Science.gov

Sample records for acetyl coa hydrolysis

  1. Mitochondrial storage form of acetyl CoA carboxylase in fasted and alloxan diabetic rats

    SciTech Connect

    Roman-Lopez, C.R.; Allred, J.B.

    1986-05-01

    Sodium dodecyl sulfate-denatured biotinyl proteins will bind (/sup 14/C)methyl avidin which remains bound through polyacrylamide gel electrophoresis. The method has been used to demonstrate the presence of two high molecular weight subunit forms of acetyl CoA carboxylase in rat liver cytoplasm, both of which are precipitated by antibody to purifed rat liver acetyl CoA carboxylase prepared from sheep serum. Rat liver mitochondria contained five distinct biotinyl protein subunits, the two largest of which have been identified as acetyl CoA carboxylase subunits on the basis of precipitation by anti-acetyl CoA carboxylase antibody. The small quantity of acetyl CoA carboxylase associated with rat liver microsomes could be attributed to cytoplasmic contamination. The binding of radioactive avidin is sufficiently tight to use as a measure of the quantity of acetyl CoA carboxylase. The quantity and activity of the cytoplasmic enzyme was reduced in fasted and in alloxan diabetic rats compared to that in fed controls but the quantity of the enzyme associated with isolated mitochondria was not reduced. The results indicate that there is a mitochondrial storage form of acetyl CoA carboxylase.

  2. ACETYL COA CARBOXYLASE, II. DEMONSTRATION OF BIOTIN-PROTEIN AND BIOTIN CARBOXYLASE SUBUNITS*

    PubMed Central

    Alberts, Alfred W.; Nervi, A. M.; Vagelos, P. R.

    1969-01-01

    Previous work has shown that Escherichia coli acetyl CoA carboxylase is composed of two dissimilar protein components, Ea which contains covalently bound biotin and forms Ea-CO2-from HCO3- and ATP, and Eb which is involved in the transfer of the carboxyl group from Ea-CO2- to acetyl CoA, forming malonyl CoA. Ea has been dissociated into two subunits at pH 9. One subunit, designated biotin carboxylase, catalyzes a model reaction, the ATP-dependent carboxylation of free (+)-biotin. The other subunit contains covalently bound which is carboxylated by the biotin carboxylase in the course of acetyl CoA carboxylation. PMID:4901473

  3. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Schnable, Patrick S.; Wen, Tsui-Jung

    2009-04-28

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.alpha. subunit of pPDH, the E1.beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.alpha. pPDH, E1.beta. pPDH, E2 pPDH, mtPDH or ALDH.

  4. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Behal, Robert; Schnable, Patrick S.; Ke, Jinshan; Johnson, Jerry L.; Allred, Carolyn C.; Fatland, Beth; Lutziger, Isabelle; Wen, Tsui-Jung

    2005-09-13

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.alpha. subunit of pPDH, the E1.beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyruvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.alpha. pPDH, E1.beta. pPDH, E2 pPDH, mtPDH or ALDH.

  5. Materials and methods for the alteration of enzyme and acetyl CoA levels in plants

    DOEpatents

    Nikolau, Basil J.; Wurtele, Eve S.; Oliver, David J.; Behal, Robert; Schnable, Patrick S.; Ke, Jinshan; Johnson, Jerry L.; Allred, Carolyn C.; Fatland, Beth; Lutziger, Isabelle; Wen, Tsui-Jung

    2004-07-20

    The present invention provides nucleic acid and amino acid sequences of acetyl CoA synthetase (ACS), plastidic pyruvate dehydrogenase (pPDH), ATP citrate lyase (ACL), Arabidopsis pyruvate decarboxylase (PDC), and Arabidopsis aldehyde dehydrogenase (ALDH), specifically ALDH-2 and ALDH-4. The present invention also provides a recombinant vector comprising a nucleic acid sequence encoding one of the aforementioned enzymes, an antisense sequence thereto or a ribozyme therefor, a cell transformed with such a vector, antibodies to the enzymes, a plant cell, a plant tissue, a plant organ or a plant in which the level of an enzyme has been altered, and a method of producing such a plant cell, plant tissue, plant organ or plant. Desirably, alteration of the level of enzyme results in an alteration of the level of acetyl CoA in the plant cell, plant tissue, plant organ or plant. In addition, the present invention provides a recombinant vector comprising an antisense sequence of a nucleic acid sequence encoding pyruvate decarboxylase (PDC), the E1.sub..alpha. subunit of pPDH, the E1.sub..beta. subunit of pPDH, the E2 subunit of pPDH, mitochondrial pyurvate dehydrogenase (mtPDH) or aldehyde dehydrogenase (ALDH) or a ribozyme that can cleave an RNA molecule encoding PDC, E1.sub..alpha. pPDH, E1.sub..beta. pPDH, E2 pPDH, mtPDH or ALDH.

  6. Leucine-684: A conserved residue of an AMP-acetyl CoA synthetase (AceCS) from Leishmania donovani is involved in substrate recognition, catalysis and acetylation.

    PubMed

    Soumya, Neelagiri; Tandan, Hitendra; Damre, Mangesh V; Gangwal, Rahul P; Sangamwar, Abhay T; Singh, Sushma

    2016-04-15

    AMP-acetyl CoA synthetase (AMP-AceCS) is a key enzyme which catalyzes the activation of acetate to acetyl CoA, an important intermediate at the cross roads of various anabolic and catabolic pathways. Multiple sequence alignment of Leishmania donovani AceCS with other organisms revealed the presence of a highly conserved leucine residue at 684 position which is known to be crucial for acetylation by protein acetyl transferases in other organisms. In an attempt to understand the role of leucine residue at 684 position in L. donovani acetyl CoA synthetase (LdAceCS), it was mutated to proline (P) by site directed mutagenesis. Kinetic analysis of the L684P-LdAceCS mutant revealed approximately two fold increased binding affinity with acetate, whereas fivefold decreased affinity was observed with ATP. There was insignificant change in secondary structure as revealed by CD however, two fold decreased fluorescence intensity was observed at an emission maxima of 340nm. Interestingly, L684P mutation abolished the acetylation of the mutant enzyme indicating the importance of L684 in acetylation of the enzyme. Changes in biochemical parameters of the mutant protein were validated by homology modeling of the wild type and mutant LdAceCS enzyme using Salmonella enterica AceCS crystal structure as template. Our data provides evidence for the role of leucine 684 residue in substrate recognition, catalysis and acetylation of the AceCS enzyme. PMID:26794803

  7. Expression of a yeast acetyl CoA hydrolase in the mitochondrion of tobacco plants inhibits growth and restricts photosynthesis.

    PubMed

    Bender-Machado, Lilia; Bäuerlein, Michael; Carrari, Fernando; Schauer, Nicolas; Lytovchenko, Anna; Gibon, Yves; Kelly, Amelie A; Loureiro, Marcello; Müller-Röber, Bernd; Willmitzer, Lothar; Fernie, Alisdair R

    2004-07-01

    Acetyl Coenzyme A (acetyl CoA) is required in the mitochondria to fuel the operation of the Krebs cycle and within the cytosolic, peroxisomal and plastidial compartments wherein it acts as the immediate precursor for a wide range of anabolic functions. Since this metabolite is impermeable to membranes it follows that discrete pathways both for its synthesis and for its utilization must be present in each of these organelles and that the size of the various compartmented pools are independently regulated. To determine the specific role of acetyl CoA in the mitochondria we exploited a transgenic approach to introduce a yeast acetyl CoA hydrolase (EC 3.1.2.1.) into this compartment in tobacco plants. Despite the facts that the introduced enzyme was correctly targeted and that there were marked reductions in the levels of citrate and malate and an increase in the acetate content of the transformants, the transgenic plants surprisingly exhibited increased acetyl CoA levels. The lines were further characterised by a severe growth retardation, abnormal leaf colouration and a dramatic reduction in photosynthetic activity correlated with a marked reduction in the levels of transcripts of photosynthesis and in the content of photosynthetic pigments. The altered rate of photosynthesis in the transgenics was also reflected by a modified carbon partitioning in leaves of these lines, however, further studies revealed that this was most likely caused by a decreased source to sink transport of carbohydrate. In summary these results suggest that the content of acetyl CoA is under tight control and that alterations in the level of this central metabolite have severe metabolic and developmental consequences in tobacco. PMID:15604707

  8. Acetyl CoA Carboxylase: Isolation and Characterization of Native Biotin Carboxyl Carrier Protein

    PubMed Central

    Fall, R. Ray; Nervi, A. M.; Alberts, Alfred W.; Vagelos, P. Roy

    1971-01-01

    A large form of biotin carboxyl carrier protein (BCCPL) has been isolated from extracts of Escherichia coli. It has a minimal molecular weight of 20,000, according to its behavior on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and contains approximately 1 mol of biotin per 22,000 g of protein. BCCPL exhibits Km values, in the biotin carboxylase and transcarboxylase half-reactions of acetyl CoA carboxylase, of 2 10-7 M and 4 10-7 M, respectively; these values are 50-100 times lower than those obtained with smaller forms of BCCP previously isolated. Electrophoresis of crude extracts of E. coli indicates that the major biotin-containing protein migrates at the same rate as BCCPL, which suggests that BCCPL is the native form of BCCP in E. coli. Images PMID:4934522

  9. Role of CoA and acetyl-CoA in regulating cardiac fatty acid and glucose oxidation.

    PubMed

    Abo Alrob, Osama; Lopaschuk, Gary D

    2014-08-01

    CoA (coenzyme A) and its derivatives have a critical role in regulating cardiac energy metabolism. This includes a key role as a substrate and product in the energy metabolic pathways, as well as serving as an allosteric regulator of cardiac energy metabolism. In addition, the CoA ester malonyl-CoA has an important role in regulating fatty acid oxidation, secondary to inhibiting CPT (carnitine palmitoyltransferase) 1, a key enzyme involved in mitochondrial fatty acid uptake. Alterations in malonyl-CoA synthesis by ACC (acetyl-CoA carboxylase) and degradation by MCD (malonyl-CoA decarboxylase) are important contributors to the high cardiac fatty acid oxidation rates seen in ischaemic heart disease, heart failure, obesity and diabetes. Additional control of fatty acid oxidation may also occur at the level of acetyl-CoA involvement in acetylation of mitochondrial fatty acid β-oxidative enzymes. We find that acetylation of the fatty acid β-oxidative enzymes, LCAD (long-chain acyl-CoA dehydrogenase) and β-HAD (β-hydroxyacyl-CoA dehydrogenase) is associated with an increase in activity and fatty acid oxidation in heart from obese mice with heart failure. This is associated with decreased SIRT3 (sirtuin 3) activity, an important mitochondrial deacetylase. In support of this, cardiac SIRT3 deletion increases acetylation of LCAD and β-HAD, and increases cardiac fatty acid oxidation. Acetylation of MCD is also associated with increased activity, decreases malonyl-CoA levels and an increase in fatty acid oxidation. Combined, these data suggest that malonyl-CoA and acetyl-CoA have an important role in mediating the alterations in fatty acid oxidation seen in heart failure. PMID:25110000

  10. Effect of alterations of the specific activity of the intracellular acetyl CoA pool on apparent rates of hepatic cholesterogenesis.

