.

  8. Digestive Enzyme Supplementation in Gastrointestinal Diseases.

    PubMed

    Ianiro, Gianluca; Pecere, Silvia; Giorgio, Valentina; Gasbarrini, Antonio; Cammarota, Giovanni

    2016-01-01

    Digestive enzymes are able to break down proteins and carbohydrates and lipids, and their supplementation may play a role in the management of digestive disorders, from lactose intolerance to cystic fibrosis. To date, several formulations of digestive enzymes are available on the market, being different each other in terms of enzyme type, source and origin, and dosage. This review, performed through a non-systematic search of the available literature, will provide an overview of the current knowledge of digestive enzyme supplementation in gastrointestinal disorders, discussion of the use of pancreatic enzymes, lactase (β-galactosidase) and conjugated bile acids, and also exploring the future perspective of digestive enzyme supplementation. Currently, the animal-derived enzymes represent an established standard of care, however the growing study of plant-based and microbe-derived enzymes offers great promise in the advancement of digestive enzyme therapy. New frontiers of enzyme replacement are being evaluated also in the treatment of diseases not specifically related to enzyme deficiency, whereas the combination of different enzymes might constitute an intriguing therapeutic option in the future.

  9. Cloning and characterization of a cell cycle-regulated gene encoding topoisomerase I from Nicotiana tabacum that is inducible by light, low temperature and abscisic acid.

    PubMed

    Mudgil, Y; Singh, B N; Upadhyaya, K C; Sopory, S K; Reddy, M K

    2002-05-01

    We have cloned a full-length 2874-bp cDNA coding for tobacco topoisomerase I, with an ORF of 2559 bp encoding a protein of 852 amino acids with a calculated molecular mass of 95 kDa and an estimated pI of 9.51. The deduced amino acid sequence shows homology to other eukaryotic topoisomerases I. Tobacco topoisomerase I was over-expressed in Escherichia coli, and the purified recombinant protein was found to relax both positively and negatively super-coiled DNA in the absence of the divalent cation Mg(2+)and ATP. These characteristic features indicate that the tobacco enzyme is a type I topoisomerase. The recombinant protein could be phosphorylated at (a) threonine residue(s) by protein kinase C. However, phosphorylation did not cause any change in its enzymatic activity. The genomic organization of the topoisomerase I gene revealed the presence of 8 exons and 7 introns in the region corresponding to the ORF and one intron in the 3' UTR region. Transcript analysis using RT-PCR showed basal constitutive expression in all organs examined, and the gene was expressed at all stages of the cell cycle--but the level of expression increased during the G1-S phase. The transcript level also increased following exposure to light, low-temperature stress and abscisic acid, a stress hormone.

  10. Acute and chronic effects of clofibrate and clofibric acid on the enzymes acetylcholinesterase, lactate dehydrogenase and catalase of the mosquitofish, Gambusia holbrooki.

    PubMed

    Nunes, B; Carvalho, F; Guilhermino, L

    2004-12-01

    The objective of this study was to investigate both acute and chronic effects of clofibrate and clofibric acid on the enzymes acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and catalase (CAT) of the mosquitofish (Gambusia holbrooki). AChE, commonly used as a biomarker of neurotoxicity, was determined in the total head. LDH, an important enzyme of anaerobic metabolism, was quantified in dorsal muscle, and CAT, enzyme which has been used as indicative parameter of peroxisome proliferation, was determined in the liver. Furthermore, alterations of body and liver weight were also determined, through the calculation of the ratios final body weight/initial body weight, liver weight/final body weight, liver weight/gills weight and liver weight/head weight. Acute exposure of G. holbrooki to both clofibrate and clofibric acid induced a decrease in liver CAT activity, an increase in muscle LDH activity, while no effects were observed on AChE activity. However, chronic exposure did not alter significantly the enzymatic activities, suggesting reduced or null effects over these pathways, relative to effects reported in other species. No effects were observed for the calculated ratios, except a significant weight reduction for males chronically exposed to clofibrate.

  11. Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids.

    PubMed

    Iyer, Lakshminarayan M; Tahiliani, Mamta; Rao, Anjana; Aravind, L

    2009-06-01

    Modified bases in nucleic acids present a layer of information that directs biological function over and beyond the coding capacity of the conventional bases. While a large number of modified bases have been identified, many of the enzymes generating them still remain to be discovered. Recently, members of the 2-oxoglutarate- and iron(II)-dependent dioxygenase super-family, which modify diverse substrates from small molecules to biopolymers, were predicted and subsequently confirmed to catalyze oxidative modification of bases in nucleic acids. Of these, two distinct families, namely the AlkB and the kinetoplastid base J binding proteins (JBP) catalyze in situ hydroxylation of bases in nucleic acids. Using sensitive computational analysis of sequences, structures and contextual information from genomic structure and protein domain architectures, we report five distinct families of 2-oxoglutarate- and iron(II)-dependent dioxygenase that we predict to be involved in nucleic acid modifications. Among the DNA-modifying families, we show that the dioxygenase domains of the kinetoplastid base J-binding proteins belong to a larger family that includes the Tet proteins, prototyped by the human oncogene Tet1, and proteins from basidiomycete fungi, chlorophyte algae, heterolobosean amoeboflagellates and bacteriophages. We present evidence that some of these proteins are likely to be involved in oxidative modification of the 5-methyl group of cytosine leading to the formation of 5-hydroxymethylcytosine. The Tet/JBP homologs from basidiomycete fungi such as Laccaria and Coprinopsis show large lineage-specific expansions and a tight linkage with genes encoding a novel and distinct family of predicted transposases, and a member of the Maelstrom-like HMG family. We propose that these fungal members are part of a mobile transposon. To the best of our knowledge, this is the first report of a eukaryotic transposable element that encodes its own DNA-modification enzyme with a

  12. Model-Assisted Analysis of Sugar Metabolism throughout Tomato Fruit Development Reveals Enzyme and Carrier Properties in Relation to Vacuole Expansion[W

    PubMed Central

    Beauvoit, Bertrand P.; Colombié, Sophie; Monier, Antoine; Andrieu, Marie-Hélène; Biais, Benoit; Bénard, Camille; Chéniclet, Catherine; Dieuaide-Noubhani, Martine; Nazaret, Christine; Mazat, Jean-Pierre; Gibon, Yves

    2014-01-01

    A kinetic model combining enzyme activity measurements and subcellular compartmentation was parameterized to fit the sucrose, hexose, and glucose-6-P contents of pericarp throughout tomato (Solanum lycopersicum) fruit development. The model was further validated using independent data obtained from domesticated and wild tomato species and on transgenic lines. A hierarchical clustering analysis of the calculated fluxes and enzyme capacities together revealed stage-dependent features. Cell division was characterized by a high sucrolytic activity of the vacuole, whereas sucrose cleavage during expansion was sustained by both sucrose synthase and neutral invertase, associated with minimal futile cycling. Most importantly, a tight correlation between flux rate and enzyme capacity was found for fructokinase and PPi-dependent phosphofructokinase during cell division and for sucrose synthase, UDP-glucopyrophosphorylase, and phosphoglucomutase during expansion, thus suggesting an adaptation of enzyme abundance to metabolic needs. In contrast, for most enzymes, flux rates varied irrespectively of enzyme capacities, and most enzymes functioned at <5% of their maximal catalytic capacity. One of the major findings with the model was the high accumulation of soluble sugars within the vacuole together with organic acids, thus enabling the osmotic-driven vacuole expansion that was found during cell division. PMID:25139005

  13. Ionizable Side Chains at Catalytic Active Sites of Enzymes

    PubMed Central

    Jimenez-Morales, David; Liang, Jie

    2012-01-01

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

  14. Ectomycorrhizal fungi enhance nitrogen and phosphorus nutrition of Nothofagus dombeyi under drought conditions by regulating assimilative enzyme activities.

    PubMed

    Alvarez, Maricel; Huygens, Dries; Olivares, Erick; Saavedra, Isabel; Alberdi, Miren; Valenzuela, Eduardo

    2009-08-01

    Drought stress conditions (DC) reduce plant growth and nutrition, restraining the sustainable reestablishment of Nothofagus dombeyi in temperate south Chilean forest ecosystems. Ectomycorrhizal symbioses have been documented to enhance plant nitrogen (N) and phosphorus (P) uptake under drought, but the regulation of involved assimilative enzymes remains unclear. We studied 1-year-old N. dombeyi (Mirb.) Oerst. plants in association with the ectomycorrhizal fungi Pisolithus tinctorius (Pers.) Coker & Couch. and Descolea antartica Sing. In greenhouse experiments, shoot and root dry weights, mycorrhizal colonization, foliar N and P concentrations, and root enzyme activities [glutamate synthase (glutamine oxoglutarate aminotransferase (GOGAT), EC 1.4.1.13-14), glutamine synthetase (GS, EC 6.3.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), nitrate reductase (NR, EC 1.6.6.1), and acid phosphomonoesterase (PME, EC 3.1.3.1-2)] were determined as a function of soil-water content. Inoculation of N. dombeyi with P. tinctorius and D. antartica significantly stimulated plant growth and increased plant foliar N and P concentrations, especially under DC. Ectomycorrhizal inoculation increased the activity of all studied enzymes relative to non-mycorrhizal plants under drought. We speculate that GDH is a key enzyme involved in the enhancement of ectomycorrhizal carbon (C) availability by fuelling the tricarboxylic acid (TCA) cycle under conditions of drought-induced carbon deficit. All studied assimilative enzymes of the ectomycorrhizal associations, involved in C, N, and P transfers, are closely interlinked and interdependent. The up-regulation of assimilative enzyme activities by ectomycorrhizal fungal root colonizers acts as a functional mechanism to increase seedling endurance to drought. We insist upon incorporating ectomycorrhizal inoculation in existing Chilean afforestation programs.

  15. Coevolution and life cycle specialization of plant cell wall degrading enzymes in a hemibiotrophic pathogen.

    PubMed

    Brunner, Patrick C; Torriani, Stefano F F; Croll, Daniel; Stukenbrock, Eva H; McDonald, Bruce A

    2013-06-01

    Zymoseptoria tritici is an important fungal pathogen on wheat that originated in the Fertile Crescent. Its closely related sister species Z. pseudotritici and Z. ardabiliae infect wild grasses in the same region. This recently emerged host-pathogen system provides a rare opportunity to investigate the evolutionary processes shaping the genome of an emerging pathogen. Here, we investigate genetic signatures in plant cell wall degrading enzymes (PCWDEs) that are likely affected by or driving coevolution in plant-pathogen systems. We hypothesize four main evolutionary scenarios and combine comparative genomics, transcriptomics, and selection analyses to assign the majority of PCWDEs in Z. tritici to one of these scenarios. We found widespread differential transcription among different members of the same gene family, challenging the idea of functional redundancy and suggesting instead that specialized enzymatic activity occurs during different stages of the pathogen life cycle. We also find that natural selection has significantly affected at least 19 of the 48 identified PCWDEs. The majority of genes showed signatures of purifying selection, typical for the scenario of conserved substrate optimization. However, six genes showed diversifying selection that could be attributed to either host adaptation or host evasion. This study provides a powerful framework to better understand the roles played by different members of multigene families and to determine which genes are the most appropriate targets for wet laboratory experimentation, for example, to elucidate enzymatic function during relevant phases of a pathogen's life cycle.

  16. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid (DNA...

  17. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid (DNA...

  18. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid (DNA...

  19. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid (DNA...

  20. 21 CFR 862.3360 - Drug metabolizing enzyme genotyping system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Drug metabolizing enzyme genotyping system. 862... Test Systems § 862.3360 Drug metabolizing enzyme genotyping system. (a) Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid (DNA...

  1. Ester-Mediated Amide Bond Formation Driven by Wet-Dry Cycles: A Possible Path to Polypeptides on the Prebiotic Earth.

    PubMed

    Forsythe, Jay G; Yu, Sheng-Sheng; Mamajanov, Irena; Grover, Martha A; Krishnamurthy, Ramanarayanan; Fernández, Facundo M; Hud, Nicholas V

    2015-08-17

    Although it is generally accepted that amino acids were present on the prebiotic Earth, the mechanism by which α-amino acids were condensed into polypeptides before the emergence of enzymes remains unsolved. Here, we demonstrate a prebiotically plausible mechanism for peptide (amide) bond formation that is enabled by α-hydroxy acids, which were likely present along with amino acids on the early Earth. Together, α-hydroxy acids and α-amino acids form depsipeptides-oligomers with a combination of ester and amide linkages-in model prebiotic reactions that are driven by wet-cool/dry-hot cycles. Through a combination of ester-amide bond exchange and ester bond hydrolysis, depsipeptides are enriched with amino acids over time. These results support a long-standing hypothesis that peptides might have arisen from ester-based precursors. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Modeling the effects of tree species and incubation temperature on soil's extracellular enzyme activity in 78-year-old tree plantations

    NASA Astrophysics Data System (ADS)

    Zhou, Xiaoqi; Wang, Shen S. J.; Chen, Chengrong

    2017-12-01

    Forest plantations have been widely used as an effective measure for increasing soil carbon (C), and nitrogen (N) stocks and soil enzyme activities play a key role in soil C and N losses during decomposition of soil organic matter. However, few studies have been carried out to elucidate the mechanisms behind the differences in soil C and N cycling by different tree species in response to climate warming. Here, we measured the responses of soil's extracellular enzyme activity (EEA) to a gradient of temperatures using incubation methods in 78-year-old forest plantations with different tree species. Based on a soil enzyme kinetics model, we established a new statistical model to investigate the effects of temperature and tree species on soil EEA. In addition, we established a tree species-enzyme-C/N model to investigate how temperature and tree species influence soil C/N contents over time without considering plant C inputs. These extracellular enzymes included C acquisition enzymes (β-glucosidase, BG), N acquisition enzymes (N-acetylglucosaminidase, NAG; leucine aminopeptidase, LAP) and phosphorus acquisition enzymes (acid phosphatases). The results showed that incubation temperature and tree species significantly influenced all soil EEA and Eucalyptus had 1.01-2.86 times higher soil EEA than coniferous tree species. Modeling showed that Eucalyptus had larger soil C losses but had 0.99-2.38 times longer soil C residence time than the coniferous tree species over time. The differences in the residual soil C and N contents between Eucalyptus and coniferous tree species, as well as between slash pine (Pinus elliottii Engelm. var. elliottii) and hoop pine (Araucaria cunninghamii Ait.), increase with time. On the other hand, the modeling results help explain why exotic slash pine can grow faster, as it has 1.22-1.38 times longer residual soil N residence time for LAP, which mediate soil N cycling in the long term, than native coniferous tree species like hoop pine and

  3. Determining soil enzyme activities for the assessment of fungi and citric acid-assisted phytoextraction under cadmium and lead contamination.

    PubMed

    Mao, Liang; Tang, Dong; Feng, Haiwei; Gao, Yang; Zhou, Pei; Xu, Lurong; Wang, Lumei

    2015-12-01

    Microorganism or chelate-assisted phytoextraction is an effective remediation tool for heavy metal polluted soil, but investigations into its impact on soil microbial activity are rarely reported. Consequently, cadmium (Cd)- and lead (Pb)-resistant fungi and citric acid (CA) were introduced to enhance phytoextraction by Solanum nigrum L. under varied Cd and Pb pollution levels in a greenhouse pot experiment. We then determined accumulation of Cd and Pb in S. nigrum and the soil enzyme activities of dehydrogenase, phosphatase, urease, catalase, sucrase, and amylase. Detrended canonical correspondence analysis (DCCA) was applied to assess the interactions between remediation strategies and soil enzyme activities. Results indicated that the addition of fungi, CA, or their combination enhanced the root biomass of S. nigrum, especially at the high-pollution level. The combined treatment of CA and fungi enhanced accumulation of Cd about 22-47 % and of Pb about 13-105 % in S. nigrum compared with the phytoextraction alone. However, S. nigrum was not shown to be a hyperaccumulator for Pb. Most enzyme activities were enhanced after remediation. The DCCA ordination graph showed increasing enzyme activity improvement by remediation in the order of phosphatase, amylase, catalase, dehydrogenase, and urease. Responses of soil enzyme activities were similar for both the addition of fungi and that of CA. In summary, results suggest that fungi and CA-assisted phytoextraction is a promising approach to restoring heavy metal polluted soil.

  4. Simulation of the pentose cycle in lactating rat mammary gland

    PubMed Central

    Haut, Michael J.; London, Jack W.; Garfinkel, David

    1974-01-01

    A computer model representing the pentose cycle, the tricarboxylic acid cycle and glycolysis in slices of lactating rat mammary glands has been constructed. This model is based primarily on the studies, with radioactive chemicals, of Abraham & Chaikoff (1959) [although some of the discrepant data of Katz & Wals (1972) could be accommodated by changing one enzyme activity]. Data obtained by using [1-14C]-, [6-14C]- and [3,4-14C]-glucose were simulated, as well as data obtained by using unlabelled glucose (for which some new experimental data are presented). Much past work on the pentose cycle has been mainly concerned with the division of glucose flow between the pentose cycle and glycolysis, and has relied on the assumption that the system is in steady state (both labelled and unlabelled). This assumption may not apply to lactating rat mammary glands, since the model shows that the percentage flow through the shunt progressively decreased for the first 2h of a 3h experiment, and we were unable to construct a completely steady-state model. The model allows examination of many quantitative features of the system, especially the amount of material passing through key enzymes, some of which appear to be regulated by NADP+ concentrations as proposed by McLean (1960). Supplementary information for this paper has been deposited as Supplementary Publication SUP 50023 at the British Museum (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5. PMID:4154746

  5. The effects of estrus cycle on drug metabolism in the rat.

    PubMed

    Brandstetter, Y; Kaplanski, J; Leibson, V; Ben-Zvi, Z

    1986-01-01

    The effect of the female rat estral cycle on microsomal drug metabolism in-vivo and in-vitro has been studied. Two microsomal enzymes, aminopyrine-N-demethylase and aniline hydroxylase showed a greater specific activity (p less than 0.01) in the diestrus phase of the estral cycle while the oxidative enzyme aryl hydrocarbon hydroxylase and the conjugative enzyme, glucuronyl transferase, were not affected. In vivo studies which included theophylline and antipyrine metabolism, and hexobarbital sleeping times showed no difference between the different phases of the estral cycle. Conflicting evidence about the effect of steroid sex hormones on hepatic drug metabolism is discussed.

  6. Effect on combined cycle efficiency of stack gas temperature constraints to avoid acid corrosion

    NASA Technical Reports Server (NTRS)

    Nainiger, J. J.

    1980-01-01

    To avoid condensation of sulfuric acid in the gas turbine exhaust when burning fuel oils contaning sulfur, the exhaust stack temperature and cold-end heat exchanger surfaces must be kept above the condensation temperature. Raising the exhaust stack temperature, however, results in lower combined cycle efficiency compared to that achievable by a combined cycle burning a sulfur-free fuel. The maximum difference in efficiency between the use of sulfur-free and fuels containing 0.8 percent sulfur is found to be less than one percentage point. The effect of using a ceramic thermal barrier coating (TBC) and a fuel containing sulfur is also evaluated. The combined-cycle efficiency gain using a TBC with a fuel containing sulfur compared to a sulfur-free fuel without TBC is 0.6 to 1.0 percentage points with air-cooled gas turbines and 1.6 to 1.8 percentage points with water-cooled gas turbines.

  7. Sodium Phenylbutyrate Decreases Plasma Branched-Chain Amino Acids in Patients with Urea Cycle Disorders

    PubMed Central

    Burrage, Lindsay C.; Jain, Mahim; Gandolfo, Laura; Lee, Brendan H.; Nagamani, Sandesh CS.

    2014-01-01

    Sodium phenylbutyrate (NaPBA) is a commonly used medication for the treatment of patients with urea cycle disorders (UCDs). Previous reports involving small numbers of patients with UCDs have shown that NaPBA treatment can result in lower plasma levels of the branched-chain amino acids (BCAA) but this has not been studied systematically. From a large cohort of patients (n=553) with UCDs enrolled in Longitudinal Study of Urea Cycle Disorders, a collaborative multicenter study of the Urea Cycle Disorders Consortium, we evaluated whether treatment with NaPBA leads to a decrease in plasma BCAA levels. Our analysis shows that NaPBA use independently affects the plasma BCAA levels even after accounting for multiple confounding covariates. Moreover, NaPBA use increases the risk for BCAA deficiency. This effect of NaPBA seems specific to plasma BCAA levels, as levels of other essential amino acids are not altered by its use. Our study, in an unselected population of UCD subjects, is the largest to analyze the effects of NaPBA on BCAA metabolism and potentially has significant clinical implications. Our results indicate that plasma BCAA levels should to be monitored in patients treated with NaPBA since patients taking the medication are at increased risk for BCAA deficiency. On a broader scale, they could open avenues to explore NaPBA as a therapy in maple syrup urine disease and other common complex disorders with dysregulation of BCAA metabolism. PMID:25042691

  8. Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

    PubMed

    Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

    2017-06-16

    Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

  9. Bagasse hydrolyzates from Agave tequilana as substrates for succinic acid production by Actinobacillus succinogenes in batch and repeated batch reactor.

    PubMed

    Corona-González, Rosa Isela; Varela-Almanza, Karla María; Arriola-Guevara, Enrique; Martínez-Gómez, Álvaro de Jesús; Pelayo-Ortiz, Carlos; Toriz, Guillermo

    2016-04-01

    The aim of this work was to obtain fermentable sugars by enzymatic or acid hydrolyses of Agave tequilana Weber bagasse in order to produce succinic acid with Actinobacillus succinogenes. Hydrolyses were carried out with mineral acids (sulfuric and hydrochloric acids) or a commercial cellulolytic enzyme, and were optimized statistically by a response surface methodology, having as factors the concentration of acid/enzyme and time of hydrolysis. The concentration of sugars obtained at optimal conditions for each hydrolysis were 21.7, 22.4y 19.8g/L for H2SO4, HCl and the enzymatic preparation respectively. Concerning succinic acid production, the enzymatic hydrolyzates resulted in the highest yield (0.446g/g) and productivity (0.57g/Lh) using A. succinogenes in a batch reactor system. Repeated batch fermentation with immobilized A. succinogenes in agar and with the enzymatic hydrolyzates resulted in a maximum concentration of succinic acid of 33.6g/L from 87.2g/L monosaccharides after 5 cycles in 40h, obtaining a productivity of 1.32g/Lh. Copyright © 2016. Published by Elsevier Ltd.

  10. The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases

    PubMed Central

    Rueda, Elda M.; Johnson, Jerry E.; Giddabasappa, Anand; Swaroop, Anand; Brooks, Matthew J.; Sigel, Irena; Chaney, Shawnta Y.

    2016-01-01

    Purpose The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utilization, synthesis, and buffering. Glucose is the major substrate for ATP synthesis through glycolysis and oxidative phosphorylation (OXPHOS), whereas intermediary metabolism through the tricarboxylic acid (TCA) cycle utilizes non-glucose-derived monocarboxylates, amino acids, and alpha ketoacids to support mitochondrial ATP and GTP synthesis. Cellular ATP is buffered by specialized equilibrium-driven high-energy phosphate (~P) transferring kinases. Our goals were twofold: 1) to characterize the gene expression, protein expression, and activity of key synthesizing and regulating enzymes of energy metabolism in the whole mouse retina, retinal compartments, and/or cells and 2) to provide an integrative analysis of the results related to function. Methods mRNA expression data of energy-related genes were extracted from our whole retinal Affymetrix microarray data. Fixed-frozen retinas from adult C57BL/6N mice were used for immunohistochemistry, laser scanning confocal microscopy, and enzymatic histochemistry. The immunoreactivity levels of well-characterized antibodies, for all major retinal cells and their compartments, were obtained using our established semiquantitative confocal and imaging techniques. Quantitative cytochrome oxidase (COX) and lactate dehydrogenase (LDH) activity was determined histochemically. Results The Affymetrix data revealed varied gene expression patterns of the ATP synthesizing and regulating enzymes found in the muscle, liver, and brain. Confocal studies showed differential cellular and compartmental distribution of isozymes involved in glucose, glutamate, glutamine, lactate, and creatine metabolism. The pattern and intensity of the antibodies and of the COX and LDH activity showed the high capacity of photoreceptors for aerobic glycolysis and OXPHOS. Competition assays with pyruvate revealed that LDH-5 was localized in the photoreceptor

  11. The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases.

    PubMed

    Rueda, Elda M; Johnson, Jerry E; Giddabasappa, Anand; Swaroop, Anand; Brooks, Matthew J; Sigel, Irena; Chaney, Shawnta Y; Fox, Donald A

    2016-01-01

    The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utilization, synthesis, and buffering. Glucose is the major substrate for ATP synthesis through glycolysis and oxidative phosphorylation (OXPHOS), whereas intermediary metabolism through the tricarboxylic acid (TCA) cycle utilizes non-glucose-derived monocarboxylates, amino acids, and alpha ketoacids to support mitochondrial ATP and GTP synthesis. Cellular ATP is buffered by specialized equilibrium-driven high-energy phosphate (~P) transferring kinases. Our goals were twofold: 1) to characterize the gene expression, protein expression, and activity of key synthesizing and regulating enzymes of energy metabolism in the whole mouse retina, retinal compartments, and/or cells and 2) to provide an integrative analysis of the results related to function. mRNA expression data of energy-related genes were extracted from our whole retinal Affymetrix microarray data. Fixed-frozen retinas from adult C57BL/6N mice were used for immunohistochemistry, laser scanning confocal microscopy, and enzymatic histochemistry. The immunoreactivity levels of well-characterized antibodies, for all major retinal cells and their compartments, were obtained using our established semiquantitative confocal and imaging techniques. Quantitative cytochrome oxidase (COX) and lactate dehydrogenase (LDH) activity was determined histochemically. The Affymetrix data revealed varied gene expression patterns of the ATP synthesizing and regulating enzymes found in the muscle, liver, and brain. Confocal studies showed differential cellular and compartmental distribution of isozymes involved in glucose, glutamate, glutamine, lactate, and creatine metabolism. The pattern and intensity of the antibodies and of the COX and LDH activity showed the high capacity of photoreceptors for aerobic glycolysis and OXPHOS. Competition assays with pyruvate revealed that LDH-5 was localized in the photoreceptor inner segments. The

  12. Enzyme-mediated bacterial biodegradation of an azo dye (C.I. Acid blue 113): reuse of treated dye wastewater in post-tanning operations.