    PubMed

    Dietschy, J M; Brown, M S

    1974-09-01

    We have previously shown significant dilution of the specific activity of the intracellular acetyl CoA pool when radiolabeled acetate is used as the precursor in liver slice experiments. In the present study, using liver from animals subjected to various manipulations known to alter the rate of cholesterogenesis, the specific activity of the intramitochondrial acetyl CoA pool was 27-49% of the theoretical specific activity expected if no endogenous dilution occurred. Because the cytosolic acetyl CoA pool that gives rise to cholesterol is not in equilibrium with the intramitochondrial pool, these values cannot be used to correct the flux of labeled carbon from [(14)C]acetate into cholesterol. However, because [(14)C]octanoate is rapidly oxidized intramitochondrially to acetyl CoA, which feeds both the intra- and extramitochondrial metabolic pathways, [(14)C]octanoate can be utilized to determine true flux rates of C(2) units into cholesterol and other products. Using this substrate in liver slices from animals subjected to a variety of experimental manipulations, the specific activity of the intracellular acetyl CoA pool was 54-71% of the expected specific activity. After correction for endogenous dilution, the C(2) flux into cholesterol varied from 335 to 459 nmoles.g(-1).hr(-1) in control animals, was suppressed 10-40-fold in animals subjected to fasting and cholesterol feeding, and increased into the range of 1500 nmoles.g(-1).hr(-1) after derepression with cholestyramine feeding or biliary diversion. Data also are presented that show very good agreement between the corrected C(2) flux rate from octanoate into cholesterol and microsomal HMG CoA reductase activity in the same liver under conditions in which the synthetic rates were varied over a 100-fold range. PMID:4413018

  11. The susceptibility of N-acetyl-S-alkyl- and N-acetyl-S-aryl-cysteine ethyl esters to chymotryptic hydrolysis

    PubMed Central

    Damoglou, A. P.; Lindley, H.; Stapleton, I. W.

    1970-01-01

    1. The preparation of the ethyl esters of some N-acetyl-S-alkyl- and N-acetyl-S-aryl-cysteine derivatives is described. 2. Kinetic parameters for the hydrolysis of these esters by chymotrypsin are reported. 3. The results are discussed in terms of the mechanism of action of chymotrypsin and also with reference to the possible application to sequence studies of cysteine-containing proteins. PMID:5529715

  12. A dinuclear nickel complex modeling of the Ni(d)(II)-Ni(p)(I) state of the active site of acetyl CoA synthase.

    PubMed

    Matsumoto, Tsuyoshi; Ito, Mikinao; Kotera, Mai; Tatsumi, Kazuyuki

    2010-03-28

    The dinuclear Ni(II)-Ni(I) complex Ni(II)(dadt(Et))Ni(I)(SDmp)(PPh(3)) was synthesized as a Ni(II)(d)-Ni(I)(p) model of the A-cluster in acetyl CoA synthase. This complex was reacted with Co(dmgBF(2))(2)(Me)(Py) and KSDmp successively to afford Ni(dadt(Et))Ni(Me)(SDmp), which further reacts with CO to afford the acetylthioester CH(3)C(O)SDmp via reductive elimination. PMID:20221531

  13. Chromium downregulates the expression of Acetyl CoA Carboxylase 1 gene in lipogenic tissues of domestic goats: a potential strategy for meat quality improvement.

    PubMed

    Najafpanah, Mohammad Javad; Sadeghi, Mostafa; Zali, Abolfazl; Moradi-Shahrebabak, Hossein; Mousapour, Hojatollah

    2014-06-15

    Acetyl CoA Carboxylase 1 (ACC1) is a biotin-dependent enzyme that catalyzes the carboxylation of Acetyl CoA to form Malonyl CoA, the key intermediate metabolite in fatty acid synthesis. In this study, the mRNA expression of the ACC1 gene was evaluated in four different tissues (liver, visceral fat, subcutaneous fat, and longissimus muscle) of the domestic goat (Capra hircus) kids feeding on four different levels of trivalent chromium (0, 0.5, 1, and 1.5mg/day) as food supplementation. RT-qPCR technique was used for expression analyses and heat shock protein 90 gene (HSP-90) was considered as reference gene for data normalization. Our results revealed that 1.5mg/day chromium significantly reduced the expression of the ACC1 gene in liver, visceral fat, and subcutaneous fat tissues, but not in longissimus muscles (P<0.05). We measured some phenotypic traits of kid's carcasses to detect their probable correlations with chromium-mediated downregulation of ACC1 expression. Interestingly, changes in ACC1 expression were accompanied with decreased accumulation of fats in adipose tissues such that the subcutaneous fat thickness and heart fat percentage decreased in kids feeding on chromium. By contrast, chromium supplemented kids showed higher percentage of muscles despite the fact that their total body weight did not differ from that of non-supplemented kids. Our study suggests that trivalent chromium alters the direction of energy accumulation towards muscles rather than fats and provides insights into application of chromium supplementation as a useful strategy for improvement of meat quality in domestic animals. PMID:24704275

  14. The role of acetyl xylan esterase in the solubilization of xylan and enzymatic hydrolysis of wheat straw and giant reed

    PubMed Central

    2011-01-01

    Background Due to the complexity of lignocellulosic materials, a complete enzymatic hydrolysis into fermentable sugars requires a variety of cellulolytic and xylanolytic enzymes. Addition of xylanases has been shown to significantly improve the performance of cellulases and to increase cellulose hydrolysis by solubilizing xylans in lignocellulosic materials. The goal of this work was to investigate the effect of acetyl xylan esterase (AXE) originating from Trichoderma reesei on xylan solubilization and enzymatic hydrolysis of cellulose. Results The solubilization of xylan in pretreated wheat straw and giant reed (Arundo donax) by xylanolytic enzymes and the impact of the sequential or simultaneous solubilization of xylan on the hydrolysis of cellulose by purified enzymes were investigated. The results showed that the removal of acetyl groups in xylan by AXE increased the accessibility of xylan to xylanase and improved the hydrolysis of xylan in pretreated wheat straw and giant reed. Solubilization of xylan led to an increased accessibility of cellulose to cellulases and thereby increased the hydrolysis extent of cellulose. A clear synergistic effect between cellulases and xylanolytic enzymes was observed. The highest hydrolysis yield of cellulose was obtained with a simultaneous use of cellulases, xylanase and AXE, indicating the presence of acetylated xylan within the cellulose matrix. Acetylated xylobiose and acetylated xylotriose were produced from xylan without AXE, as confirmed by atmospheric pressure matrix-assisted laser desorption/ionization ion trap mass spectrometry. Conclusions The results in this paper demonstrate that supplementation of xylanase with AXE enhances the solubilization of xylan to some extent and, consequently, increases the subsequent hydrolysis of cellulose. The highest hydrolysis yield was, however, obtained by simultaneous hydrolysis of xylan and cellulose, indicating a layered structure of cellulose and xylan chains in the cell wall substrate. AXE has an important role in the hydrolysis of lignocellulosic materials containing acetylated xylan. PMID:22185437

  15. Heat activation of rat epididymal fat tissue acetyl-coa carboxylase is due to dephosphorylation by its endogenous phosphatase.

    PubMed

    Krakower, G R; Kim, K H

    1980-12-01

    Acetyl-CoA carboxylase from rat epididymal fat tissue is activated by incubation at 30 C in the absence of citrate or metal ions. This activation is accompanied by a corresponding loss of 32P from the labeled enzyme, and it is not blocked by the heat-stable phosphorylase phosphatase inhibitor proteins from rabbit muscle. We have succeeded in separating an activity which activates and dephosphorylates acetyl-CoA carboxylase from the carboxylase using polyethylene glycol-6000. These results suggest that the temperature-dependent activation of acetyl-CoA carboxylase in crude or partially purified preparations results from dephosphorylation of the carboxylase by bound phosphatase. PMID:6111734

  16. Properties of retrograded and acetylated starch produced via starch extrusion or starch hydrolysis with pullulanase.

    PubMed

    Kapelko, M; Zięba, T; Gryszkin, A; Styczyńska, M; Wilczak, A

    2013-09-12

    The aim of the present study was to determine the impact of serial modifications of starch, including firstly starch extrusion or hydrolysis with pullulanase, followed by retrogradation (through freezing and defrosting of pastes) and acetylation (under industrial conditions), on its susceptibility to amylolysis. The method of production had a significant effect on properties of the resultant preparations, whilst the direction and extent of changes depended on the type of modification applied. In the produced starch esters, the degree of substitution, expressed by the per cent of acetylation, ranged from 3.1 to 4.4 g/100 g. The acetylation had a significant impact on contents of elements determined with the atomic emission spectrometry, as it contributed to an increased Na content and decreased contents of Ca and K. The DSC thermal characteristics enabled concluding that the modifications caused an increase in temperatures and a decrease in heat of transition (or its lack). The acetylation of retrograded starch preparations increased their solubility in water and water absorbability. The modifications were found to exert various effects on the rheological properties of pastes determined based on the Brabender's pasting characteristics and flow curves determined with the use of an oscillatory-rotating viscosimeter. All starch acetates produced were characterized by ca. 40% resistance to amylolysis. PMID:23911484

  17. [3H]Indole-3-acetyl-myo-inositol hydrolysis by extracts of Zea mays L. vegetative tissue

    NASA Technical Reports Server (NTRS)

    Hall, P. J.; Bandurski, R. S.

    1986-01-01

    [3H]Indole-3-acetyl-myo-inositol was hydrolyzed by buffered extracts of acetone powders prepared from 4 day shoots of dark grown Zea mays L. seedlings. The hydrolytic activity was proportional to the amount of extract added and was linear for up to 6 hours at 37 degrees C. Boiled or alcohol denatured extracts were inactive. Analysis of reaction mixtures by high performance liquid chromatography demonstrated that not all isomers of indole-3-acetyl-myo-inositol were hydrolyzed at the same rate. Buffered extracts of acetone powders were prepared from coleoptiles and mesocotyls. The rates of hydrolysis observed with coleoptile extracts were greater than those observed with mesocotyl extracts. Active extracts also catalyzed the hydrolysis of esterase substrates such as alpha-naphthyl acetate and the methyl esters of indoleacetic acid and naphthyleneacetic acid. Attempts to purify the indole-3-acetyl-myo-inositol hydrolyzing activity by chromatographic procedures resulted in only slight purification with large losses of activity. Chromatography over hydroxylapatite allowed separation of two enzymically active fractions, one of which catalyzed the hydrolysis of both indole-3-acetyl-myo-inositol and esterase substrates. With the other enzymic hydrolysis of esterase substrates was readily demonstrated, but no hydrolysis of indole-3-acetyl-myo-inositol was ever detected.

  18. Hydrolysis of wheat arabinoxylan by two acetyl xylan esterases from Chaetomium thermophilum.

    PubMed

    Tong, Xiaoxue; Lange, Lene; Grell, Morten Nedergaard; Busk, Peter Kamp

    2015-01-01

    The thermophilic filamentous ascomycete Chaetomium thermophilum produces functionally diverse hemicellulases when grown on hemicellulose as carbon source. Acetyl xylan esterase (EC 3.1.1.72) is an important accessory enzyme in hemicellulose biodegradation. Although the genome of C. thermophilum has been sequenced, its carbohydrate esterases are not annotated yet. We applied peptide pattern recognition (PPR) tool for sequence analysis of the C. thermophilum genome, and 11 carbohydrate esterase genes were discovered. Furthermore, we cloned and heterologously expressed two putative acetyl xylan esterase genes, CtAxeA and CtAxeB, in Pichia pastoris. The recombinant proteins, rCtAxeA and rCtAxeB, released acetic acids from p-nitrophenyl acetate and water-insoluble wheat arabinoxylan. These results indicate that CtAxeA and CtAxeB are true acetyl xylan esterases. For both recombinant esterases, over 93 % of the initial activity was retained after 24 h of incubation at temperatures up to 60 C, and over 90 % of the initial activity was retained after 24 h of incubation in different buffers from pH 4.0 to 9.0 at 4 and 50 C. The overall xylose yield from wheat arabinoxylan hydrolysis was 8 % with xylanase treatment and increased to 34 % when xylanase was combined with rCtAxeA and rCtAxeB. In sum, the present study first report the biochemical characterization of two acetyl xylan esterases from C. thermophilum, which are efficient in hydrolyzing hemicellulose with potential application in biomass bioconversion to high value chemicals or biofuels. PMID:25369895

  19. Methods for measuring CoA and CoA derivatives in biological samples.

    PubMed

    Tsuchiya, Yugo; Pham, Uyen; Gout, Ivan

    2014-08-01

    CoA (coenzyme A) is a ubiquitous and essential cofactor that acts as an acyl group carrier in biochemical reactions. Apart from participating in numerous metabolic pathways as substrates and intermediates, CoA and a number of its thioester derivatives, such as acetyl-CoA, can also directly regulate the activity of proteins by allosteric mechanisms and by affecting protein acetylation reactions. Cellular levels of CoA and CoA thioesters change under various physiological and pathological conditions. Defective CoA biosynthesis is implicated in NBIA (neurodegeneration with brain iron accumulation). However, the exact role of CoA in the pathogenesis of NBIA is not well understood. Accurate and reliable assays for measuring CoA species in biological samples are essential for studying the roles of CoA and CoA derivatives in health and disease. The present mini-review discusses methods that are commonly used to measure CoA species in biological samples. PMID:25110010

  20. Enzymatic hydrolysis of chitin pretreated by rapid depressurization from supercritical 1,1,1,2-tetrafluoroethane toward highly acetylated oligosaccharides.