    PubMed

    Senthilvelan, T; Kanagaraj, J; Panda, R C

    2014-11-01

    "Dyeing" is a common practice used to color the hides during the post-tanning operations in leather processing generating plenty of wastewater. The waste stream containing dye as pollutant is severely harmful to living beings. An azo dye (C.I. Acid Blue 113) has been biodegraded effectively by bacterial culture mediated with azoreductase enzyme to reduce the pollution load in the present investigation. The maximum rate of dye degradation was found to be 96 ± 4 and 92 ± 4 % for the initial concentrations of 100 and 200 mg/l, respectively. The enzyme activity was measured using NADH as a substrate. Fourier transform infrared spectroscopy (FT-IR) analysis was confirmed that the transformation of azo linkage could be transformed into N2 or NH3 or incorporated into complete biomass. Breaking down of dye molecules to various metabolites (such as aniline, naphthalene-1,4-diamine, 3-aminobenzenesulfonic acid, naphthalene-1-sulfonic acid, 8-aminonaphthalene-1-sulfonic acid, 5,8-diaminonaphthalene-1-sulfonic acid) was confirmed by gas chromatography and mass spectra (GC-MS) and mass (electrospray ionization (ESI)) spectra analysis. The treated wastewater could be reused for dyeing operation in the leather processing, and the properties of produced leather were evaluated by conventional methods that revealed to have improved dye penetration into the grain layer of experimental leather sample and resulted in high levelness of dyeing, which helps to obtain the desired smoothness and soft leather properties.

  13. Unsuspected task for an old team: succinate, fumarate and other Krebs cycle acids in metabolic remodeling.

    PubMed

    Bénit, Paule; Letouzé, Eric; Rak, Malgorzata; Aubry, Laetitia; Burnichon, Nelly; Favier, Judith; Gimenez-Roqueplo, Anne-Paule; Rustin, Pierre

    2014-08-01

    Seventy years from the formalization of the Krebs cycle as the central metabolic turntable sustaining the cell respiratory process, key functions of several of its intermediates, especially succinate and fumarate, have been recently uncovered. The presumably immutable organization of the cycle has been challenged by a number of observations, and the variable subcellular location of a number of its constitutive protein components is now well recognized, although yet unexplained. Nonetheless, the most striking observations have been made in the recent period while investigating human diseases, especially a set of specific cancers, revealing the crucial role of Krebs cycle intermediates as factors affecting genes methylation and thus cell remodeling. We review here the recent advances and persisting incognita about the role of Krebs cycle acids in diverse aspects of cellular life and human pathology. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Impact of temperature on sea bass, Dicentrarchus labrax, retina: Fatty acid composition, expression of rhodopsin and enzymes of lipid and melatonin metabolism.

    PubMed

    Bouaziz, Mehdi; Bejaoui, Safa; Rabeh, Imen; Besbes, Raouf; El Cafsi, M 'Hamed; Falcon, Jack

    2017-06-01

    Teleost fish are ectothermic vertebrates. Their metabolism, physiology and behavior rely on the external temperature. This study, on the retina of the sea bass Dicentrarchus labrax, reports on the impact of temperature on the fatty acid composition and mRNA abundance of key enzymes of lipid metabolism: fatty acid desaturase-2 (FADS2), fatty acid elongase-5 (ELOVL5), sterol regulatory element-binding protein-1 (SREBP-1), triglyceride lipase and phospholipase A2 (PLA2). We also report on the effects on the photopigment molecule rhodopsin and on enzymes of the melatonin synthesis pathway, namely arylalkylamine N-acetyltransferases 1a and 1b and acetylserotonin methyltransferase. Juvenile fish were placed for 30 days at 18, 23 or 28 °C. At 23 °C, the fatty acid composition of D. labrax retina showed, as generally reported for the retina of other fish species, particularly high amounts of docosahexaenoic (DHA), palmitic and oleic acids. The fatty acids composition was not significantly (P > 0.05) altered between 23 and 28 °C, but did increase at 18 °C compared to 23 and 28 °C. At 18 °C there were noticeable increases in total DHA, ecosapentaenoic, arachidonic, oleic, linoleic, palmitoleic and stearic acids. A negative correlation was found in the abundance of neutral (NL) vs. polar (PL) lipids: 18 °C induced an increase in NL and a decrease in PL, while 28 °C induced higher PL with decreased NL. In NL the changes affected mainly triglycerides. FADS2 and ELOVL5 mRNA abundance decreased from 18° to 28 °C while SREBP-1 and triglyceride lipase mRNA remained stable. Conversely PLA2 mRNA was more abundant at 23 than at 18 and 28 °C. Temperature increased and decreased rhodopsin mRNA abundance, at 28 °C and 18 °C respectively, while there was no effect on mRNA from the melatonin synthesis enzymes. In conclusion the data indicate a temperature induced redistribution of fatty acids among the lipid classes that might affect the physical properties of

  15. Phenylalanine ammonia lyase from Arabidopsis thaliana (AtPAL2): A potent MIO-enzyme for the synthesis of non-canonical aromatic alpha-amino acids: Part I: Comparative characterization to the enzymes from Petroselinum crispum (PcPAL1) and Rhodosporidium toruloides (RtPAL).

    PubMed

    Dreßen, Alana; Hilberath, Thomas; Mackfeld, Ursula; Billmeier, Arne; Rudat, Jens; Pohl, Martina

    2017-09-20

    Phenylalanine ammonia lyase (PAL) from Arabidopsis thaliana (AtPAL2) was comparatively characterized to the well-studied enzyme from parsley (PcPAL1) and Rhodosporidium toruloides (RtPAL) with respect to kinetic parameters for the deamination and the amination reaction, pH- and temperature optima and the substrate range of the amination reaction. Whereas both plant enzymes are specific for phenylalanine, the bifunctional enzyme from Rhodosporidium toruloides shows K M -values for L-Phe and L-Tyr in the same order of magnitude and, compared to both plant enzymes, a 10-15-fold higher activity. At 30°C all enzymes were sufficiently stable with half-lives of 3.4days (PcPAL1), 4.6days (AtPAL2) and 9.7days (RtPAL/TAL). Very good results for the amination of various trans-cinnamic acid derivatives were obtained using E. coli cells as whole cell biocatalysts in ammonium carbonate buffer. Investigation of the substrate ranges gave interesting results for the newly tested enzymes from A. thaliana and R. toruloides. Only the latter accepts besides 4-hydroxy-CA also 3-methoxy-4-hydroxy-CA as a substrate, which is an interesting intermediate for the formation of pharmaceutically relevant L-Dopa. AtPAL2 is a very good catalyst for the formation of (S)-3-F-Phe, (S)-4-F-Phe and (S)-2-Cl-Phe. Such non-canonical amino acids are valuable building blocks for the formation of various drug molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. ORENZA: a web resource for studying ORphan ENZyme activities

    PubMed Central

    Lespinet, Olivier; Labedan, Bernard

    2006-01-01

    Background Despite the current availability of several hundreds of thousands of amino acid sequences, more than 36% of the enzyme activities (EC numbers) defined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) are not associated with any amino acid sequence in major public databases. This wide gap separating knowledge of biochemical function and sequence information is found for nearly all classes of enzymes. Thus, there is an urgent need to explore these sequence-less EC numbers, in order to progressively close this gap. Description We designed ORENZA, a PostgreSQL database of ORphan ENZyme Activities, to collate information about the EC numbers defined by the NC-IUBMB with specific emphasis on orphan enzyme activities. Complete lists of all EC numbers and of orphan EC numbers are available and will be periodically updated. ORENZA allows one to browse the complete list of EC numbers or the subset associated with orphan enzymes or to query a specific EC number, an enzyme name or a species name for those interested in particular organisms. It is possible to search ORENZA for the different biochemical properties of the defined enzymes, the metabolic pathways in which they participate, the taxonomic data of the organisms whose genomes encode them, and many other features. The association of an enzyme activity with an amino acid sequence is clearly underlined, making it easy to identify at once the orphan enzyme activities. Interactive publishing of suggestions by the community would provide expert evidence for re-annotation of orphan EC numbers in public databases. Conclusion ORENZA is a Web resource designed to progressively bridge the unwanted gap between function (enzyme activities) and sequence (dataset present in public databases). ORENZA should increase interactions between communities of biochemists and of genomicists. This is expected to reduce the number of orphan enzyme activities by allocating gene

  17. Helicase-dependent amplification of nucleic acids.

    PubMed

    Cao, Yun; Kim, Hyun-Jin; Li, Ying; Kong, Huimin; Lemieux, Bertrand

    2013-10-11

    Helicase-dependent amplification (HDA) is a novel method for the isothermal in vitro amplification of nucleic acids. The HDA reaction selectively amplifies a target sequence by extension of two oligonucleotide primers. Unlike the polymerase chain reaction (PCR), HDA uses a helicase enzyme to separate the deoxyribonucleic acid (DNA) strands, rather than heat denaturation. This allows DNA amplification without the need for thermal cycling. The helicase used in HDA is a helicase super family II protein obtained from a thermophilic organism, Thermoanaerobacter tengcongensis (TteUvrD). This thermostable helicase is capable of unwinding blunt-end nucleic acid substrates at elevated temperatures (60° to 65°C). The HDA reaction can also be coupled with reverse transcription for ribonucleic acid (RNA) amplification. The products of this reaction can be detected during the reaction using fluorescent probes when incubations are conducted in a fluorimeter. Alternatively, products can be detected after amplification using a disposable amplicon containment device that contains an embedded lateral flow strip. Copyright © 2013 John Wiley & Sons, Inc.

  18. The activity of salivary aldehyde dehydrogenase during the menstrual cycle and pregnancy.

    PubMed

    Giebułtowicz, Joanna; Wroczyński, Piotr; Kosiński, Przemysław; Pietrzak, Bronisława

    2013-03-01

    The aim of the present study was to describe the changes in the activity of ALDH3A1 in saliva in relation to the menstrual cycle and pregnancy. We also measured major salivary antioxidants, salivary peroxidase (SPO) activity and uric acid (UA) concentration. Fasting saliva samples were collected from 63 women with uncomplicated pregnancies and from 39 healthy women of reproductive age, but not pregnant. Saliva samples were also collected from 10 healthy women with regular menstrual cycles in the early follicular, the mid-cycle and the mid-luteal phase during one menstrual cycle. SPO and ALDH3A1 activity was determined fluorimetrically, whereas UA concentration photometrically. The ALDH3A1 did not vary significantly among phases of menstrual cycle. However, the enzyme activity decreased with the length of pregnancy and in the third trimester is significantly lower than that in the saliva of non-pregnant women. Lower concentration of UA and in the third trimester the activity of ALDH3A1 in saliva of pregnant women could be a risk factor of, e.g. oral pathologies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Oxygenated monoterpenes citral and carvacrol cause oxidative damage in Escherichia coli without the involvement of tricarboxylic acid cycle and Fenton reaction.