    PubMed

    Villa-Lerma, Guadalupe; González-Márquez, Humberto; Gimeno, Miquel; Trombotto, Stéphane; David, Laurent; Ifuku, Shinsuke; Shirai, Keiko

    2016-06-01

    The hydrolysis of chitin treated under supercritical conditions was successfully carried out using chitinases obtained by an optimized fermentation of the fungus Lecanicillium lecanii. The biopolymer was subjected to a pretreatment based on suspension in supercritical 1,1,1,2-tetrafluoroethane (scR134a), which possesses a critical temperature and pressure of 101°C and 40bar, respectively, followed by rapid depressurization to atmospheric pressure and further fibrillation. This methodology was compared to control untreated chitins and chitin subjected to steam explosion showing improved production of reducing sugars (0.18mg/mL), enzymatic hydrolysis and high acetylation (FA of 0.45) in products with degrees of polymerization between 2 and 5. PMID:26970920

  1. Transition-state analysis of 2-O-acetyl-ADP-ribose hydrolysis by human macrodomain 1.

    PubMed

    Hirsch, Brett M; Burgos, Emmanuel S; Schramm, Vern L

    2014-10-17

    Macrodomains, including the human macrodomain 1 (MacroD1), are erasers of the post-translational modification of monoadenosinediphospho-ribosylation and hydrolytically deacetylate the sirtuin product O-acetyl-ADP-ribose (OAADPr). OAADPr has been reported to play a role in cell signaling based on oocyte microinjection studies, and macrodomains affect an array of cell processes including transcription and response to DNA damage. Here, we investigate human MacroD1 by transition-state (TS) analysis based on kinetic isotope effects (KIEs) from isotopically labeled OAADPr substrates. Competitive radiolabeled-isotope effects and mass spectrometry were used to obtain KIE data to yield intrinsic KIE values. Intrinsic KIEs were matched to a quantum chemical structure of the TS that includes the active site residues Asp184 and Asn174 and a structural water molecule. Transition-state analysis supports a concerted mechanism with an early TS involving simultaneous nucleophilic water attack and leaving group bond cleavage where the breaking C-O ester bond=1.60 and the C-O bond to the attacking water nucleophile=2.30 . The MacroD1 TS provides mechanistic understanding of the OAADPr esterase chemistry. PMID:25051211

  2. Transition-State Analysis of 2-O-Acetyl-ADP-Ribose Hydrolysis by Human Macrodomain 1

    PubMed Central

    2015-01-01

    Macrodomains, including the human macrodomain 1 (MacroD1), are erasers of the post-translational modification of monoadenosinediphospho-ribosylation and hydrolytically deacetylate the sirtuin product O-acetyl-ADP-ribose (OAADPr). OAADPr has been reported to play a role in cell signaling based on oocyte microinjection studies, and macrodomains affect an array of cell processes including transcription and response to DNA damage. Here, we investigate human MacroD1 by transition-state (TS) analysis based on kinetic isotope effects (KIEs) from isotopically labeled OAADPr substrates. Competitive radiolabeled-isotope effects and mass spectrometry were used to obtain KIE data to yield intrinsic KIE values. Intrinsic KIEs were matched to a quantum chemical structure of the TS that includes the active site residues Asp184 and Asn174 and a structural water molecule. Transition-state analysis supports a concerted mechanism with an early TS involving simultaneous nucleophilic water attack and leaving group bond cleavage where the breaking CO ester bond = 1.60 and the CO bond to the attacking water nucleophile = 2.30 . The MacroD1 TS provides mechanistic understanding of the OAADPr esterase chemistry. PMID:25051211

  3. Regulation of Acetate Metabolism and Acetyl Co-a Synthetase 1 (ACS1) Expression by Methanol Expression Regulator 1 (Mxr1p) in the Methylotrophic Yeast Pichia pastoris.

    PubMed

    Sahu, Umakant; Rangarajan, Pundi N

    2016-02-12

    Methanol expression regulator 1 (Mxr1p) is a zinc finger protein that regulates the expression of genes encoding enzymes of the methanol utilization pathway in the methylotrophic yeast Pichia pastoris by binding to Mxr1p response elements (MXREs) present in their promoters. Here we demonstrate that Mxr1p is a key regulator of acetate metabolism as well. Mxr1p is cytosolic in cells cultured in minimal medium containing a yeast nitrogen base, ammonium sulfate, and acetate (YNBA) but localizes to the nucleus of cells cultured in YNBA supplemented with glutamate or casamino acids as well as nutrient-rich medium containing yeast extract, peptone, and acetate (YPA). Deletion of Mxr1 retards the growth of P. pastoris cultured in YNBA supplemented with casamino acids as well as YPA. Mxr1p is a key regulator of ACS1 encoding acetyl-CoA synthetase in cells cultured in YPA. A truncated Mxr1p comprising 400 N-terminal amino acids activates ACS1 expression and enhances growth, indicating a crucial role for the N-terminal activation domain during acetate metabolism. The serine 215 residue, which is known to regulate the expression of Mxr1p-activated genes in a carbon source-dependent manner, has no role in the Mxr1p-mediated activation of ACS1 expression. The ACS1 promoter contains an Mxr1p response unit (MxRU) comprising two MXREs separated by a 30-bp spacer. Mutations that abrogate MxRU function in vivo abolish Mxr1p binding to MxRU in vitro. Mxr1p-dependent activation of ACS1 expression is most efficient in cells cultured in YPA. The fact that MXREs are conserved in genes outside of the methanol utilization pathway suggests that Mxr1p may be a key regulator of multiple metabolic pathways in P. pastoris. PMID:26663080

  4. Prebiotic Fiber Increases Hepatic Acetyl CoA Carboxylase Phosphorylation and Suppresses Glucose-Dependent Insulinotropic Polypeptide Secretion More Effectively When Used with Metformin in Obese Rats1,2

    PubMed Central

    Pyra, Kim A.; Saha, Dolan C.; Reimer, Raylene A.

    2013-01-01

    Independently, metformin (MET) and the prebiotic, oligofructose (OFS), have been shown to increase glucagon-like peptide (GLP-1) secretion. Our objective was to determine whether using OFS as an adjunct with MET augments GLP-1 secretion in obese rats. Male, diet-induced obese Sprague Dawley rats were randomized to: 1) high-fat/-sucrose diet [HFHS; control (C); 20% fat, 50% sucrose wt:wt]; 2) HFHS+10% OFS (OFS); 3) HFHS + MET [300 mg/kg/d (MET)]; 4) HFHS+10% OFS+MET (OFS +MET). Body composition, glycemia, satiety hormones, and mechanisms related to dipeptidyl peptidase 4 (DPP4) activity in plasma, hepatic AMP-activated protein kinase (AMPK; Western blots), and gut microbiota (qPCR) were examined. Direct effects of MET and SCFA were examined in human enteroendocrine cells. The interaction between OFS and MET affected fat mass, hepatic TG, secretion of glucose-dependent insulinotropic polypeptide (GIP) and leptin, and AMPKα2 mRNA and phosphorylated acetyl CoA carboxylase (pACC) levels (P < 0.05). Combined, OFS and MET reduced GIP secretion to a greater extent than either treatment alone (P < 0.05). The hepatic pACC level was increased by OFS+MET by at least 50% above all other treatments, which did not differ from each other (P < 0.05). OFS decreased plasma DPP4 activity (P < 0.001). Cecal Bifidobacteria (P < 0.001) were markedly increased and C. leptum decreased (P < 0.001) with OFS consumption. In human enteroendocrine cells, the interaction between MET and SCFA affected GLP-1 secretion (P < 0.04) but was not associated with higher GLP-1 than the highest individual doses. In conclusion, the combined actions of OFS and MET were associated with important interaction effects that have the potential to improve metabolic outcomes associated with obesity. PMID:22223580

  5. Accurate, quantitative assays for the hydrolysis of soluble type I, II, and III /sup 3/H-acetylated collagens by bacterial and tissue collagenases

    SciTech Connect

    Mallya, S.K.; Mookhtiar, K.A.; Van Wart, H.E.

    1986-11-01

    Accurate and quantitative assays for the hydrolysis of soluble /sup 3/H-acetylated rat tendon type I, bovine cartilage type II, and human amnion type III collagens by both bacterial and tissue collagenases have been developed. The assays are carried out at any temperature in the 1-30/sup 0/C range in a single reaction tube and the progress of the reaction is monitored by withdrawing aliquots as a function of time, quenching with 1,10-phenanthroline, and quantitation of the concentration of hydrolysis fragments. The latter is achieved by selective denaturation of these fragments by incubation under conditions described in the previous paper of this issue. The assays give percentages of hydrolysis of all three collagen types by neutrophil collagenase that agree well with the results of gel electrophoresis experiments. The initial rates of hydrolysis of all three collagens are proportional to the concentration of both neutrophil or Clostridial collagenases over a 10-fold range of enzyme concentrations. All three assays can be carried out at collagen concentrations that range from 0.06 to 2 mg/ml and give linear double reciprocal plots for both tissue and bacterial collagenases that can be used to evaluate the kinetic parameters K/sub m/ and k/sub cat/ or V/sub max/. The assay developed for the hydrolysis of rat type I collagen by neutrophil collagenase is shown to be more sensitive by at least one order of magnitude than comparable assays that use rat type I collagen fibrils or gels as substrate.

  6. In vivo assessment of captopril selectivity of angiotensin I-converting enzyme inhibition: differential inhibition of acetyl-ser-asp-lys-pro and angiotensin I hydrolysis.

    PubMed

    Junot, C; Menard, J; Gonzales, M F; Michaud, A; Corvol, P; Ezan, E

    1999-06-01

    Angiotensin I-converting enzyme (ACE) is a zinc metallopeptidase that plays a major role in blood pressure regulation. The demonstration that the hemoregulatory peptide acetyl-Ser-Asp-Lys-Pro (AcSDKP) is a natural and specific substrate of the N-active site of ACE suggests that this enzyme may have a new physiological role such as the modulation of hematopoietic stem cells. In vitro studies have shown that ACE inhibitors displayed various potencies in inhibiting the degradation of different natural or synthetic substrates of ACE, among which captopril inhibits AcSDKP hydrolysis more potently than angiotensin I hydrolysis. To look for this selectivity in vivo, we investigated the pharmacodynamic effect of increasing doses of captopril (0.01-10 mg/kg) during the 90 min after i.v. administration to spontaneously hypertensive rats. Plasma and urinary AcSDKP levels were measured. The renin-angiotensin system was evaluated by measurements of ACE activity in plasma samples, using the synthetic substrate Hip-His-Leu, by determinations of plasma renin concentrations and measurements of arterial blood pressure. The results showed that captopril (0.01-0.3 mg/kg) selectively inhibited AcSDKP hydrolysis, with limited effects on the renin-angiotensin system. AcSDKP levels in plasma and urine rose to a plateau 4 times the basal level for doses more than 0.3 mg/kg. All of the parameters reflecting the renin-angiotensin system were significantly affected at doses of 1 and 10 mg/kg. The present study therefore confirms that captopril can be used to protect hematopoietic stem cells during antitumor chemotherapy while having only a limited effect on cardiovascular homeostasis. PMID:10336514

  7. Proton inventory of the water-catalyzed hydrolysis of 1-acetyl-1,2,4-triazole. Examination of ionic strength effects

    SciTech Connect

    Patterson, J.F.; Huskey, W.P.; Hogg, J.L.