    PubMed

    Chueca, Beatriz; Pagán, Rafael; García-Gonzalo, Diego

    2014-10-17

    Oxygenated monoterpenes citral and carvacrol are common constituents of many essential oils (EOs) that have been extensively studied as antimicrobial agents but whose mechanisms of microbial inactivation have not been totally elucidated. A recent study described a mechanism of Escherichia coli death for (+)-limonene, a hydrocarbon monoterpene also frequently present in EOs, similar to the common mechanism proposed for bactericidal antibiotics. This mechanism involves the formation of Fenton-mediated hydroxyl radical, a reactive oxygen species (ROS), via tricarboxylic acid (TCA) cycle, which would ultimately inactivate cells. Our objective was to determine whether E. coli MG1655 inactivation by citral and carvacrol follows a similar mechanism of cell death. Challenging experiments with 300μL/L citral and 100μL/L carvacrol inactivated at least 2.5log10cycles of exponentially growing cells in 3h under aerobic conditions. The presence of thiourea (an ROS scavenger) reduced cell inactivation in 2log10cycles, demonstrating the role of ROS in cell death. Decreased resistance of a ΔrecA mutant (deficient in an enzyme involved in SOS response to DNA damage) indicated that citral and carvacrol caused oxidative damage to DNA. Although the mechanism of E. coli inactivation by carvacrol and citral was similarly mediated by ROS, their formation did not follow the same pathways described for (+)-limonene and bactericidal drugs because neither Fenton reaction nor NADH production via the TCA cycle was involved in cell death. Moreover, further experiments demonstrated antimicrobial activity of citral and carvacrol in anaerobic environments without the involvement of ROS. As a consequence, cell death by carvacrol and citral in anaerobiosis follows a different mechanism than that observed under aerobic conditions. These results demonstrated a different mechanism of inactivation by citral and carvacrol with regard to (+)-limonene and bactericidal antibiotics, indicating the

  20. Lipid sensing by mTOR complexes via de novo synthesis of phosphatidic acid

    PubMed Central

    Menon, Deepak; Salloum, Darin; Bernfeld, Elyssa; Gorodetsky, Elizabeth; Akselrod, Alla; Frias, Maria A.; Sudderth, Jessica; Chen, Pei-Hsuan; DeBerardinis, Ralph; Foster, David A.

    2017-01-01

    mTOR, the mammalian target of rapamycin, integrates growth factor and nutrient signals to promote a transformation from catabolic to anabolic metabolism, cell growth, and cell cycle progression. Phosphatidic acid (PA) interacts with the FK506-binding protein–12-rapamycin-binding (FRB) domain of mTOR, which stabilizes both mTOR complexes: mTORC1 and mTORC2. We report here that mTORC1 and mTORC2 are activated in response to exogenously supplied fatty acids via the de novo synthesis of PA, a central metabolite for membrane phospholipid biosynthesis. We examined the impact of exogenously supplied fatty acids on mTOR in KRas-driven cancer cells, which are programmed to utilize exogenous lipids. The induction of mTOR by oleic acid was dependent upon the enzymes responsible for de novo synthesis of PA. Suppression of the de novo synthesis of PA resulted in G1 cell cycle arrest. Although it has long been appreciated that mTOR is a sensor of amino acids and glucose, this study reveals that mTOR also senses the presence of lipids via production of PA. PMID:28223357

  1. Lipid sensing by mTOR complexes via de novo synthesis of phosphatidic acid.

    PubMed

    Menon, Deepak; Salloum, Darin; Bernfeld, Elyssa; Gorodetsky, Elizabeth; Akselrod, Alla; Frias, Maria A; Sudderth, Jessica; Chen, Pei-Hsuan; DeBerardinis, Ralph; Foster, David A

    2017-04-14

    mTOR, the mammalian target of rapamycin, integrates growth factor and nutrient signals to promote a transformation from catabolic to anabolic metabolism, cell growth, and cell cycle progression. Phosphatidic acid (PA) interacts with the FK506-binding protein-12-rapamycin-binding (FRB) domain of mTOR, which stabilizes both mTOR complexes: mTORC1 and mTORC2. We report here that mTORC1 and mTORC2 are activated in response to exogenously supplied fatty acids via the de novo synthesis of PA, a central metabolite for membrane phospholipid biosynthesis. We examined the impact of exogenously supplied fatty acids on mTOR in KRas-driven cancer cells, which are programmed to utilize exogenous lipids. The induction of mTOR by oleic acid was dependent upon the enzymes responsible for de novo synthesis of PA. Suppression of the de novo synthesis of PA resulted in G 1 cell cycle arrest. Although it has long been appreciated that mTOR is a sensor of amino acids and glucose, this study reveals that mTOR also senses the presence of lipids via production of PA. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Changes in element accumulation, phenolic metabolism, and antioxidative enzyme activities in the red-skin roots of Panax ginseng.

    PubMed

    Zhou, Ying; Yang, Zhenming; Gao, Lingling; Liu, Wen; Liu, Rongkun; Zhao, Junting; You, Jiangfeng

    2017-07-01

    Red-skin root disease has seriously decreased the quality and production of Panax ginseng (ginseng). To explore the disease's origin, comparative analysis was performed in different parts of the plant, particularly the epidermis, cortex, and/or fibrous roots of 5-yr-old healthy and diseased red-skin ginseng. The inorganic element composition, phenolic compound concentration, reactive oxidation system, antioxidant concentrations such as ascorbate and glutathione, activities of enzymes related to phenolic metabolism and oxidation, and antioxidative system particularly the ascorbate-glutathione cycle were examined using conventional methods. Aluminum (Al), iron (Fe), magnesium, and phosphorus were increased, whereas manganese was unchanged and calcium was decreased in the epidermis and fibrous root of red-skin ginseng, which also contained higher levels of phenolic compounds, higher activities of the phenolic compound-synthesizing enzyme phenylalanine ammonia-lyase and the phenolic compound oxidation-related enzymes guaiacol peroxidase and polyphenoloxidase. As the substrate of guaiacol peroxidase, higher levels of H 2 O 2 and correspondingly higher activities of superoxide dismutase and catalase were found in red-skin ginseng. Increased levels of ascorbate and glutathione; increased activities of l-galactose 1-dehydrogenase, ascorbate peroxidase, ascorbic acid oxidase, and glutathione reductase; and lower activities of dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione peroxidase were found in red-skin ginseng. Glutathione- S -transferase activity remained constant. Hence, higher element accumulation, particularly Al and Fe, activated multiple enzymes related to accumulation of phenolic compounds and their oxidation. This might contribute to red-skin symptoms in ginseng. It is proposed that antioxidant and antioxidative enzymes, especially those involved in ascorbate-glutathione cycles, are activated to protect against phenolic compound

  3. Enzyme-linked immunosorbent assay (ELISA) for the anthropogenic marker isolithocholic acid in water.

    PubMed

    Baldofski, Stefanie; Hoffmann, Holger; Lehmann, Andreas; Breitfeld, Stefan; Garbe, Leif-Alexander; Schneider, Rudolf J

    2016-11-01

    Bile acids are promising chemical markers to assess the pollution of water samples with fecal material. This study describes the optimization and validation of a direct competitive enzyme-linked immunosorbent assay for the bile acid isolithocholic acid (ILA). The quantification range of the optimized assay was between 0.09 and 15 μg/L. The assay was applied to environmental water samples. Most studies until now were focused on bile acid fractions in the particulate phase of water samples. In order to avoid tedious sample preparation, we undertook to evaluate the dynamics and significance of ILA levels in the aqueous phase. Very low concentrations in tap and surface water samples made a pre-concentration step necessary for this matrix as well as for wastewater treatment plant (WWTP) effluent. Mean recoveries for spiked water samples were between 97% and 109% for tap water and WWTP influent samples and between 102% and 136% for WWTP effluent samples. 90th percentiles of intra-plate and inter-plate coefficients of variation were below 10% for influents and below 20% for effluents and surface water. ILA concentrations were quantified in the range of 33-72 μg/L in influent, 21-49 ng/L in effluent and 18-48 ng/L in surface water samples. During wastewater treatment the ILA levels were reduced by more than 99%. ILA concentrations of influents determined by ELISA and LC-MS/MS were in good agreement. However, findings in LC-ELISA experiments suggest that the true ILA levels in concentrated samples are lower due to interfering effects of matrix compounds and/or cross-reactants. Yet, the ELISA will be a valuable tool for the performance check and comparison of WWTPs and the localization of fecal matter input into surface waters. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Ionizable side chains at catalytic active sites of enzymes.

    PubMed

    Jimenez-Morales, David; Liang, Jie; Eisenberg, Bob

    2012-05-01

    Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1,072 Å(3). The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes.

  5. Molecular modeling and simulation of FabG, an enzyme involved in the fatty acid pathway of Streptococcus pyogenes.

    PubMed

    Shafreen, Rajamohmed Beema; Pandian, Shunmugiah Karutha

    2013-09-01

    Streptococcus pyogenes (SP) is the major cause of pharyngitis accompanied by strep throat infections in humans. 3-keto acyl reductase (FabG), an important enzyme involved in the elongation cycle of the fatty acid pathway of S. pyogenes, is essential for synthesis of the cell-membrane, virulence factors and quorum sensing-related mechanisms. Targeting SPFabG may provide an important aid for the development of drugs against S. pyogenes. However, the absence of a crystal structure for FabG of S. pyogenes limits the development of structure-based drug designs. Hence, in the present study, a homology model of FabG was generated using the X-ray crystallographic structure of Aquifex aeolicus (PDB ID: 2PNF). The modeled structure was refined using energy minimization. Furthermore, active sites were predicted, and a large dataset of compounds was screened against SPFabG. The ligands were docked using the LigandFit module that is available from Discovery Studio version 2.5. From this list, 13 best hit ligands were chosen based on the docking score and binding energy. All of the 13 ligands were screened for Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties. From this, the two best descriptors, along with one descriptor that lay outside the ADMET plot, were selected for molecular dynamic (MD) simulation. In vitro testing of the ligands using biological assays further substantiated the efficacy of the ligands that were screened based on the in silico methods. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Carbon metabolism of peach fruit after harvest: changes in enzymes involved in organic acid and sugar level modifications.

    PubMed

    Borsani, Julia; Budde, Claudio O; Porrini, Lucía; Lauxmann, Martin A; Lombardo, Verónica A; Murray, Ricardo; Andreo, Carlos S; Drincovich, María F; Lara, María V

    2009-01-01

    Peach (Prunus persica L. Batsch) is a climacteric fruit that ripens after harvest, prior to human consumption. Organic acids and soluble sugars contribute to the overall organoleptic quality of fresh peach; thus, the integrated study of the metabolic pathways controlling the levels of these compounds is of great relevance. Therefore, in this work, several metabolites and enzymes involved in carbon metabolism were analysed during the post-harvest ripening of peach fruit cv 'Dixiland'. Depending on the enzyme studied, activity, protein level by western blot, or transcript level by quantitative real time-PCR were analysed. Even though sorbitol did not accumulate at a high level in relation to sucrose at harvest, it was rapidly consumed once the fruit was separated from the tree. During the ripening process, sucrose degradation was accompanied by an increase of glucose and fructose. Specific transcripts encoding neutral invertases (NIs) were up-regulated or down-regulated, indicating differential functions for each putative NI isoform. Phosphoenolpyruvate carboxylase was markedly induced, and may participate as a glycolytic shunt, since the malate level did not increase during post-harvest ripening. The fermentative pathway was highly induced, with increases in both the acetaldehyde level and the enzymes involved in this process. In addition, proteins differentially expressed during the post-harvest ripening process were also analysed. Overall, the present study identified enzymes and pathways operating during the post-harvest ripening of peach fruit, which may contribute to further identification of varieties with altered levels of enzymes/metabolites or in the evaluation of post-harvest treatments to produce fruit of better organoleptic attributes.

  7. Omega-3 polyunsaturated fatty acid docosahexaenoic acid and its role in exhaustive-exercise-induced changes in female rat ovulatory cycle.

    PubMed

    Mostafa, Abeer F; Samir, Shereen M; Nagib, R M

    2018-04-01

    Exhaustive exercises can cause delayed menarche or menstrual cycle irregularities in females. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) are incorporated into a wide range of benefits in many physiological systems. Our work aimed to assess the role of ω-3 PUFA docosahexaenoic acid (DHA) on the deleterious effects of exhaustive exercise on the female reproductive system in rats. Virgin female rats were randomly divided into 4 groups (12 rats in each): control group, omega-3 group treated with DHA, exhaustive exercise group, and exhaustive exercised rats treated with DHA. Omega-3 was given orally to the rats once daily for 4 estrous cycles. Exhaustive exercises revealed lower levels in progesterone and gonadotropins together with histopathological decrease in number of growing follicles and corpora lutea. Moreover, the exercised rats showed low levels of ovarian antioxidants with high level of caspase-3 and plasma cortisol level that lead to disruption of hypothalamic-pituitary-gonadal axis. ω-3 PUFA DHA has beneficial effects on the number of newly growing follicles in both sedentary and exercised rats with decreasing the level of caspase-3 and increasing the antioxidant activity in ovaries. Exhaustive exercises can cause ovulatory problems in female rats that can be improved by ω-3 supplementation.

  8. Preparation of crosslinked enzyme aggregates (CLEAs) of acid urease with urethanase activity and their application.

    PubMed

    Zhang, Qian; Zha, Xiaohong; Zhou, Nandi; Tian, Yaping

    2016-04-01

    An acid urease from Providencia rettgeri JN-B815 was purified via ultrasonication, ethanol precipitation, and DEAE ion-exchange column chromatography. It was found that the enzyme exhibits not only urease activity, but also urethanase activity, which made it possible to reduce EC already existed or would produce and its precursor urea at the same time. Then, crosslinked enzyme aggregates of P. rettgeri urease (PRU-CLEAs) were prepared using genipin as crosslinking agent. The purification process of acid urease, the effects of genipin concentration, and crosslinking time on PRU-CLEAs activity were investigated. The crosslinking was performed at pH 4.5 for 2.5 h, using 0.3% genipin as crosslinking agent, and 0.3 g · L(-1) bovine serum albumin as protein feeder. Using the obtained PRU-CLEAs, the removal rate of urea was up to 9.31 mg · L(-1) · h(-1). The removal rate of urea was still up to 7.56 mg · L(-1) · h(-1) after PRU-CLEAs was re-used for 6 times. When PRU-CLEAs were applied in a batch stirred and membrane reactor, the removal rate of urea in rice wine reached 5.16 mg · L(-1) · h(-1) and the removal rate of EC was 9.21 μg · L(-1) · h(-1). Furthermore, the treatment with PRU-CLEAs revealed no significant change of volatile flavor substances in Chinese rice wine. Thus PRU-CLEAs have great potential in the elimination of EC in Chinese rice wine. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. pKa cycling of the general acid/base in glycoside hydrolase families 33 and 34.

    PubMed

    Yu, Haibo; Griffiths, Thomas M

    2014-03-28

    Glycoside hydrolase families 33 and 34 catalyse the hydrolysis of terminal sialic acid residues from sialyl oligosaccharides and glycoconjugates with a net retention of the stereochemistry at the anomeric centre. It is generally believed that the conserved aspartic acid in the active site functions as a general acid to protonate the hydroxyl group of the departing aglycone during glycosylation, and then as a general base to facilitate the nucleophilic attack of the water molecule on the intermediate state during the deglycosylation reaction. The dual role of the general acid/base places specific demands upon its protonation state, and thus pKa values. However, it is not fully understood how this catalytic residue can achieve such pKa cycling during catalysis. We present both MM and combined QM/MM simulations to characterise the pKa values of the proposed catalytic general acid/base in the glycoside hydrolase families 33 and 34. Collectively, our study suggests that the binding of anionic substrates and the local solvation properties along with the neutralisation of the nearby glutamic acid upon glycosylation modulate the electrostatic environment around the general acid/base to achieve its proper protonation states.

  10. Ser/Thr Phosphorylation Regulates the Fatty Acyl-AMP Ligase Activity of FadD32, an Essential Enzyme in Mycolic Acid Biosynthesis*

    PubMed Central

    Le, Nguyen-Hung; Molle, Virginie; Eynard, Nathalie; Miras, Mathieu; Stella, Alexandre; Bardou, Fabienne; Galandrin, Ségolène; Guillet, Valérie; André-Leroux, Gwenaëlle; Bellinzoni, Marco; Alzari, Pedro; Mourey, Lionel; Burlet-Schiltz, Odile; Daffé, Mamadou; Marrakchi, Hedia

    2016-01-01

    Mycolic acids are essential components of the mycobacterial cell envelope, and their biosynthetic pathway is a well known source of antituberculous drug targets. Among the promising new targets in the pathway, FadD32 is an essential enzyme required for the activation of the long meromycolic chain of mycolic acids and is essential for mycobacterial growth. Following the in-depth biochemical, biophysical, and structural characterization of FadD32, we investigated its putative regulation via post-translational modifications. Comparison of the fatty acyl-AMP ligase activity between phosphorylated and dephosphorylated FadD32 isoforms showed that the native protein is phosphorylated by serine/threonine protein kinases and that this phosphorylation induced a significant loss of activity. Mass spectrometry analysis of the native protein confirmed the post-translational modifications and identified Thr-552 as the phosphosite. Phosphoablative and phosphomimetic FadD32 mutant proteins confirmed both the position and the importance of the modification and its correlation with the negative regulation of FadD32 activity. Investigation of the mycolic acid condensation reaction catalyzed by Pks13, involving FadD32 as a partner, showed that FadD32 phosphorylation also impacts the condensation activity. Altogether, our results bring to light FadD32 phosphorylation by serine/threonine protein kinases and its correlation with the enzyme-negative regulation, thus shedding a new horizon on the mycolic acid biosynthesis modulation and possible inhibition strategies for this promising drug target. PMID:27590338

  11. Bisphenol A Alters n-6 Fatty Acid Composition and Decreases Antioxidant Enzyme Levels in Rat Testes: A LC-QTOF-Based Metabolomics Study

    PubMed Central

    Qiao, Shanlei; Hu, Nan; Hu, Yanhui; Wu, Wei; Qiu, Lianglin; Zhang, Ruyang; Wang, Yubang; Wang, Shoulin; Zhou, Zuomin; Xia, Yankai; Wang, Xinru

    2012-01-01

    Background Male reproductive toxicity induced by exposure to bisphenol A (BPA) has been widely reported. The testes have proven to be a major target organ of BPA toxicity, so studying testicular metabolite variation holds promise for the discovery of mechanisms linked to the toxic effects of BPA on reproduction. Methodology/Principal Findings Male Sprague-Dawley rats were orally administered doses of BPA at the levels of 0, 50 mg/kg/d for 8 weeks. We used an unbiased liquid chromatography-quadrupole time-of-flight (LC-QTOF)-based metabolomics approach to discover, identify, and analyze the variation of testicular metabolites. Two n-6 fatty acids, linoleic acid (LA) and arachidonic acid (AA) were identified as potential testicular biomarkers. Decreased levels of LA and increased levels of AA as well as AA/LA ratio were observed in the testes of the exposed group. According to these suggestions, testicular antioxidant enzyme levels were detected. Testicular superoxide dismutase (SOD) declined significantly in the exposed group compared with that in the non-exposed group, and the glutathione peroxidase (GSH-Px) as well as catalase (CAT) also showed a decreasing trend in BPA treated group. Conclusions/Significance BPA caused testicular n-6 fatty acid composition variation and decreased antioxidant enzyme levels. This study emphasizes that metabolomics brings the promise of biomarkers identification for the discovery of mechanisms underlying reproductive toxicity. PMID:23024759

  12. Bisphenol A alters n-6 fatty acid composition and decreases antioxidant enzyme levels in rat testes: a LC-QTOF-based metabolomics study.

    PubMed

    Chen, Minjian; Xu, Bin; Ji, Wenliang; Qiao, Shanlei; Hu, Nan; Hu, Yanhui; Wu, Wei; Qiu, Lianglin; Zhang, Ruyang; Wang, Yubang; Wang, Shoulin; Zhou, Zuomin; Xia, Yankai; Wang, Xinru

    2012-01-01

    Male reproductive toxicity induced by exposure to bisphenol A (BPA) has been widely reported. The testes have proven to be a major target organ of BPA toxicity, so studying testicular metabolite variation holds promise for the discovery of mechanisms linked to the toxic effects of BPA on reproduction. Male Sprague-Dawley rats were orally administered doses of BPA at the levels of 0, 50 mg/kg/d for 8 weeks. We used an unbiased liquid chromatography-quadrupole time-of-flight (LC-QTOF)-based metabolomics approach to discover, identify, and analyze the variation of testicular metabolites. Two n-6 fatty acids, linoleic acid (LA) and arachidonic acid (AA) were identified as potential testicular biomarkers. Decreased levels of LA and increased levels of AA as well as AA/LA ratio were observed in the testes of the exposed group. According to these suggestions, testicular antioxidant enzyme levels were detected. Testicular superoxide dismutase (SOD) declined significantly in the exposed group compared with that in the non-exposed group, and the glutathione peroxidase (GSH-Px) as well as catalase (CAT) also showed a decreasing trend in BPA treated group. BPA caused testicular n-6 fatty acid composition variation and decreased antioxidant enzyme levels. This study emphasizes that metabolomics brings the promise of biomarkers identification for the discovery of mechanisms underlying reproductive toxicity.

  13. Nanoarmored Enzymes for Organic Enzymology: Synthesis and Characterization of Poly(2-Alkyloxazoline)-Enzyme Conjugates.

    PubMed

    Leurs, Melanie; Tiller, Joerg C

    2017-01-01

    The properties of enzymes can be altered significantly by modification with polymers. Numerous different methods are known to obtain such polymer-enzyme conjugates (PECs). However, there is no universal method to render enzymes into PECs that are fully soluble in organic solvents. Here, we present a method, which achieves such high degree of modification of proteins that the majority of modified enzymes will be soluble in organic solvents. This is achieved by preparing poly(2-alkyloxazoline)s (POx) with an NH 2 end group and coupling this functional polymer via pyromellitic acid dianhydride onto the amino groups of the respective protein. The resulting PECs are capable of serving as surfactants for unmodified proteins, rendering the whole mixture organosoluble. Depending on the nature of the POx and the molecular weight and the nature of the enzyme, the PECs are soluble in chloroform or even toluene. Another advantage of this method is that the poly(2-alkyloxazoline) can be activated with the coupling agent and used for the enzyme conjugation without further purification. The POx-enzyme conjugates generated by this modification strategy show modulated catalytic activity in both, aqueous and organic, systems. © 2017 Elsevier Inc. All rights reserved.

  14. Optimizing Immobilized Enzyme Performance in Cell-Free Environments to Produce Liquid Fuels

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Belfort, Georges; Grimaldi, Joseph J.

    2015-01-27

    Limitations on biofuel production using cell culture (Escherichia coli, Clostridium, Saccharomyces cerevisiae, brown microalgae, blue-green algae and others) include low product (alcohol) concentrations (≤0.2 vol%) due to feedback inhibition, instability of cells, and lack of economical product recovery processes. To overcome these challenges, an alternate simplified biofuel production scheme was tested based on a cell-free immobilized enzyme system. Using this cell free system, we were able to obtain about 2.6 times higher concentrations of iso-butanol using our non-optimized system as compared with live cell systems. This process involved two steps: (i) converts acid to aldehyde using keto-acid decarboxylase (KdcA), andmore » (ii) produces alcohol from aldehyde using alcohol dehydrogenase (ADH) with a cofactor (NADH) conversion from inexpensive formate using a third enzyme, formate dehydrogenase (FDH). To increase stability and conversion efficiency with easy separations, the first two enzymes were immobilized onto methacrylate resin. Fusion proteins of labile KdcA (fKdcA) were expressed to stabilize the covalently immobilized KdcA. Covalently immobilized ADH exhibited long-term stability and efficient conversion of aldehyde to alcohol over multiple batch cycles without fusions. High conversion rates and low protein leaching were achieved by covalent immobilization of enzymes on methacrylate resin. The complete reaction scheme was demonstrated by immobilizing both ADH and fKdcA and using FDH free in solution. The new system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.5 % (v/v). Further increases in titer will require continuous removal of the isobutanol using our novel brush membrane system that exhibits a 1.5 fold increase in the separation factor of isobutanol from water versus that obtained for commercial silicone rubber membranes. These bio-inspired brush membranes are based

  15. The alpha glycerophosphate cycle in Drosophila melanogaster VI. structure and evolution of enzyme paralogs in the genus Drosophila.