    1980-11-07

    Proton inventories of the water-catalyzed hydrolysis of 1-acetyl-1,2-4-triazole have been completed under a variety of conditions. The solvent deuterium isotope effect, k/sub H/sub 2/O/k/sub D/sub 2/O/, determined at pH 4.7 or the equivalent point on the pD rate profile at 25/sup 0/C by using acetic acid-acetate buffers at 1 M ionic strength was 3.18. The solvent deuterium isotope effects determined at ionic strenghs of 1 and 0.5 M by using 10/sup -3/ M HCl (DCl) to control the pH(D) were 3.13 and 3.07, respectively. In all cases the proton inventories exhibit significant downward curvature and are, within experimental error, consistent with a cyclic transition state structure involving four water molecules. The equation k/sub n/ = k/sub 0/(1 - n + 0.75n) describes the proton inventories where the value of the isotope fractionation factor for the four in-flight protons is 0.75. These inventories are compared to an earlier study done with no ionic strength control, and several alternative transition states are considered in detail.

  8. Identification of a novel CoA synthase isoform, which is primarily expressed in the brain.

    PubMed

    Nemazanyy, Ivan; Panasyuk, Ganna; Breus, Oksana; Zhyvoloup, Alexander; Filonenko, Valeriy; Gout, Ivan T

    2006-03-24

    CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy beta and originally identified CoA synthase, CoASy alpha. The transcript specific for CoASy beta was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy beta. In contrast to CoASy alpha, which shows ubiquitous expression, CoASy beta is primarily expressed in the brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in the brain, requires further elucidation. PMID:16460672

  9. Identification of a novel CoA synthase isoform, which is primarily expressed in Brain

    SciTech Connect

    Nemazanyy, Ivan . E-mail: nemazanyy@imbg.org.ua; Panasyuk, Ganna; Breus, Oksana; Zhyvoloup, Alexander; Filonenko, Valeriy; Gout, Ivan T. . E-mail: i.gout@ucl.ac.uk

    2006-03-24

    CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy {beta} and originally identified CoA synthase, CoASy {alpha}. The transcript specific for CoASy {beta} was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy {beta}. In contrast to CoASy {alpha}, which shows ubiquitous expression, CoASy {beta} is primarily expressed in Brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in Brain, requires further elucidation.

  10. Protein Acetylation and Acetyl Coenzyme A Metabolism in Budding Yeast

    PubMed Central

    Galdieri, Luciano; Zhang, Tiantian; Rogerson, Daniella; Lleshi, Rron

    2014-01-01

    Cells sense and appropriately respond to the physical conditions and availability of nutrients in their environment. This sensing of the environment and consequent cellular responses are orchestrated by a multitude of signaling pathways and typically involve changes in transcription and metabolism. Recent discoveries suggest that the signaling and transcription machineries are regulated by signals which are derived from metabolism and reflect the metabolic state of the cell. Acetyl coenzyme A (CoA) is a key metabolite that links metabolism with signaling, chromatin structure, and transcription. Acetyl-CoA is produced by glycolysis as well as other catabolic pathways and used as a substrate for the citric acid cycle and as a precursor in synthesis of fatty acids and steroids and in other anabolic pathways. This central position in metabolism endows acetyl-CoA with an important regulatory role. Acetyl-CoA serves as a substrate for lysine acetyltransferases (KATs), which catalyze the transfer of acetyl groups to the epsilon-amino groups of lysines in histones and many other proteins. Fluctuations in the concentration of acetyl-CoA, reflecting the metabolic state of the cell, are translated into dynamic protein acetylations that regulate a variety of cell functions, including transcription, replication, DNA repair, cell cycle progression, and aging. This review highlights the synthesis and homeostasis of acetyl-CoA and the regulation of transcriptional and signaling machineries in yeast by acetylation. PMID:25326522

  11. Hydrolysis of low concentrations of the acetylthiocholine analogs acetyl(homo)thiocholine and acetyl(nor)thiocholine by acetylcholinesterase may be limited by selective gating at the enzyme peripheral site

    PubMed Central

    Beri, Veena; Auletta, Jeffrey T.; Maharvi, Ghulam M.; Wood, Juanita F.; Fauq, Abdul H.; Rosenberry, Terrone L.

    2012-01-01

    Hydrolysis of acetylcholine by acetylcholinesterase (AChE) is extremely rapid, with a second-order hydrolysis rate constant kE (often denoted kcat/KM) that approaches 108 M?1s?1. AChE contains a deep active site gorge with two sites of ligand binding, an acylation site (or A-site) containing the catalytic triad at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site is known to contribute to catalytic efficiency with acetylthiocholine (AcSCh) by transiently trapping the substrate in a low affinity complex on its way to the A-site, where a short-lived acyl enzyme intermediate is produced. Here we ask whether the P-site does more than simply trap the substrate but in fact selectively gates entry to the A-site to provide specificity for AcSCh (and acetylcholine) relative to the close structural analogs acetylhomothiocholine (Ac-hSCh, which adds one additional methylene group to thiocholine) and acetylnorthiocholine (Ac-nSCh, which deletes one methylene group from thiocholine). We synthesized Ac-hSCh and Ac-nSCh and overcame technical difficulties associated with instability of the northiocholine hydrolysis product. We then compared the catalytic parameters of these substrates with AChE to those of AcSCh. Values of kE for Ac-hSCh and Ac-nSCh were about 2% of that for AcSCh. The kE for AcSCh is close to the theoretical diffusion-controlled limit for the substrate association rate constant, but kE values for Ac-hSCh or Ac-nSCh are too low to be limited by diffusion control. However, analyses of kinetic solvent isotope effects and inhibition patterns for P-site inhibitors indicate that these two analogs also do not equilibrate with the A-site prior to the initial acylation step of catalysis. We propose that kE for these substrates is partially rate-limited by a gating step that involves the movement of bound substrate from the P-site to the A-site. PMID:23047027

  12. Synthesis of radiolabeled acetyl-coenzyme A from sodium acetate

    SciTech Connect

    Clough, R.C.; Barnum, S.R.; Jaworski, J.G.

    1989-01-01

    The synthesis of high specific radioactivity (/sup 14/C)-acetyl-Coenzyme A from (/sup 14/C)sodium acetate, 2,6-dichlorobenzoic acid, 1,1'-carbonyldiimidazole, and CoA is reported. Starting with 1 mumol of (/sup 14/C)sodium acetate, this method yields pure (/sup 14/C)acetyl-CoA in yields approaching 40%. Chromatography on a reversed-phase ODS column was used to separate acetyl-CoA from Coenzyme A and side products. The acetylating agent is apparently a reaction intermediate, acetylimidazole.

  13. SIRT4 coordinates the balance between lipid synthesis and catabolism by repressing malonyl CoA decarboxylase.

    PubMed

    Laurent, Galle; German, Natalie J; Saha, Asish K; de Boer, Vincent C J; Davies, Michael; Koves, Timothy R; Dephoure, Noah; Fischer, Frank; Boanca, Gina; Vaitheesvaran, Bhavapriya; Lovitch, Scott B; Sharpe, Arlene H; Kurland, Irwin J; Steegborn, Clemens; Gygi, Steven P; Muoio, Deborah M; Ruderman, Neil B; Haigis, Marcia C

    2013-06-01

    Lipid metabolism is tightly controlled by the nutritional state of the organism. Nutrient-rich conditions increase lipogenesis, whereas nutrient deprivation promotes fat oxidation. In this study, we identify the mitochondrial sirtuin, SIRT4, as a regulator of lipid homeostasis. SIRT4 is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), an enzyme that produces acetyl CoA from malonyl CoA. Malonyl CoA provides the carbon skeleton for lipogenesis and also inhibits fat oxidation. Mice lacking SIRT4 display elevated MCD activity and decreased malonyl CoA in skeletal muscle and white adipose tissue. Consequently, SIRT4 KO mice display deregulated lipid metabolism, leading to increased exercise tolerance and protection against diet-induced obesity. In sum, this work elucidates SIRT4 as an important regulator of lipid homeostasis, identifies MCD as a SIRT4 target, and deepens our understanding of the malonyl CoA regulatory axis. PMID:23746352

  14. The Fasted/Fed Mouse Metabolic Acetylome: N6-Acetylation Differences Suggest Acetylation Coordinates Organ-Specific Fuel Switching

    PubMed Central

    Yang, Li; Vaitheesvaran, Bhavapriya; Hartil, Kirsten; Robinson, Alan J.; Hoopmann, Michael R.; Eng, Jimmy K.; Kurland, Irwin J.; Bruce, James E.

    2011-01-01

    The elucidation of extra-nuclear lysine acetylation has been of growing interest, as the co-substrate for acetylation, acetyl CoA, is at a key metabolic intersection. Our hypothesis was that mitochondrial and cytoplasmic protein acetylation may be part of a fasted/re-fed feedback control system for the regulation of the metabolic network in fuel switching, where acetyl CoA would be provided by fatty acid oxidation, or glycolysis, respectively. To test this we characterized the mitochondrial and cytoplasmic acetylome in various organs that have a high metabolic rate relative to their mass, and/or switch fuels, under fasted and re-fed conditions (brain, kidney, liver, skeletal muscle, heart muscle, white and brown adipose tissues). Using immunoprecipitation, coupled with LC-MSMS label free quantification, we show there is a dramatic variation in global quantitative profiles of acetylated proteins from different organs. In total, 733 acetylated peptides from 337 proteins were identified and quantified, out of which 31 acetylated peptides from the metabolic proteins that may play organ-specific roles were analyzed in detail. Results suggest that fasted/re-fed acetylation changes coordinated by organ-specific (de-)acetylases in insulin-sensitive versus insensitive organs may underlie fuel use and switching. Characterization of the tissue-specific acetylome should increase understanding of metabolic conditions wherein normal fuel switching is disrupted, such as in Type II diabetes. PMID:21728379

  15. Antagonism of P2Y1-induced vasorelaxation by acyl CoA: a critical role for palmitate and 3?-phosphate

    PubMed Central

    Alefishat, E; Alexander, SPH; Ralevic, V

    2013-01-01

    Background and Purpose Acyl derivatives of CoA have been shown to act as antagonists at human platelet and recombinant P2Y1 receptors, but little is known about their effects in the cardiovascular system. This study evaluated the effect of these endogenous nucleotide derivatives at P2Y1 receptors natively expressed in rat and porcine blood vessels. Experimental Approach Isometric tension recordings were used to evaluate the effects of CoA, acetyl CoA, palmitoyl CoA (PaCoA) and 3?-dephospho-palmitoyl-CoA on concentration relaxationresponse curves to ADP and uridine triphosphate (UTP). A FlexStation monitored ADP- and UTP-evoked calcium responses in HEK293 cells. Key Results Acetyl CoA and PaCoA, but not CoA, inhibited endothelium-dependent relaxations to ADP with apparent selectivity for P2Y1 receptors (over P2Y2/4 receptors) in rat thoracic aorta; PaCoA was more potent than acetyl CoA (331-fold vs. fivefold shift of ADP response curve evoked by 10 ?M PaCoA and acetyl CoA, respectively); the apparent pA2 value for PaCoA was 6.44. 3?-dephospho-palmitoyl-CoA (10 ?M) was significantly less potent than PaCoA (20-fold shift). In porcine mesenteric arteries, PaCoA and the P2Y1 receptor antagonist MRS2500 blocked ADP-mediated endothelium-dependent relaxations; in contrast, they were ineffective against ADP-mediated endothelium-independent relaxation in porcine coronary arteries (which does not involve P2Y1 receptors). Calcium responses evoked by ADP activation of endogenous P2Y1 receptors in HEK293 cells were inhibited in the presence of PaCoA, which failed to alter responses to UTP (acting at endogenous P2Y2/4 receptors). Conclusions and Implications Acyl derivatives of CoA can act as endogenous selective antagonists of P2Y1 receptors in blood vessels, and this inhibitory effect critically depends on the palmitate and 3?-ribose phosphate substituents on CoA. PMID:23215951

  16. Differences among Adult COAs and Adult Non-COAs on Levels of Self-Esteem, Depression, and Anxiety.

    ERIC Educational Resources Information Center

    Dodd, David T.; Roberts, Richard L.

    1994-01-01

    Examined self-esteem, depression, and anxiety among 60 adult children of alcoholics (COAs) and 143 adult non-COAs. Subjects completed Children of Alcoholics Screening Test, demographic questionnaire, Beck Depression Inventory, State-Trait Anxiety Inventory, and Coopersmith Self-Esteem Inventory. Found no significant differences between COAs and

  17. Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine.

    PubMed

    Starai, V J; Celic, I; Cole, R N; Boeke, J D; Escalante-Semerena, J C

    2002-12-20

    Acetyl-coenzyme A (CoA) synthetase (Acs) is an enzyme central to metabolism in prokaryotes and eukaryotes. Acs synthesizes acetyl CoA from acetate, adenosine triphosphate, and CoA through an acetyl-adenosine monophosphate (AMP) intermediate. Immunoblotting and mass spectrometry analysis showed that Salmonella enterica Acs enzyme activity is posttranslationally regulated by acetylation of lysine-609. Acetylation blocks synthesis of the adenylate intermediate but does not affect the thioester-forming activity of the enzyme. Activation of the acetylated enzyme requires the nicotinamide adenine dinucleotide-dependent protein deacetylase activity of the CobB Sir2 protein from S. enterica. We propose that acetylation modulates the activity of all the AMP-forming family of enzymes, including nonribosomal peptide synthetases, luciferase, and aryl- and acyl-CoA synthetases. These findings extend our knowledge of the roles of Sir2 proteins in gene silencing, chromosome stability, and cell aging and imply that lysine acetylation is a common regulatory mechanism in eukaryotes and prokaryotes. PMID:12493915

  18. Identification and Characterization of Mitochondrial Acetyl-Coenzyme A Hydrolase from Pisum sativum L. Seedlings 1

    PubMed Central

    Zeiher, Carolyn A.; Randall, Douglas D.

    1990-01-01

    Mitochondria from Pisum sativum seedlings purified free of peroxisomal and chlorophyll contamination were examined for acetyl-coenzyme A (CoA) hydrolase activity. Acetyl-CoA hydrolase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of the mitochondria. The products, acetate and CoA, were quantified by two independent methods and verified that the observed activity was an acetyl-CoA hydrolase. The pea mitochondrial acetyl-CoA hydrolase showed a Km for acetyl-CoA of 74 micromolar and a Vmax of 6.1 nanomoles per minute per milligram protein. CoA was a linear competitive inhibitor of the enzyme with a Kis of 16 micromolar. The sensitivity of the enzyme to changes in mole fraction of acetyl-CoA suggested that the changes in the intramitochondrial acetyl-CoA/CoA ratio may be an effective mechanism of control. The widespread distribution of mitochondrial acetyl-CoA hydrolase activity among different plant species indicated that this may be a general mechanism in plants for synthesizing acetate. PMID:16667687

  19. Acetyl chloride

    Integrated Risk Information System (IRIS)

    Acetyl chloride ; CASRN 75 - 36 - 5 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  20. Acetylation of woody lignocellulose: significance and regulation

    PubMed Central

    Pawar, Prashant Mohan-Anupama; Koutaniemi, Sanna; Tenkanen, Maija; Mellerowicz, Ewa J.

    2013-01-01

    Non-cellulosic cell wall polysaccharides constitute approximately one quarter of usable biomass for human exploitation. In contrast to cellulose, these components are usually substituted by O-acetyl groups, which affect their properties and interactions with other polymers, thus affecting their solubility and extractability. However, details of these interactions are still largely obscure. Moreover, polysaccharide hydrolysis to constituent monosaccharides is hampered by the presence of O-acetyl groups, necessitating either enzymatic (esterase) or chemical de-acetylation, increasing the costs and chemical consumption. Reduction of polysaccharide acetyl content in planta is a way to modify lignocellulose toward improved saccharification. In this review we: (1) summarize literature on lignocellulose acetylation in different tree species, (2) present data and current hypotheses concerning the role of O-acetylation in determining woody lignocellulose properties, (3) describe plant proteins involved in lignocellulose O-acetylation, (4) give examples of microbial enzymes capable to de-acetylate lignocellulose, and (5) discuss prospects for exploiting these enzymes in planta to modify xylan acetylation. PMID:23734153

  1. Intracellular Acetyl Unit Transport in Fungal Carbon Metabolism ▿

    PubMed Central

    Strijbis, Karin; Distel, Ben

    2010-01-01

    Acetyl coenzyme A (acetyl-CoA) is a central metabolite in carbon and energy metabolism. Because of its amphiphilic nature and bulkiness, acetyl-CoA cannot readily traverse biological membranes. In fungi, two systems for acetyl unit transport have been identified: a shuttle dependent on the carrier carnitine and a (peroxisomal) citrate synthase-dependent pathway. In the carnitine-dependent pathway, carnitine acetyltransferases exchange the CoA group of acetyl-CoA for carnitine, thereby forming acetyl-carnitine, which can be transported between subcellular compartments. Citrate synthase catalyzes the condensation of oxaloacetate and acetyl-CoA to form citrate that can be transported over the membrane. Since essential metabolic pathways such as fatty acid β-oxidation, the tricarboxylic acid (TCA) cycle, and the glyoxylate cycle are physically separated into different organelles, shuttling of acetyl units is essential for growth of fungal species on various carbon sources such as fatty acids, ethanol, acetate, or citrate. In this review we summarize the current knowledge on the different systems of acetyl transport that are operational during alternative carbon metabolism, with special focus on two fungal species: Saccharomyces cerevisiae and Candida albicans. PMID:20889721

  2. Synthesis of acetyl coenzyme A by carbon monoxide dehydrogenase complex from acetate-grown Methanosarcina thermophila.

    PubMed Central

    Abbanat, D R; Ferry, J G

    1990-01-01

    The carbon monoxide dehydrogenase (CODH) complex from Methanosarcina thermophila catalyzed the synthesis of acetyl coenzyme A (acetyl-CoA) from CH3I, CO, and coenzyme A (CoA) at a rate of 65 nmol/min/mg at 55 degrees C. The reaction ended after 5 min with the synthesis of 52 nmol of acetyl-CoA per nmol of CODH complex. The optimum temperature for acetyl-CoA synthesis in the assay was between 55 and 60 degrees C; the rate of synthesis at 55 degrees C was not significantly different between pHs 5.5 and 8.0. The rate of acetyl-CoA synthesis was independent of CoA concentrations between 20 microM and 1 mM; however, activity was inhibited 50% with 5 mM CoA. Methylcobalamin did not substitute for CH3I in acetyl-CoA synthesis; no acetyl-CoA or propionyl coenzyme A was detected when sodium acetate or CH3CH2I replaced CH3I in the assay mixture. CO could be replaced with CO2 and titanium(III) citrate. When CO2 and 14CO were present in the assay, the specific activity of the acetyl-CoA synthesized was 87% of the specific activity of 14CO, indicating that CO was preferentially incorporated into acetyl-CoA without prior oxidation to free CO2. Greater than 100 microM potassium cyanide was required to significantly inhibit acetyl-CoA synthesis, and 500 microM was required for 50% inhibition; in contrast, oxidation of CO by the CODH complex was inhibited 50% by approximately 10 microM potassium cyanide. PMID:2123865

  3. A type III polyketide synthase from Rhizobium etli condenses malonyl CoAs to a heptaketide pyrone with unusually high catalytic efficiency.

    PubMed

    Jeya, Marimuthu; Kim, Tae-Su; Kumar Tiwari, Manish; Li, Jinglin; Zhao, Huimin; Lee, Jung-Kul

    2012-10-30

    A novel type III polyketide synthase (RePKS) from Rhizobium etli produced a heptaketide pyrone using acetyl-CoA and six molecules of malonyl-CoA. Its catalytic efficiency (k(cat)/K(m) = 5230 mM(-1) min(-1)) for malonyl CoA was found to be the highest ever reported. Molecular dynamics studies revealed the unique features of RePKS. PMID:23059854

  4. Doping Induced Itinerant Ferromagnetism in CoAs

    NASA Astrophysics Data System (ADS)

    Chen, Chih-Wei; Morosan, Emilia

    2013-03-01

    The magnetism in ?-CoAs is dominated by strong spin fluctuations. In this study, we explore the effects of Phosphorus doping in ?-CoAs. Phosphorus is isovalent with Arsenic, and the resulting doping introduces disorder and chemical pressure. In CoAs1-xPx, a cross-over from the spin fluctuation-dominated regime to an itinerant ferromagnetic (IFM) state take places around x = 0.04. The IFM state persists up to x <= 0.27. For compositions between x = 0.28 and 0.40, the magnetization data suggests a possible Stoner enhanced state. We acknowledge the support from DOD PECASE.

  5. A spectrophotometric assay for measuring acetyl-coenzyme A carboxylase.

    PubMed

    Kroeger, Jasmin K; Zarzycki, Jan; Fuchs, Georg

    2011-04-01

    Acetyl-coenzyme A (CoA) carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacteria and eukaryota. This enzyme is the target of drug design for treatment of human metabolic diseases and of herbicides acting specifically on the eukaryotic form of the enzyme in grasses. Acetyl-CoA carboxylase activity screening in drug and herbicide design depends mostly on a time-consuming enzyme assay that is based on the incorporation of radiolabeled bicarbonate into the product malonyl-CoA. Here we describe a new simple, continuous, and quick photometric assay avoiding radioactive substrate. It couples the carboxylation of acetyl-CoA to the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of malonyl-CoA, which is catalyzed by recombinant malonyl-CoA reductase of Chloroflexus aurantiacus. This assay can be adapted for high-throughput screening. PMID:21138728

  6. CAPTAN HYDROLYSIS

    EPA Science Inventory

    Captan (N-(trichloromethylthio)-4-cyclohexene-1,2-dicarboximide) undergoes hydrolysis readily in water with a maximum half-life of 710 min. Over the pH range 2-6, the reaction is pH independent and the pseudo-first-order rate constant is (1.8 + or - 0.1) x 10 to the -5th power/s....