    PubMed

    Carmon, Amber; MacIntyre, Ross

    2010-01-01

    The genome sequences of 12 Drosophila species contain 3 paralogs for alpha glycerophosphate dehydrogenase (GPDH) and for the mitochondrial alpha glycerophosphate oxidase (GPO). These 2 enzymes participate in the alpha glycerophosphate cycle in the adult thoracic flight muscles. The flight muscle enzymes are encoded by gpdh-1 at 26A2 and gpo-1 at 52C8. In this paper, we show that the GPDH paralogs share the same evolutionarily conserved functional domains and most intron positions, whereas the GPO paralogs share only some of the functional domains of mitochondrial oxidoreductases. The GPO paralogs not expressed in the flight muscles essentially lack introns. GPDH paralogs encoded by gpdh-2 and gpdh-3 and the GPO paralogs encoded by gpo-2 and gpo-3 are expressed only in the testes. Gene trees for the GPDH and GPO paralogs indicate that the genes expressed in the flight muscles are evolving very slowly presumably under strong purifying selection whereas the paralogs expressed in the testes are evolving more rapidly. The concordance between species and gene trees, d(N)/d(S) ratios, phylogenetic analysis by maximum likelihood-based tests, and analyses of radical and conservative substitutions all indicate that the additional GPDH and GPO paralogs are also evolving under purifying selection.

  16. Enhancing polyphenol extraction from unripe apples by carbohydrate-hydrolyzing enzymes.

    PubMed

    Zheng, Hu-zhe; Hwang, In-Wook; Chung, Shin-Kyo

    2009-12-01

    The effects of process variables such as enzyme types, enzyme ratio, reaction temperature, pH, time, and ethanol concentration on the extraction of unripe apple polyphenol were investigated. The results indicated that Viscozyme L had the strongest effect on polyphenols extraction and was selected to study the polyphenol composition. The ratio of enzyme (Viscozyme L) to substrate (2 fungal beta-glucanase units (FBG)) at 0.02, reaction at pH 3.7, 50 degrees C for 12 h, and ethanol concentration of 70% were chosen as the most favorable extraction condition. Total phenolic content (TPC), reducing sugar content (RSC), and extraction yield increased by about 3, 1.5, and 2 times, respectively, compared with control. The contents of p-coumaric acid, ferulic acid, and caffeic acid increased to 8, 4, and 32 times, respectively. The enzyme-aided polyphenol extraction process from unripe apples might be applied to food industry for enhancing bioactive compound production.

  17. Enhancing polyphenol extraction from unripe apples by carbohydrate-hydrolyzing enzymes*

    PubMed Central

    Zheng, Hu-zhe; Hwang, In-Wook; Chung, Shin-Kyo

    2009-01-01

    The effects of process variables such as enzyme types, enzyme ratio, reaction temperature, pH, time, and ethanol concentration on the extraction of unripe apple polyphenol were investigated. The results indicated that Viscozyme L had the strongest effect on polyphenols extraction and was selected to study the polyphenol composition. The ratio of enzyme (Viscozyme L) to substrate (2 fungal beta-glucanase units (FBG)) at 0.02, reaction at pH 3.7, 50 °C for 12 h, and ethanol concentration of 70% were chosen as the most favorable extraction condition. Total phenolic content (TPC), reducing sugar content (RSC), and extraction yield increased by about 3, 1.5, and 2 times, respectively, compared with control. The contents of p-coumaric acid, ferulic acid, and caffeic acid increased to 8, 4, and 32 times, respectively. The enzyme-aided polyphenol extraction process from unripe apples might be applied to food industry for enhancing bioactive compound production. PMID:19946955

  18. Enzyme cofactors: Double-edged sword for catalysis

    NASA Astrophysics Data System (ADS)

    Ivanov, Ivaylo

    2013-01-01

    The metal cofactors responsible for the activity of CDK2 -- a representative member of the kinase superfamily of enzymes -- have now been shown to also have inhibitory effects during the catalytic cycle.

  19. Oxysterol biosynthetic enzymes.

    PubMed

    Russell, D W

    2000-12-15

    Oxysterols, herein defined as derivatives of cholesterol with a hydroxyl group on the side chain, play several roles in lipid metabolism. Members of this class regulate the expression of genes that participate in both sterol and fat metabolism, serve as substrates for the synthesis of bile acids, and are intermediates in the transfer of sterols from the periphery to the liver. Three abundant naturally occurring oxysterols are 24-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. The cholesterol hydroxylase enzymes that synthesize each of these have been isolated over the last several years and their study has produced insight into the biology of oxysterols. This article focuses on the properties of these enzymes.

  20. Molecular basis of HHQ biosynthesis: molecular dynamics simulations, enzyme kinetic and surface plasmon resonance studies

    PubMed Central

    2013-01-01

    Background PQS (PseudomonasQuinolone Signal) and its precursor HHQ are signal molecules of the P. aeruginosa quorum sensing system. They explicate their role in mammalian pathogenicity by binding to the receptor PqsR that induces virulence factor production and biofilm formation. The enzyme PqsD catalyses the biosynthesis of HHQ. Results Enzyme kinetic analysis and surface plasmon resonance (SPR) biosensor experiments were used to determine mechanism and substrate order of the biosynthesis. Comparative analysis led to the identification of domains involved in functionality of PqsD. A kinetic cycle was set up and molecular dynamics (MD) simulations were used to study the molecular bases of the kinetics of PqsD. Trajectory analysis, pocket volume measurements, binding energy estimations and decompositions ensured insights into the binding mode of the substrates anthraniloyl-CoA and β-ketodecanoic acid. Conclusions Enzyme kinetics and SPR experiments hint at a ping-pong mechanism for PqsD with ACoA as first substrate. Trajectory analysis of different PqsD complexes evidenced ligand-dependent induced-fit motions affecting the modified ACoA funnel access to the exposure of a secondary channel. A tunnel-network is formed in which Ser317 plays an important role by binding to both substrates. Mutagenesis experiments resulting in the inactive S317F mutant confirmed the importance of this residue. Two binding modes for β-ketodecanoic acid were identified with distinct catalytic mechanism preferences. PMID:23916145

  1. Gibberellic acid promoting phytic acid degradation in germinating soybean under calcium lactate treatment.

    PubMed

    Hui, Qianru; Wang, Mian; Wang, Pei; Ma, Ya; Gu, Zhenxin; Yang, Runqiang

    2018-01-01

    Phytic acid as a phosphorus storage vault provides phosphorus for plant development. It is an anti-nutritional factor for humans and some animals. However, its degradation products lower inositol phosphates have positive effects on human health. In this study, the effect of gibberellic acid (GA) on phytic acid degradation under calcium lactate (Ca) existence was investigated. The results showed that Ca + GA treatment promoted the growth status, hormone metabolism and phytic acid degradation in germinating soybean. At the same time, the availability of phosphorus, the activity of phytic acid degradation-associated enzyme and phosphoinositide-specific phospholipase C (PI-PLC) increased. However, the relative genes expression of phytic acid degradation-associated enzymes did not vary in accordance with their enzymes activity. The results revealed that GA could mediate the transport and function of calcium and a series of physiological and biochemical changes to regulate phytic acid degradation of soybean sprouts. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  2. Retinoic acid catabolizing enzyme CYP26C1 is a genetic modifier in SHOX deficiency.

    PubMed

    Montalbano, Antonino; Juergensen, Lonny; Roeth, Ralph; Weiss, Birgit; Fukami, Maki; Fricke-Otto, Susanne; Binder, Gerhard; Ogata, Tsutomu; Decker, Eva; Nuernberg, Gudrun; Hassel, David; Rappold, Gudrun A

    2016-12-01

    Mutations in the homeobox gene SHOX cause SHOX deficiency, a condition with clinical manifestations ranging from short stature without dysmorphic signs to severe mesomelic skeletal dysplasia. In rare cases, individuals with SHOX deficiency are asymptomatic. To elucidate the factors that modify disease severity/penetrance, we studied a three-generation family with SHOX deficiency. The variant p.Phe508Cys of the retinoic acid catabolizing enzyme CYP26C1 co-segregated with the SHOX variant p.Val161Ala in the affected individuals, while the SHOX mutant alone was present in asymptomatic individuals. Two further cases with SHOX deficiency and damaging CYP26C1 variants were identified in a cohort of 68 individuals with LWD The identified CYP26C1 variants affected its catabolic activity, leading to an increased level of retinoic acid. High levels of retinoic acid significantly decrease SHOX expression in human primary chondrocytes and zebrafish embryos. Individual morpholino knockdown of either gene shortens the pectoral fins, whereas depletion of both genes leads to a more severe phenotype. Together, our findings describe CYP26C1 as the first genetic modifier for SHOX deficiency. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  3. Enzymes involved in a novel anaerobic cyclohexane carboxylic acid degradation pathway.

    PubMed

    Kung, Johannes W; Meier, Anne-Katrin; Mergelsberg, Mario; Boll, Matthias

    2014-10-01

    The anaerobic degradation of cyclohexane carboxylic acid (CHC) has so far been studied only in Rhodopseudomonas palustris, in which CHC is activated to cyclohexanoyl coenzyme A (cyclohexanoyl-CoA [CHCoA]) and then dehydrogenated to cyclohex-1-ene-1-carboxyl-CoA (CHeneCoA). This intermediate is further degraded by reactions of the R. palustris-specific benzoyl-CoA degradation pathway of aromatic compounds. However, CHeneCoA is not an intermediate in the degradation of aromatic compounds in all other known anaerobic bacteria; consequently, degradation of CHC was mostly unknown in anaerobic bacteria. We identified a previously unknown CHC degradation pathway in the Fe(III)-reducing Geobacter metallireducens by determining the following CHC-induced in vitro activities: (i) the activation of CHC to CHCoA by a succinyl-CoA:CHC CoA transferase, (ii) the 1,2-dehydrogenation of CHCoA to CHeneCoA by CHCoA dehydrogenase, and (iii) the unusual 1,4-dehydrogenation of CHeneCoA to cyclohex-1,5-diene-1-carboxyl-CoA. This last represents a previously unknown joint intermediate of the CHC and aromatic compound degradation pathway in bacteria other than R. palustris. The enzymes catalyzing the three reactions were purified and characterized as specific enzymes after heterologous expression of the encoding genes. Quantitative reverse transcription-PCR revealed that expression of these genes was highly induced during growth with CHC but not with benzoate. The newly identified CHC degradation pathway is suggested to be present in nearly all CHC-degrading anaerobic bacteria, including denitrifying, Fe(III)-reducing, sulfate-reducing, and fermenting bacteria. Remarkably, all three CHC degradation pathways always link CHC catabolism to the catabolic pathways of aromatic compounds. We propose that the capacity to use CHC as a carbon source evolved from already-existing aromatic compound degradation pathways. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  4. Enzymes Involved in a Novel Anaerobic Cyclohexane Carboxylic Acid Degradation Pathway

    PubMed Central

    Kung, Johannes W.; Meier, Anne-Katrin; Mergelsberg, Mario

    2014-01-01

    The anaerobic degradation of cyclohexane carboxylic acid (CHC) has so far been studied only in Rhodopseudomonas palustris, in which CHC is activated to cyclohexanoyl coenzyme A (cyclohexanoyl-CoA [CHCoA]) and then dehydrogenated to cyclohex-1-ene-1-carboxyl-CoA (CHeneCoA). This intermediate is further degraded by reactions of the R. palustris-specific benzoyl-CoA degradation pathway of aromatic compounds. However, CHeneCoA is not an intermediate in the degradation of aromatic compounds in all other known anaerobic bacteria; consequently, degradation of CHC was mostly unknown in anaerobic bacteria. We identified a previously unknown CHC degradation pathway in the Fe(III)-reducing Geobacter metallireducens by determining the following CHC-induced in vitro activities: (i) the activation of CHC to CHCoA by a succinyl-CoA:CHC CoA transferase, (ii) the 1,2-dehydrogenation of CHCoA to CHeneCoA by CHCoA dehydrogenase, and (iii) the unusual 1,4-dehydrogenation of CHeneCoA to cyclohex-1,5-diene-1-carboxyl-CoA. This last represents a previously unknown joint intermediate of the CHC and aromatic compound degradation pathway in bacteria other than R. palustris. The enzymes catalyzing the three reactions were purified and characterized as specific enzymes after heterologous expression of the encoding genes. Quantitative reverse transcription-PCR revealed that expression of these genes was highly induced during growth with CHC but not with benzoate. The newly identified CHC degradation pathway is suggested to be present in nearly all CHC-degrading anaerobic bacteria, including denitrifying, Fe(III)-reducing, sulfate-reducing, and fermenting bacteria. Remarkably, all three CHC degradation pathways always link CHC catabolism to the catabolic pathways of aromatic compounds. We propose that the capacity to use CHC as a carbon source evolved from already-existing aromatic compound degradation pathways. PMID:25112478

  5. Non-growing Rhodopseudomonas palustris Increases the Hydrogen Gas Yield from Acetate by Shifting from the Glyoxylate Shunt to the Tricarboxylic Acid Cycle*

    PubMed Central

    McKinlay, James B.; Oda, Yasuhiro; Rühl, Martin; Posto, Amanda L.; Sauer, Uwe; Harwood, Caroline S.