  7. Polymorphic acetylation of nitrazepam.

    PubMed Central

    Karim, A K; Evans, D A

    1976-01-01

    Nitrazepam is metabolized in part by nitro-reduction to an amine followed by acetylation. This acetylation step has been shown to be under the control of the same genetic polymorphism as sulphamethazine (syn: sulphadimidine). PMID:775091

  8. Acute hemolytic transfusion reaction caused by anti-Coa.

    PubMed

    Covin, R B; Evans, K S; Olshock, R; Thompson, H W

    2001-01-01

    Coa is a high-frequency blood group antigen in the Colton blood group system expressed on red blood cells (RBCs) of approximately 99.8 percent of random persons. Anti-Coa has been reported to cause delayed hemolytic transfusion reactions, hemolytic disease of the newborn, and accelerated clearance of RBCs in vivo. Acute hemolytic transfusion reactions (AHTRs) have not previously been reported. A 58-year-old man was hospitalized for vascular surgery. Initial blood bank evaluation revealed anti-Fya. The patient received six units of RBCs during his initial hospitalization and developed anti-E. A subsequent sample was sent to the reference laboratory when all units of RBCs appeared incompatible. Additional studies, including alloadsorptions, revealed the presence of anti-E, anti-Fya, and an apparent warm autoantibody. One unit of least-incompatible RBCs was transfused during surgery. The patient had an increase in temperature. Hemoglobinuria and a decrease in hematocrit were also noted. Due to the clinical impression of an AHTR, the pre- and postreaction samples were reevaluated in the reference laboratory and demonstrated the presence of anti-Coa in both. Based on clinical and laboratory evaluation this patient appears to have had an AHTR due to anti-Coa. This is the first known reported case of an AHTR caused by anti-Coa. PMID:15373591

  9. Benorylate hydrolysis by human plasma and human liver.

    PubMed Central

    Williams, F M; Moore, U; Seymour, R A; Mutch, E M; Nicholson, E; Wright, P; Wynne, H; Blain, P G; Rawlins, M D

    1989-01-01

    1. Benorylate (4-acetamido phenyl-O-acetylsalicylate) hydrolysis in vitro by human plasma and by human liver microsomes and cytosol has been investigated. 2. Benorylate was hydrolysed by a route involving initial hydrolysis of the acetyl group to yield phenetsal followed by hydrolysis to paracetamol and salicylate. Hydrolysis via acetylsalicylate was minor. 3. Benorylate was more actively hydrolysed by liver cytosol than microsomes and about 10 times faster than plasma. 4. Following a single oral dose benorylate (4 g) to volunteers only salicylate and paracetamol were detected in the plasma. 5. The therapeutic effects of benorylate appear to be mediated by salicylate and paracetamol. PMID:2575401

  10. Benorylate hydrolysis by human plasma and human liver.

    PubMed

    Williams, F M; Moore, U; Seymour, R A; Mutch, E M; Nicholson, E; Wright, P; Wynne, H; Blain, P G; Rawlins, M D

    1989-12-01

    1. Benorylate (4-acetamido phenyl-O-acetylsalicylate) hydrolysis in vitro by human plasma and by human liver microsomes and cytosol has been investigated. 2. Benorylate was hydrolysed by a route involving initial hydrolysis of the acetyl group to yield phenetsal followed by hydrolysis to paracetamol and salicylate. Hydrolysis via acetylsalicylate was minor. 3. Benorylate was more actively hydrolysed by liver cytosol than microsomes and about 10 times faster than plasma. 4. Following a single oral dose benorylate (4 g) to volunteers only salicylate and paracetamol were detected in the plasma. 5. The therapeutic effects of benorylate appear to be mediated by salicylate and paracetamol. PMID:2575401

  11. Global Hawk Pacific (GloPac) COA and Mission Coordination

    NASA Technical Reports Server (NTRS)

    Dillon, Mark; Hall, Philip

    2010-01-01

    This slide presentation reviews the science objectives of the Global Hawk unmanned aircraft system (UAS) in the Pacific region, shows examp le flight tracks, the satellite under-flight requirement, the flight planning, and the agencies coordination of the airspace required for the Certificate of Authorization (COA).

  12. Effect of (L-Carnitine) on acetyl-L-carnitine production by heart mitochondria

    SciTech Connect

    Bieber, L.L.; Lilly, K.; Lysiak, W.

    1986-05-01

    The authors recently reported a large efflux of acetyl-L-carnitine from rat heart mitochondria during state 3 respiration with pyruvate as substrate both in the presence and absence of malate. In this series of experiments, the effect of the concentration of L-carnitine on the efflux of acetyl-L-carnitine and on the production of /sup 14/CO/sub 2/ from 2-/sup 14/C-pyruvate was determined. Maximum acetylcarnitine production (approximately 25 n moles/min/mg protein) was obtained at 3-5 mM L-carnitine in the absence of added malate. /sup 14/CO/sub 2/ production decreased as the concentration of L-carnitine increased; it plateaued at 3-5 mM L-carnitine. These data indicate carnitine can stimulate flux of pyruvate through pyruvate dehydrogenase and can reduce flux of acetyl CoA through the Krebs cycle by acting as an acceptor of the acetyl moieties of acetyl CoA generated by pyruvate dehydrogenase.

  13. A Multisubunit Acetyl Coenzyme A Carboxylase from Soybean1

    PubMed Central

    Reverdatto, Sergei; Beilinson, Vadim; Nielsen, Niels C.

    1999-01-01

    A multisubunit form of acetyl coenzyme A (CoA) carboxylase (ACCase) from soybean (Glycine max) was characterized. The enzyme catalyzes the formation of malonyl CoA from acetyl CoA, a rate-limiting step in fatty acid biosynthesis. The four known components that constitute plastid ACCase are biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and the ?- and ?-subunits of carboxyltransferase (?- and ?-CT). At least three different cDNAs were isolated from germinating soybean seeds that encode BC, two that encode BCCP, and four that encode ?-CT. Whereas BC, BCCP, and ?-CT are products of nuclear genes, the DNA that encodes soybean ?-CT is located in chloroplasts. Translation products from cDNAs for BC, BCCP, and ?-CT were imported into isolated pea (Pisum sativum) chloroplasts and became integrated into ACCase. Edman microsequence analysis of the subunits after import permitted the identification of the amino-terminal sequence of the mature protein after removal of the transit sequences. Antibodies specific for each of the chloroplast ACCase subunits were generated against products from the cDNAs expressed in bacteria. The antibodies permitted components of ACCase to be followed during fractionation of the chloroplast stroma. Even in the presence of 0.5 m KCl, a complex that contained BC plus BCCP emerged from Sephacryl 400 with an apparent molecular mass greater than about 800 kD. A second complex, which contained ?- and ?-CT, was also recovered from the column, and it had an apparent molecular mass of greater than about 600 kD. By mixing the two complexes together at appropriate ratios, ACCase enzymatic activity was restored. Even higher ACCase activities were recovered by mixing complexes from pea and soybean. The results demonstrate that the active form of ACCase can be reassembled and that it could form a high-molecular-mass complex. PMID:10069834

  14. Metabolic engineering of Clostridium tyrobutyricum for n-butanol production: effects of CoA transferase.

    PubMed

    Yu, Le; Zhao, Jingbo; Xu, Mengmeng; Dong, Jie; Varghese, Saju; Yu, Mingrui; Tang, I-Ching; Yang, Shang-Tian

    2015-06-01

    The overexpression of CoA transferase (ctfAB), which catalyzes the reaction: acetate/butyrate + acetoacetyl-CoA ? acetyl/butyryl-CoA + acetoacetate, was studied for its effects on acid reassimilation and butanol biosynthesis in Clostridium tyrobutyricum (?ack, adhE2). The plasmid pMTL007 was used to co-express adhE2 and ctfAB from Clostridium acetobutylicum ATCC 824. In addition, the sol operon containing ctfAB, adc (acetoacetate decarboxylase), and ald (aldehyde dehydrogenase) was also cloned from Clostridium beijerinckii NCIMB 8052 and expressed in C. tyrobutyricum (?ack, adhE2). Mutants expressing these genes were evaluated for their ability to produce butanol from glucose in batch fermentations at pH5.0 and 6.0. Compared to C. tyrobutyricum (?ack, adhE2) without expressing ctfAB, all mutants with ctfAB overexpression produced more butanol, with butanol yield increased to 0.22?-?0.26g/g (vs. 0.10?-?0.13g/g) and productivity to 0.35g/lh (vs. 0.13g/lh) because of the reduced acetate and butyrate production. The expression of ctfAB also resulted in acetone production from acetoacetate through a non-enzymatic decarboxylation. PMID:25851718

  15. Synthesis and magnetic properties of superparamagnetic CoAs nanostructures

    NASA Astrophysics Data System (ADS)

    Desai, P.; Ashokaan, N.; Masud, J.; Pariti, A.; Nath, M.

    2015-03-01

    This article provides a comprehensive guide on the synthesis and characterization of superparamagnetic CoAs nanoparticles and elongated nanostructures with high blocking temperature, (TB), via hot-injection precipitation and solvothermal methods. Cobalt arsenides constitute an important family of magnetically active solids that find a variety of applications ranging from magnetic semiconductors to biomedical imaging. While the higher temperature hot-injection precipitation technique (300 C) yields pure CoAs nanostructures, the lower temperature solvothermal method (200 C) yields a mixture of CoAs nanoparticles along with other Co-based impurity phases. The synthesis in all these cases involved usage of triphenylarsine ((C6H5)3As) as the As precursor which reacts with solid Co2(CO)8 by ligand displacement to yield a single source precursor. The surfactant, hexadecylamine (HDA) further assists in controlling the morphology of the nanostructures. HDA also provides a basic medium and molten flux-like conditions for the redox chemistry to occur between Co and As at elevated temperatures. The influence of the length of reaction time was investigated by studying the evolution of product morphology over time. It was observed that while spontaneous nucleation at higher temperature followed by controlled growth led to the predominant formation of short nanorods, with longer reaction time, the nanorods were further converted to nanoparticles. The size of the nanoparticles obtained, was mostly in the range of 10-15 nm. The key finding of this work is exceptionally high coercivity in CoAs nanostructures for the first time. Coercivity observed was as high as 0.1 T (1000 Oe) at 2 K. These kinds of magnetic nanostructures find multiple applications in spintronics, whereas the superparamagnetic nanoparticles are viable for use in magnetic storage, ferrofluids and as contrast enhancing agents in MRI.

  16. Acetyl-coenzyme A

    PubMed Central

    Schroeder, Sabrina; Pendl, Tobias; Zimmermann, Andreas; Eisenberg, Tobias; Carmona-Gutierrez, Didac; Ruckenstuhl, Christoph; Mario, Guillermo; Pietrocola, Federico; Harger, Alexandra; Magnes, Christoph; Sinner, Frank; Pieber, Thomas R; Dengjel, Jrn; Sigrist, Stephan J; Kroemer, Guido; Madeo, Frank

    2014-01-01

    As the major lysosomal degradation pathway, autophagy represents the guardian of cellular homeostasis, removing damaged and potentially harmful material and replenishing energy reserves in conditions of starvation. Given its vast physiological importance, autophagy is crucially involved in the process of aging and associated pathologies. Although the regulation of autophagy strongly depends on nutrient availability, specific metabolites that modulate autophagic responses are poorly described. Recently, we revealed nucleo-cytosolic acetyl-coenzyme A (AcCoA) as a phylogenetically conserved inhibitor of starvation-induced and age-associated autophagy. AcCoA is the sole acetyl-group donor for protein acetylation, explaining why pharmacological or genetic manipulations that modify the concentrations of nucleo-cytosolic AcCoA directly affect the levels of protein acetylation. The acetylation of histones and cytosolic proteins inversely correlates with the rate of autophagy in yeast and mammalian cells, respectively, despite the fact that the routes of de novo AcCoA synthesis differ across phyla. Thus, we propose nucleo-cytosolic AcCoA to act as a conserved metabolic rheostat, linking the cellular metabolic state to the regulation of autophagy via effects on protein acetylation. PMID:24904996

  17. The extended reductive acetyl-CoA pathway: ATPases in metal cluster maturation and reductive activation.

    PubMed

    Jeoung, Jae-Hun; Goetzl, Sebastian; Hennig, Sandra Elisabeth; Fesseler, Jochen; Wörmann, Christina; Dendra, Julia; Dobbek, Holger

    2014-05-01

    The reductive acetyl-coenzyme A (acetyl-CoA) pathway, also known as the Wood-Ljungdahl pathway, allows reduction and condensation of two molecules of carbon dioxide (CO2) to build the acetyl-group of acetyl-CoA. Productive utilization of CO2 relies on a set of oxygen sensitive metalloenzymes exploiting the metal organic chemistry of nickel and cobalt to synthesize acetyl-CoA from activated one-carbon compounds. In addition to the central catalysts, CO dehydrogenase and acetyl-CoA synthase, ATPases are needed in the pathway. This allows the coupling of ATP binding and hydrolysis to electron transfer against a redox potential gradient and metal incorporation to (re)activate one of the central players of the pathway. This review gives an overview about our current knowledge on how these ATPases achieve their tasks of maturation and reductive activation. PMID:24477517

  18. Biochemical and Kinetic Characterization of the Recombinant ADP-Forming Acetyl Coenzyme A Synthetase from the Amitochondriate Protozoan Entamoeba histolytica

    PubMed Central

    Jones, Cheryl P.

    2014-01-01

    Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PPi-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PPi were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 0.17 mM (mean standard deviation) and 0.75 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PPi displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed. PMID:25303954

  19. The hydrolysis of polyimides

    NASA Technical Reports Server (NTRS)

    Hoagland, P. D.; Fox, S. W.

    1973-01-01

    Thermal polymerization of aspartic acid produces a polysuccinimide (I), a chain of aspartoyl residues. An investigation was made of the alkaline hydrolysis of the imide rings of (I) which converts the polyimide to a polypeptide. The alkaline hydrolysis of polyimides can be expected to be kinetically complex due to increasing negative charge generated by carboxylate groups. For this reason, a diimide, phthaloyl-DL-aspartoyl-beta-alanine (IIA) was synthesized for a progressive study of the hydrolysis of polyimides. In addition, this diimide (IIA) can be related to thalidomide and might be expected to exhibit similar reactivity during hydrolysis of the phthalimide ring.

  20. Mercury methylation independent of the acetyl-coenzyme A pathway in sulfate-reducing bacteria.

    PubMed

    Ekstrom, Eileen B; Morel, François M M; Benoit, Janina M

    2003-09-01

    Sulfate-reducing bacteria (SRB) in anoxic waters and sediments are the major producers of methylmercury in aquatic systems. Although a considerable amount of work has addressed the environmental factors that control methylmercury formation and the conditions that control bioavailability of inorganic mercury to SRB, little work has been undertaken analyzing the biochemical mechanism of methylmercury production. The acetyl-coenzyme A (CoA) pathway has been implicated as being key to mercury methylation in one SRB strain, Desulfovibrio desulfuricans LS, but this result has not been extended to other SRB species. To probe whether the acetyl-CoA pathway is the controlling biochemical process for methylmercury production in SRB, five incomplete-oxidizing SRB strains and two Desulfobacter strains that do not use the acetyl-CoA pathway for major carbon metabolism were assayed for methylmercury formation and acetyl-CoA pathway enzyme activities. Three of the SRB strains were also incubated with chloroform to inhibit the acetyl-CoA pathway. So far, all species that have been found to have acetyl-CoA activity are complete oxidizers that require the acetyl-CoA pathway for basic metabolism, as well as methylate mercury. Chloroform inhibits Hg methylation in these species either by blocking the methylating enzyme or by indirect effects on metabolism and growth. However, we have identified four incomplete-oxidizing strains that clearly do not utilize the acetyl-CoA pathway either for metabolism or mercury methylation (as confirmed by the absence of chloroform inhibition). Hg methylation is thus independent of the acetyl-CoA pathway and may not require vitamin B(12) in some and perhaps many incomplete-oxidizing SRB strains. PMID:12957930

  1. Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase.

    PubMed Central

    Guranowski, A; Günther Sillero, M A; Sillero, A

    1994-01-01

    Yeast (Saccharomyces cerevisiae) acetyl coenzyme A (CoA) synthetase (EC 6.2.1.1) catalyzes the synthesis of adenosine 5'-tetraphosphate (P4A) and adenosine 5'-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate (P3 or P4), with relative velocities of 7:1, respectively. Of 12 nucleotides tested as potential donors of nucleotidyl moiety, only ATP, adenosine-5'-O-[3-thiotriphosphate], and acetyl-AMP were substrates, with relative velocities of 100, 62, and 80, respectively. The Km values for ATP, P3, and acetyl-AMP were 0.16, 4.7, and 1.8 mM, respectively. The synthesis of p4A could proceed in the absence of exogenous acetate but was stimulated twofold by acetate, with an apparent Km value of 0.065 mM. CoA did not participate in the synthesis of p4A (p5A) and inhibited the reaction (50% inhibitory concentration of 0.015 mM). At pH 6.3, which was optimum for formation of p4A (p5A), the rate of acetyl-CoA synthesis (1.84 mumol mg-1 min-1) was 245 times faster than the rate of synthesis of p4A measured in the presence of acetate. The known formation of p4A (p5A) in yeast sporulation and the role of acetate may therefore be related to acetyl-CoA synthetase. Images PMID:7910605

  2. ATP-Citrate Lyase Is Required for Production of Cytosolic Acetyl Coenzyme A and Development in Aspergillus nidulans▿

    PubMed Central

    Hynes, Michael J.; Murray, Sandra L.

    2010-01-01

    Acetyl coenzyme A (CoA) is a central metabolite in carbon and energy metabolism and in the biosynthesis of cellular molecules. A source of cytoplasmic acetyl-CoA is essential for the production of fatty acids and sterols and for protein acetylation, including histone acetylation in the nucleus. In Saccharomyces cerevisiae and Candida albicans acetyl-CoA is produced from acetate by cytoplasmic acetyl-CoA synthetase, while in plants and animals acetyl-CoA is derived from citrate via ATP-citrate lyase. In the filamentous ascomycete Aspergillus nidulans, tandem divergently transcribed genes (aclA and aclB) encode the subunits of ATP-citrate lyase, and we have deleted these genes. Growth is greatly diminished on carbon sources that do not result in cytoplasmic acetyl-CoA, such as glucose and proline, while growth is not affected on carbon sources that result in the production of cytoplasmic acetyl-CoA, such as acetate and ethanol. Addition of acetate restores growth on glucose or proline, and this is dependent on facA, which encodes cytoplasmic acetyl-CoA synthetase, but not on the regulatory gene facB. Transcription of aclA and aclB is repressed by growth on acetate or ethanol. Loss of ATP-citrate lyase results in severe developmental effects, with the production of asexual spores (conidia) being greatly reduced and a complete absence of sexual development. This is in contrast to Sordaria macrospora, in which fruiting body formation is initiated but maturation is defective in an ATP-citrate lyase mutant. Addition of acetate does not repair these defects, indicating a specific requirement for high levels of cytoplasmic acetyl-CoA during differentiation. Complementation in heterokaryons between aclA and aclB deletions for all phenotypes indicates that the tandem gene arrangement is not essential. PMID:20495057

  3. Simultaneous pretreatment and enzymatic hydrolysis of forage biomass

    SciTech Connect

    Henk, L.; Linden, J.C.

    1993-12-31

    Sweet sorghum is an attractive fermentation feedstock because as much as 40% of the dry weight consists of readily femented sugars such as sucrose, glucose and frutose. Cellulose and hemicellulose comprise another 50%. However, if this material is to be used a year-round feedstock for ethanol production, a stable method of storage must be developed to maintain the sugar content. A modified version of the traditional ensiling process is made effective by the addition of cellulolytic/hemicellulolytic enzymes and lactic acid bacteria to freshly chopped sweet sorghum prior to the production of silage. In situ hydrolysis of cellulose and hemicellulose occurs concurrently with the acidic ensiling fementation. By hydolyzing the acetyl groups using acetyl xylan esterase and 3-0-methyl glucuronyl side chains using pectinase from hemicellulose, cellulose becomes accessible to hydrolysis by cellulase, both during in situ ensiling with enzymes and in the simultaneous saccharification and fermentation (SSF) to ethanol.

  4. N-Acetylaspartate reductions in brain injury: impact on post-injury neuroenergetics, lipid synthesis, and protein acetylation

    PubMed Central

    Moffett, John R.; Arun, Peethambaran; Ariyannur, Prasanth S.; Namboodiri, Aryan M. A.

    2013-01-01

    N-Acetylaspartate (NAA) is employed as a non-invasive marker for neuronal health using proton magnetic resonance spectroscopy (MRS). This utility is afforded by the fact that NAA is one of the most concentrated brain metabolites and that it produces the largest peak in MRS scans of the healthy human brain. NAA levels in the brain are reduced proportionately to the degree of tissue damage after traumatic brain injury (TBI) and the reductions parallel the reductions in ATP levels. Because NAA is the most concentrated acetylated metabolite in the brain, we have hypothesized that NAA acts in part as an extensive reservoir of acetate for acetyl coenzyme A synthesis. Therefore, the loss of NAA after TBI impairs acetyl coenzyme A dependent functions including energy derivation, lipid synthesis, and protein acetylation reactions in distinct ways in different cell populations. The enzymes involved in synthesizing and metabolizing NAA are predominantly expressed in neurons and oligodendrocytes, respectively, and therefore some proportion of NAA must be transferred between cell types before the acetate can be liberated, converted to acetyl coenzyme A and utilized. Studies have indicated that glucose metabolism in neurons is reduced, but that acetate metabolism in astrocytes is increased following TBI, possibly reflecting an increased role for non-glucose energy sources in response to injury. NAA can provide additional acetate for intercellular metabolite trafficking to maintain acetyl CoA levels after injury. Here we explore changes in NAA, acetate, and acetyl coenzyme A metabolism in response to brain injury. PMID:24421768

  5. N-Acetyl-4-aminophenol (paracetamol), N-acetyl-2-aminophenol and acetanilide in urine samples from the general population, individuals exposed to aniline and paracetamol users.

    PubMed

    Dierkes, Georg; Weiss, Tobias; Modick, Hendrik; Käfferlein, Heiko Udo; Brüning, Thomas; Koch, Holger M

    2014-01-01

    Epidemiological studies suggest associations between the use of N-acetyl-4-aminophenol (paracetamol) during pregnancy and increased risks of reproductive disorders in the male offspring. Previously we have reported a ubiquitous urinary excretion of N-acetyl-4-aminophenol in the general population. Possible sources are (1) direct intake of paracetamol through medication, (2) paracetamol residues in the food chain and (3) environmental exposure to aniline or related substances that are metabolized into N-acetyl-4-aminophenol. In order to elucidate the origins of the excretion of N-acetyl-4-aminophenol in urine and to contribute to the understanding of paracetamol and aniline metabolism in humans we developed a rapid, turbulent-flow HPLC-MS/MS method with isotope dilution for the simultaneous quantification of N-acetyl-4-aminophenol and two other aniline related metabolites, N-acetyl-2-aminophenol and acetanilide. We applied this method to three sets of urine samples: (1) individuals with no known exposure to aniline and also no recent paracetamol medication; (2) individuals after occupational exposure to aniline but no paracetamol medication and (3) paracetamol users. We confirmed the omnipresent excretion of N-acetyl-4-aminophenol. Additionally we revealed an omnipresent excretion of N-acetyl-2-aminophenol. In contrast, acetanilide was only found after occupational exposure to aniline, not in the general population or after paracetamol use. The results lead to four preliminary conclusions: (1) other sources than aniline seem to be responsible for the major part of urinary N-acetyl-4-aminophenol in the general population; (2) acetanilide is a metabolite of aniline in man and a valuable biomarker for aniline in occupational settings; (3) aniline baseline levels in the general population measured after chemical hydrolysis do not seem to originate from acetanilide and hence not from a direct exposure to aniline itself and (4) N-acetyl-2-aminophenol does not seem to be related to aniline nor to N-acetyl-4-aminophenol in man. PMID:24370547

  6. Purification and characterization of the acetyl-CoA synthetase from Mycobacterium tuberculosis.

    PubMed

    Li, Ru; Gu, Jing; Chen, Peng; Zhang, Zhiping; Deng, Jiaoyu; Zhang, Xianen

    2011-11-01

    Acetyl-CoA (AcCoA) synthetase (Acs) catalyzes the conversion of acetate into AcCoA, which is involved in many catabolic and anabolic pathways. Although this enzyme has been studied for many years in many organisms, the properties of Mycobacterium tuberculosis Acs and the regulation of its activity remain unknown. Here, the putative acs gene of M. tuberculosis H37Rv (Mt-Acs) was expressed as a fusion protein with 6×His-tag on the C-terminus in Escherichia coli. The recombinant Mt-Acs protein was successfully purified and then its enzymatic characteristics were analyzed. The optimal pH and temperature, and the kinetic parameters of Mt-Acs were determined. To investigate whether Mt-Acs is regulated by lysine acetylation as reported for Salmonella enterica Acs, its mutant K617R was also generated. Determination of the enzymatic activity suggests that Lys-617 is critical for its function. We further demonstrated that Mt-Acs underwent auto-acetylation with acetate but not with AcCoA as the acetyl donor, which resulted in the decrease of its activity. CoA, the substrate for AcCoA formation, inhibited the auto-acetylation. Furthermore, the silent information regulator (Sir2) of M. tuberculosis (Mt-Sir2) could catalyze Mt-Acs deacetylation, which resulted in activation of Acs. These results may provide more insights into the physiological roles of Mt-Acs in M. tuberculosis central metabolism. PMID:21896569

  7. Progressing batch hydrolysis process

    DOEpatents

    Wright, J.D.