    2014-01-01

    When starved for nitrogen, non-growing cells of the photosynthetic bacterium Rhodopseudomonas palustris continue to metabolize acetate and produce H2, an important industrial chemical and potential biofuel. The enzyme nitrogenase catalyzes H2 formation. The highest H2 yields are obtained when cells are deprived of N2 and thus use available electrons to synthesize H2 as the exclusive product of nitrogenase. To understand how R. palustris responds metabolically to increase H2 yields when it is starved for N2, and thus not growing, we tracked changes in biomass composition and global transcript levels. In addition to a 3.5-fold higher H2 yield by non-growing cells we also observed an accumulation of polyhydroxybutyrate to over 30% of the dry cell weight. The transcriptome of R. palustris showed down-regulation of biosynthetic processes and up-regulation of nitrogen scavenging mechanisms in response to N2 starvation but gene expression changes did not point to metabolic activities that could generate the reductant necessary to explain the high H2 yield. We therefore tracked 13C-labeled acetate through central metabolic pathways. We found that non-growing cells shifted their metabolism to use the tricarboxylic acid cycle to metabolize acetate in contrast to growing cells, which used the glyoxylate cycle exclusively. This shift enabled cells to more fully oxidize acetate, providing the necessary reducing power to explain the high H2 yield. PMID:24302724

  6. Effects of different dwarfing interstocks on key enzyme activities and the expression of genes related to malic acid metabolism in Red Fuji apples.

    PubMed

    Shi, J; Li, F F; Ma, H; Li, Z Y; Xu, J Z

    2015-12-22

    In this experiment, the test materials were 'Red Fuji' apple trees grafted onto three interstocks (No. 53, No. 111, and No. 236), which were chosen from SH40 seeding interstocks. The content of malic acid, the enzyme activities, and the expression of genes related to malic acid metabolism were determined during fruit development.The results showed that malic acid content in the ripe fruit on interstock No. 53 was higher than that in the interstock No. 111 fruit. The malate dehydrogenase (NAD-MDH) activity in apples on interstock No. 53 was highest on Day 30, Day 100, and Day 160 after bloom, and the malic enzyme (NADP-ME) activity in apples on interstock No. 111 was higher than in the interstock No. 53 fruit from Day 70 to Day 100 after bloom. The relative expression of NAD-MDH genes in interstock No. 53 fruit was higher than in No. 236 fruit on Day 100 after bloom, but the relative expression of NADP-ME in No. 236 interstock fruit was lower than in No. 53 fruit. The relative expression of NAD-MDH genes in No. 53 interstock fruit was highest on Day 160 after bloom. This might have been the main reason for the difference in the accumulation of malic acid in the ripe apples.There was a positive correlation between the relative expression of phosphoenolpyruvate carboxylase (PEPC) and the malic acid content of the fruit, and the content of malic acid in the apples was affected by the PEPC activity during the early developmental stage.

  7. Sodium phenylbutyrate decreases plasma branched-chain amino acids in patients with urea cycle disorders.

    PubMed

    Burrage, Lindsay C; Jain, Mahim; Gandolfo, Laura; Lee, Brendan H; Nagamani, Sandesh C S

    2014-01-01

    Sodium phenylbutyrate (NaPBA) is a commonly used medication for the treatment of patients with urea cycle disorders (UCDs). Previous reports involving small numbers of patients with UCDs have shown that NaPBA treatment can result in lower plasma levels of the branched-chain amino acids (BCAA) but this has not been studied systematically. From a large cohort of patients (n=553) with UCDs enrolled in the Longitudinal Study of Urea Cycle Disorders, a collaborative multicenter study of the Urea Cycle Disorders Consortium, we evaluated whether treatment with NaPBA leads to a decrease in plasma BCAA levels. Our analysis shows that NaPBA use independently affects the plasma BCAA levels even after accounting for multiple confounding covariates. Moreover, NaPBA use increases the risk for BCAA deficiency. This effect of NaPBA seems specific to plasma BCAA levels, as levels of other essential amino acids are not altered by its use. Our study, in an unselected population of UCD subjects, is the largest to analyze the effects of NaPBA on BCAA metabolism and potentially has significant clinical implications. Our results indicate that plasma BCAA levels should to be monitored in patients treated with NaPBA since patients taking the medication are at increased risk for BCAA deficiency. On a broader scale, these findings could open avenues to explore NaPBA as a therapy in maple syrup urine disease and other common complex disorders with dysregulation of BCAA metabolism. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Towards cell-free isobutanol production: Development of a novel immobilized enzyme system.

    PubMed

    Grimaldi, Joseph; Collins, Cynthia H; Belfort, Georges

    2016-01-01

    Producing fuels and chemical intermediates with cell cultures is severely limited by low product concentrations (≤0.2%(v/v)) due to feedback inhibition, cell instability, and lack of economical product recovery processes. We have developed an alternate simplified production scheme based on a cell-free immobilized enzyme system. Two immobilized enzymes (keto-acid decarboxylase (KdcA) and alcohol dehydrogenase (ADH)) and one enzyme in solution (formate dehydrogenase (FDH) for NADH recycle) produced isobutanol titers 8 to 20 times higher than the highest reported titers with S. cerevisiae on a mol/mol basis. These high conversion rates and low protein leaching were achieved by covalent immobilization of enzymes (ADH) and enzyme fusions (fKdcA) on methacrylate resin. The new enzyme system without in situ removal of isobutanol achieved a 55% conversion of ketoisovaleric acid to isobutanol at a concentration of 0.135 (mole isobutanol produced for each mole ketoisovaleric acid consumed). Further increasing titer will require continuous removal of the isobutanol using an in situ recovery system. © 2015 American Institute of Chemical Engineers.

  9. Conservation, fiber digestibility, and nutritive value of corn harvested at 2 cutting heights and ensiled with fibrolytic enzymes, either alone or with a ferulic acid esterase-producing inoculant.

    PubMed

    Lynch, J P; Baah, J; Beauchemin, K A

    2015-02-01

    The aim of this study was to determine the effects of the use of a fibrolytic enzyme product, applied at ensiling either alone or in combination with a ferulic acid esterase-producing bacterial additive, on the chemical composition, conservation characteristics, and in vitro degradability of corn silage harvested at either conventional or high cutting height. Triplicate samples of corn were harvested to leave stubble of either a conventional (15cm; NC) or high (45cm; HC) height above ground. Sub-samples of chopped herbage were ensiled untreated or with a fibrolytic enzyme product containing xylanases and cellulases applied either alone (ENZ) or in combination with a ferulic acid esterase-producing silage inoculant (ENZ+FAEI). The fibrolytic enzyme treatment was applied at 2mL of enzyme product/kg of herbage dry matter (DM), and the inoculant was applied at 1.3×10(5) cfu/g of fresh herbage. Samples were packed into laboratory-scale silos, stored for 7, 28, or 70 d, and analyzed for fermentation characteristics, and samples ensiled for 70 d were also analyzed for DM losses, chemical composition, and in vitro ruminal degradability. After 70 d of ensiling, the fermentation characteristics of corn silages were generally unaffected by cutting height, whereas the neutral detergent fiber, acid detergent fiber, and ash concentrations were lower and the starch concentration greater for silages made with crops harvested at HC compared with NC. After 70 d of ensiling, the acetic acid, ethanol concentrations, and the number of yeasts were greater, and the pH and neutral detergent fiber concentrations were lower, in silages produced using ENZ or ENZ+FAEI than the untreated silages, whereas ENZ+FAEI silages also incurred higher DM losses. No effect of additive treatment was observed on in vitro degradability indices after 48h ruminal incubation. The use of a fibrolytic enzyme product, either alone or in combination with a ferulic acid esterase-producing inoculant, at ensiling

  10. Identification and Characterization of the Genes and Enzymes Belonging to the Bile Acid Catabolic Pathway in Pseudomonas.

    PubMed

    Luengo, José M; Olivera, Elías R

    2017-01-01

    The study of the catabolic potential of microbial species isolated from different habitats has allowed the identification and characterization of bacteria able to assimilate bile acids and other steroids (e.g., testosterone and 4-androsten-3,17-dione). From soil samples, we have isolated several strains belonging to genus Pseudomonas that grow efficiently in chemical defined media containing some cyclopentane-perhydro-phenantrene derivatives as carbon sources. Genetic and biochemical studies performed with one of these bacteria (P. putida DOC21) allowed the identification of the genes and enzymes belonging to the 9,10-seco pathway, the route involved in the aerobic assimilation of steroids. In this manuscript, we describe the most relevant methods required for (1) isolation and characterization of these species; (2) determining the chromosomal location, nucleotide sequence, and functional analysis of the catabolic genes (or gene clusters) encoding the enzymes from this pathway; and (3) the tools employed to establish the role of some of the proteins that participate in this route.

  11. The dimerization domain in DapE enzymes is required for catalysis.

    PubMed

    Nocek, Boguslaw; Starus, Anna; Makowska-Grzyska, Magdalena; Gutierrez, Blanca; Sanchez, Stephen; Jedrzejczak, Robert; Mack, Jamey C; Olsen, Kenneth W; Joachimiak, Andrzej; Holz, Richard C

    2014-01-01

    The emergence of antibiotic-resistant bacterial strains underscores the importance of identifying new drug targets and developing new antimicrobial compounds. Lysine and meso-diaminopimelic acid are essential for protein production and bacterial peptidoglycan cell wall remodeling and are synthesized in bacteria by enzymes encoded within dap operon. Therefore dap enzymes may serve as excellent targets for developing a new class of antimicrobial agents. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) converts N-succinyl-L,L-diaminopimelic acid to L,L-diaminopimelic acid and succinate. The enzyme is composed of catalytic and dimerization domains, and belongs to the M20 peptidase family. To understand the specific role of each domain of the enzyme we engineered dimerization domain deletion mutants of DapEs from Haemophilus influenzae and Vibrio cholerae, and characterized these proteins structurally and biochemically. No activity was observed for all deletion mutants. Structural comparisons of wild-type, inactive monomeric DapE enzymes with other M20 peptidases suggest that the dimerization domain is essential for DapE enzymatic activity. Structural analysis and molecular dynamics simulations indicate that removal of the dimerization domain increased the flexibility of a conserved active site loop that may provide critical interactions with the substrate.

  12. Modifications of hepatic drug metabolizing enzyme activities in rats fed baobab seed oil containing cyclopropenoid fatty acids.

    PubMed

    Andrianaivo-Rafehivola, A A; Siess, M H; Gaydou, E M

    1995-05-01

    The effects on drug metabolizing enzymes of cyclopropenoid fatty acids present in baobab seed oil were evaluated in rats fed either a diet with baobab seed oil (1.27% cyclopropenoid fatty acids in the diet) or a diet with heated baobab seed oil (0.046% cyclopropenoid fatty acids in the diet). Comparison was made with rats fed a mixture of oils that contained no cyclopropenoid fatty acid. Rats fed baobab oil showed retarded growth. In comparison with the other groups, the relative liver weights were markedly increased whereas cytochrome P-450 content and NADPH cytochrome c reductase and NADH cytochrome c reductase activities were decreased. In rats fed the heated baobab oil the relative liver weight was decreased and the cytochrome P-450 level and reductase activities were increased relative to levels in rats fed the unheated oil. Ethoxycoumarin deethylase, ethoxyresorufin deethylase and pentoxyresorufin depentylase activities, expressed on the basis of cytochrome P-450, were greater in the group fed unheated baobab seed oil. Cytosolic glutathione transferase activity was markedly decreased in rats fed fresh baobab seed oil and heating the oil, which reduced the content of cyclopropenoid fatty acids, led to a considerable increase of this activity. UDP-glucuronyl transferase activities were not modified by the type of oil included in the diet. It is possible that the mechanisms of action of cyclopropenoid fatty acids are related to alterations of membrane lipid composition or microsomal proteins.