    1985-01-10

    A progressive batch hydrolysis process is disclosed for producing sugar from a lignocellulosic feedstock. It comprises passing a stream of dilute acid serially through a plurality of percolation hydrolysis reactors charged with feed stock, at a flow rate, temperature and pressure sufficient to substantially convert all the cellulose component of the feed stock to glucose. The cooled dilute acid stream containing glucose, after exiting the last percolation hydrolysis reactor, serially fed through a plurality of pre-hydrolysis percolation reactors, charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the hemicellulose component of said feedstock to glucose. The dilute acid stream containing glucose is cooled after it exits the last prehydrolysis reactor.

  8. Pretreatment and Enzymatic Hydrolysis

    SciTech Connect

    2006-06-01

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

  9. Mitochondrial disease genes COA6, COX6B and SCO2 have overlapping roles in COX2 biogenesis.

    PubMed

    Ghosh, Alok; Pratt, Anthony T; Soma, Shivatheja; Theriault, Sarah G; Griffin, Aaron T; Trivedi, Prachi P; Gohil, Vishal M

    2016-02-15

    Biogenesis of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain, is a complex process facilitated by several assembly factors. Pathogenic mutations were recently reported in one such assembly factor, COA6, and our previous work linked Coa6 function to mitochondrial copper metabolism and expression of Cox2, a copper-containing subunit of CcO. However, the precise role of Coa6 in Cox2 biogenesis remained unknown. Here we show that yeast Coa6 is an orthologue of human COA6, and like Cox2, is regulated by copper availability, further implicating it in copper delivery to Cox2. In order to place Coa6 in the Cox2 copper delivery pathway, we performed a comprehensive genetic epistasis analysis in the yeast Saccharomyces cerevisiae and found that simultaneous deletion of Coa6 and Sco2, a mitochondrial copper metallochaperone, or Coa6 and Cox12/COX6B, a structural subunit of CcO, completely abrogates Cox2 biogenesis. Unlike Coa6 deficient cells, copper supplementation fails to rescue Cox2 levels of these double mutants. Overexpression of Cox12 or Sco proteins partially rescues the coa6? phenotype, suggesting their overlapping but non-redundant roles in copper delivery to Cox2. These genetic data are strongly corroborated by biochemical studies demonstrating physical interactions between Coa6, Cox2, Cox12 and Sco proteins. Furthermore, we show that patient mutations in Coa6 disrupt Coa6-Cox2 interaction, providing the biochemical basis for disease pathogenesis. Taken together, these results place COA6 in the copper delivery pathway to CcO and, surprisingly, link it to a previously unidentified function of CcO subunit Cox12 in Cox2 biogenesis. PMID:26669719

  10. A method of COA based on multi-agent co-evolutionary algorithm

    NASA Astrophysics Data System (ADS)

    Yu, Xin; Dong, Shuaijun; Wang, Hui

    2011-11-01

    In the complex situation of the battlefield, COA (course of action) places an important role. It is required to coordinate many resources in some actions to achieve the desired purpose in the battle. The main goal of COA is to arrange the action in the right order and to put the right resource in the right action. The task which is composed of many actions is always extremely complex. Therefore, COA is actually NP-Hard and a multi-objective optimization problem. It is difficult to solve this problem by common methods. In this paper, a mechanism of co-evolutionary is introduced to solve the problem of COA. It deals well with the problems of resource management and action scheduling.

  11. Genetics Home Reference: Succinyl-CoA:3-ketoacid CoA transferase deficiency

    MedlinePLUS

    ... and Families Resources for Health Professionals What glossary definitions help with understanding succinyl-CoA:3-ketoacid CoA ... lethargy ; mitochondria ; prevalence ; recessive ; transferase You may find definitions for these and many other terms in the ...

  12. 3-Hydroxy-3-methylglutaryl CoA lyase (HL): Mouse and human HL gene (HMGCL) cloning and detection of large gene deletions in two unrelated HL-deficient patients

    SciTech Connect

    Wang, S.P.; Robert, M.F.; Mitchell, G.A.

    1996-04-01

    3-hydroxy-3-methylglutaryl CoA lyase (HL, EC 4.1.3.4) catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetic acid and acetyl CoA, the final reaction of both ketogenesis and leucine catabolism. Autosomal-recessive HL deficiency in humans results in episodes of hypoketotic hypoglycemia and coma. Using a mouse HL cDNA as a probe, we isolated a clone containing the full-length mouse HL gene that spans about 15 kb of mouse chromosome 4 and contains nine exons. The promoter region of the mouse HL gene contains elements characteristic of a housekeeping gene: a CpG island containing multiple Sp1 binding sites surrounds exon 1, and neither a TATA nor a CAAT box are present. We identified multiple transcription start sites in the mouse HL gene, 35 to 9 bases upstream of the translation start codon. We also isolated two human HL genomic clones that include HL exons 2 to 9 within 18 kb. The mouse and human HL genes (HGMW-approved symbol HMGCL) are highly homologous, with identical locations of intron-exon junctions. By genomic Southern blot analysis and exonic PCR, was found 2 of 33 HL-deficient probands to be homozygous for large deletions in the HL gene. 26 refs., 4 figs., 2 tabs.

  13. Structure of the p300 Histone Acetyltransferase Bound to Acetyl-Coenzyme A and Its Analogues

    PubMed Central

    2015-01-01

    The p300 and CBP transcriptional coactivator paralogs (p300/CBP) regulate a variety of different cellular pathways, in part, by acetylating histones and more than 70 non-histone protein substrates. Mutation, chromosomal translocation, or other aberrant activities of p300/CBP are linked to many different diseases, including cancer. Because of its pleiotropic biological roles and connection to disease, it is important to understand the mechanism of acetyl transfer by p300/CBP, in part so that inhibitors can be more rationally developed. Toward this goal, a structure of p300 bound to a Lys-CoA bisubstrate HAT inhibitor has been previously elucidated, and the enzymes catalytic mechanism has been investigated. Nonetheless, many questions underlying p300/CBP structure and mechanism remain. Here, we report a structural characterization of different reaction states in the p300 activity cycle. We present the structures of p300 in complex with an acetyl-CoA substrate, a CoA product, and an acetonyl-CoA inhibitor. A comparison of these structures with the previously reported p300/Lys-CoA complex demonstrates that the conformation of the enzyme active site depends on the interaction of the enzyme with the cofactor, and is not apparently influenced by protein substrate lysine binding. The p300/CoA crystals also contain two poly(ethylene glycol) moieties bound proximal to the cofactor binding site, implicating the path of protein substrate association. The structure of the p300/acetonyl-CoA complex explains the inhibitory and tight binding properties of the acetonyl-CoA toward p300. Together, these studies provide new insights into the molecular basis of acetylation by p300 and have implications for the rational development of new small molecule p300 inhibitors. PMID:24819397

  14. Mechanistic insight with HBCH2CoA as a probe to polyhydroxybutyrate (PHB) synthases.

    PubMed

    Zhang, Wei; Shrestha, Ruben; Buckley, Rachael M; Jewell, Jamie; Bossmann, Stefan H; Stubbe, JoAnne; Li, Ping

    2014-08-15

    Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of 3-(R)-hydroxybutyrate coenzyme A (HBCoA) to produce polyoxoesters of 1-2 MDa. A substrate analogue HBCH2CoA, in which the S in HBCoA is replaced with a CH2 group, was synthesized in 13 steps using a chemoenzymatic approach in a 7.5% overall yield. Kinetic studies reveal it is a competitive inhibitor of a class I and a class III PHB synthases, with Kis of 40 and 14 ?M, respectively. To probe the elongation steps of the polymerization, HBCH2CoA was incubated with a synthase acylated with a [(3)H]-saturated trimer-CoA ([(3)H]-sTCoA). The products of the reaction were shown to be the methylene analogue of [(3)H]-sTCoA ([(3)H]-sT-CH2-CoA), saturated dimer-([(3)H]-sD-CO2H), and trimer-acid ([(3)H]-sT-CO2H), distinct from the expected methylene analogue of [(3)H]-saturated tetramer-CoA ([(3)H]-sTet-CH2-CoA). Detection of [(3)H]-sT-CH2-CoA and its slow rate of formation suggest that HBCH2CoA may be reporting on the termination and repriming process of the synthases, rather than elongation. PMID:24896226

  15. CoA protects against the deleterious effects of caloric overload in Drosophila.

    PubMed

    Palanker Musselman, Laura; Fink, Jill L; Baranski, Thomas J

    2016-03-01

    We developed a Drosophila model of T2D in which high sugar (HS) feeding leads to insulin resistance. In this model, adipose TG storage is protective against fatty acid toxicity and diabetes. Initial biochemical and gene expression studies suggested that deficiency in CoA might underlie reduced TG synthesis in animals during chronic HS feeding. Focusing on the Drosophila fat body (FB), which is specialized for TG storage and lipolysis, we undertook a series of experiments to test the hypothesis that CoA could protect against the deleterious effects of caloric overload. Quantitative metabolomics revealed a reduction in substrate availability for CoA synthesis in the face of an HS diet. Further reducing CoA synthetic capacity by expressing FB-specific RNAi targeting pantothenate kinase (PK orfumble) or phosphopantothenoylcysteine synthase (PPCS) exacerbated HS-diet-induced accumulation of FFAs. Dietary supplementation with pantothenic acid (vitamin B5, a precursor of CoA) was able to ameliorate HS-diet-induced FFA accumulation and hyperglycemia while increasing TG synthesis. Taken together, our data support a model where free CoA is required to support fatty acid esterification and to protect against the toxicity of HS diets. PMID:26805007

  16. Molecular cloning of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli

    PubMed Central

    Walker, Kevin; Croteau, Rodney

    2000-01-01

    The cDNA clone for a 10-deacetylbaccatin III-10-O-acetyl transferase, which catalyzes formation of the last diterpene intermediate in the Taxol biosynthetic pathway, has been isolated from Taxus cuspidata. By using consensus sequences from an assembly of transacylases of plant origin and from many deduced proteins of unknown function, a homology-based PCR cloning strategy was employed to amplify initially a 911-bp gene fragment of the putative taxane C-10 hydroxyl acetyl transferase from Taxus. This amplicon was used to screen a cDNA library constructed from mRNA isolated from methyl jasmonate-induced Taxus cells, from which the full-length 10-deacetylbaccatin III-10-O-transacetylase sequence was obtained. Expression of the ORF from pCWori+ in Escherichia coli JM109 afforded a functional enzyme, as determined by 1H-NMR and MS verification of the product baccatin III derived from 10-deacetylbaccatin III and acetyl CoA. The