  13. Ketoacylsynthase Domains of a Polyunsaturated Fatty Acid Synthase in Thraustochytrium sp. Strain ATCC 26185 Can Effectively Function as Stand-Alone Enzymes in Escherichia coli.

    PubMed

    Xie, Xi; Meesapyodsuk, Dauenpen; Qiu, Xiao

    2017-05-01

    Thraustochytrium sp. strain ATCC 26185 accumulates a high level of docosahexaenoic acid (DHA), a nutritionally important ω-3 very-long-chain polyunsaturated fatty acid (VLCPUFA) synthesized primarily by polyunsaturated fatty acid (PUFA) synthase, a type I polyketide synthase-like megaenzyme. The PUFA synthase in this species comprises three large subunits, each with multiple catalytic domains. It was hypothesized that among these domains, ketoacylsynthase (KS) domains might be critical for catalyzing the condensation of specific unsaturated acyl-acyl carrier proteins (ACPs) with malonyl-ACP, thereby retaining double bonds in an extended acyl chain. To investigate the functions of these putative KS domains, two segment sequences from subunit A (KS-A) and subunit B (KS-B) of the PUFA synthase were dissected and then expressed as stand-alone enzymes in Escherichia coli The results showed that both KS-A and KS-B domains could complement the defective phenotypes of both E. coli fabB and fabF mutants. Overexpression of these domains in wild-type E. coli led to increases in total fatty acid production. KS-B produced a higher ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs), while KS-A could improve the overall production of fatty acids more effectively, particularly for the production of SFAs, implying that KS-A is more comparable to FabF, while KS-B is more similar to FabB in catalytic functions. Successful complementation and functional expression of the embedded KS domains in E. coli are the first step forward in studying the molecular mechanism of the PUFA synthase for the biosynthesis of VLCPUFAs in Thraustochytrium IMPORTANCE Very-long-chain polyunsaturated fatty acids (VLCPUFAs) are important for human health. They can be biosynthesized in either an aerobic pathway or an anaerobic pathway in nature. However, abundant VLCPUFAs in marine microorganisms are primarily synthesized by polyunsaturated fatty acid (PUFA) synthase, a megaenzyme with

  14. Ketoacylsynthase Domains of a Polyunsaturated Fatty Acid Synthase in Thraustochytrium sp. Strain ATCC 26185 Can Effectively Function as Stand-Alone Enzymes in Escherichia coli

    PubMed Central

    Xie, Xi; Meesapyodsuk, Dauenpen

    2017-01-01

    ABSTRACT Thraustochytrium sp. strain ATCC 26185 accumulates a high level of docosahexaenoic acid (DHA), a nutritionally important ω-3 very-long-chain polyunsaturated fatty acid (VLCPUFA) synthesized primarily by polyunsaturated fatty acid (PUFA) synthase, a type I polyketide synthase-like megaenzyme. The PUFA synthase in this species comprises three large subunits, each with multiple catalytic domains. It was hypothesized that among these domains, ketoacylsynthase (KS) domains might be critical for catalyzing the condensation of specific unsaturated acyl-acyl carrier proteins (ACPs) with malonyl-ACP, thereby retaining double bonds in an extended acyl chain. To investigate the functions of these putative KS domains, two segment sequences from subunit A (KS-A) and subunit B (KS-B) of the PUFA synthase were dissected and then expressed as stand-alone enzymes in Escherichia coli. The results showed that both KS-A and KS-B domains could complement the defective phenotypes of both E. coli fabB and fabF mutants. Overexpression of these domains in wild-type E. coli led to increases in total fatty acid production. KS-B produced a higher ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs), while KS-A could improve the overall production of fatty acids more effectively, particularly for the production of SFAs, implying that KS-A is more comparable to FabF, while KS-B is more similar to FabB in catalytic functions. Successful complementation and functional expression of the embedded KS domains in E. coli are the first step forward in studying the molecular mechanism of the PUFA synthase for the biosynthesis of VLCPUFAs in Thraustochytrium. IMPORTANCE Very-long-chain polyunsaturated fatty acids (VLCPUFAs) are important for human health. They can be biosynthesized in either an aerobic pathway or an anaerobic pathway in nature. However, abundant VLCPUFAs in marine microorganisms are primarily synthesized by polyunsaturated fatty acid (PUFA) synthase, a

  15. [Effects of different tillage methods on phospholipid fatty acids and enzyme activities in calcareous cinnamon soil].

    PubMed

    Pei, Xue-Xia; Dang, Jian-You; Zhang, Ding-Yi; Wang, Jiao-Ai; Zhang, Jing

    2014-08-01

    In order to study changes of physical and chemical characteristics and microbial activities in soil under different tillage methods, effects of four tillage methods, rotary tillage (RT), subsoil tillage (ST), conventional tillage (CT) with corn straw returned to soil, and rotary tillage with no corn straw returned to soil (CK), on phospholipid fatty acids (PLFA) characteristics and hydrolase enzymes activities in calcareous cinnamon soil were investigated. The results showed that soil hydrolase enzymes activities, nutrient contents, microbial diversity varied greatly with the different tillage methods. Returning corn straw to soil increased the kinds, amount of soil total PLFAs, bacteria PLFAs and actonomycetes PLFAs, while decreased the fungi PLFAs, indicating that fungi was more adaptable than bacteria to an infertile environment. ST and CT resulted in higher amounts of total PLFAs, which were 74.7% and 53.3% higher than that of CK, indicating they were more beneficial to the growth of plants. They could also improve soil physical and chemical properties, increase alk-phosphatase, protease and urease activities, which would provide a favorable soil condition for high and stable crop yields.

  16. Structure of the Mitochondrial Aminolevulinic Acid Synthase, a Key Heme Biosynthetic Enzyme.

    PubMed

    Brown, Breann L; Kardon, Julia R; Sauer, Robert T; Baker, Tania A

    2018-04-03

    5-Aminolevulinic acid synthase (ALAS) catalyzes the first step in heme biosynthesis. We present the crystal structure of a eukaryotic ALAS from Saccharomyces cerevisiae. In this homodimeric structure, one ALAS subunit contains covalently bound cofactor, pyridoxal 5'-phosphate (PLP), whereas the second is PLP free. Comparison between the subunits reveals PLP-coupled reordering of the active site and of additional regions to achieve the active conformation of the enzyme. The eukaryotic C-terminal extension, a region altered in multiple human disease alleles, wraps around the dimer and contacts active-site-proximal residues. Mutational analysis demonstrates that this C-terminal region that engages the active site is important for ALAS activity. Our discovery of structural elements that change conformation upon PLP binding and of direct contact between the C-terminal extension and the active site thus provides a structural basis for investigation of disruptions in the first step of heme biosynthesis and resulting human disorders. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. Development of an indirect enzyme linked immunoassay for abscisic acid. [Pisum sativum

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ross, G.S.; Elder, P.A.; McWha, J.A.

    1987-09-01

    AN INDIRECT METHOD OF ENZYME-LINKED-IMMUNOSORBENT-ASSAY (ELISA) IS REPORTED FOR ABSCISIC ACID (ABA), UTILIZING A THYROGLOBULIN-ABA CONJUGATE FOR COATING WELLS. THE ASSAY CAN USE COMMERCIALLY AVAILABLE MONOCLONAL ANTIBODIES, IS SENSITIVE TO AS LITTLE AS 20 PICOGRAMS ABA PER WELL, AND IS MUCH MORE CONSERVATIVE OF ANTIBODY THAN DIRECT METHODS. THE MOST DILUTE ABA STANDARDS DID NOT RETAIN THEIR ANTIGENICITY DURING STORAGE, SO ABA STANDARD SETS WERE DILUTED IMMEDIATELY PRIOR TO USE. THE INDIRECT ELISA WAS USED SUCCESSFULLY TO ESTIMATE ABA CONCENTRATIONS IN DEVELOPING COTYLEDONS OF PISUM SATIVUM L., AFTER ONLY LITTLE PRELIMINARY PURIFICATION. IT WAS VALIDATED FOR THIS TISSUE THROUGH THEmore » USE OF GAS CHROMATOGRAPHY-ELECTRON CAPTURE DETECTION (GC-EC), AND CAPILLARY GC-SELECTED ION MONITORING (GC-MS-SIM) USING LABELLED ABA AS AN INTERNAL STANDARD. FULL SPECTRUM GC-MASS SPECTROMETRY WAS ALSO USED TO VERIFY THAT ABA WAS PRESENT IN A SAMPLE ASSAYED QUANTITATIVELY BY BOTH ELISA AND GC-MS-SIM.« less

  18. Appearance and accumulation of C/sub 4/ carbon pathway enzymes in developing maize leaves and differentiating maize A188 callus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aoyagi, K.; Bassham, J.A.

    1986-02-01

    Regenerating maize A188 tissue cultures were examined for the presence of enzymes involved in C/sub 4/ photosynthesis, for cell morphology, and for /sup 14/C labeling kinetics to study the implementation of this pathway during plant development. For comparison, sections of maize seedling leaves were examined. Protein blot analysis using antibodies to leaf enzymes showed a different profile of these enzymes during the early stages of shoot regeneration from callus from the closely-coordinated profile observed in seedling leaves. Pyruvate orthophosphate dikinase (PPDK) (EC 2.7.9.1) and phosphoenolpyruvate carboxylase (PEPC) (EC 4.1.1.31) were found in nonchlorophyllous callus while ribulose 1,5-bisphosphate carboxylase (RuBPC, ECmore » 4.1.1.39) and malic enzyme, NADP-specific (ME-NADP) (EC 1.3.1.37) were not detectable until later. Enzyme activity assays showed the presence of ME-NADP as well as PEPC and PPDK in nonchlorophyllous callus. However, the activities of ME-NADP and PEPC had properties similar to those of the enzymes from C/sub 3/ leaves and from etiolated C/sub 4/ leaf tissues, but differing from the corresponding enzymes in the mature leaf. Immunoprecipitation of in vitro translation products of poly(A)RNA extracted from embryoid-forming callus showed both the 110 kilodalton precursor to chloroplast PPDK and the 94 kilodalton polypeptide. Therefore, the chloroplast tye of PPDK mRNA is present prior to the appearance of leaf morphology. Analysis of the labeled products of /sup 14/CO/sub 2/ fixation by nonchlorophyllous calli indicated ..beta..-carboxylation to give acids of the tricarboxylic acid cycle, but no incorporation into phosphoglycerate. With greening of the callus, some incorporation into phosphoglycerate and sugar phosphates occurred, and this increased in shoots as they developed, although with older shoots the increase in ..beta..-carboxylation products was even greater.« less

  19. Self-organizing biochemical cycles

    NASA Technical Reports Server (NTRS)

    Orgel, L. E.; Bada, J. L. (Principal Investigator)

    2000-01-01

    I examine the plausibility of theories that postulate the development of complex chemical organization without requiring the replication of genetic polymers such as RNA. One conclusion is that theories that involve the organization of complex, small-molecule metabolic cycles such as the reductive citric acid cycle on mineral surfaces make unreasonable assumptions about the catalytic properties of minerals and the ability of minerals to organize sequences of disparate reactions. Another conclusion is that data in the Beilstein Handbook of Organic Chemistry that have been claimed to support the hypothesis that the reductive citric acid cycle originated as a self-organized cycle can more plausibly be interpreted in a different way.

  20. Targeted metabolomics connects thioredoxin-interacting protein (TXNIP) to mitochondrial fuel selection and regulation of specific oxidoreductase enzymes in skeletal muscle.

    PubMed

    DeBalsi, Karen L; Wong, Kari E; Koves, Timothy R; Slentz, Dorothy H; Seiler, Sarah E; Wittmann, April H; Ilkayeva, Olga R; Stevens, Robert D; Perry, Christopher G R; Lark, Daniel S; Hui, Simon T; Szweda, Luke; Neufer, P Darrell; Muoio, Deborah M

    2014-03-21

    Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIP(SKM-/-)) Txnip deficiency. Compared with littermate controls, both TKO and TXNIP(SKM-/-) mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of β-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability.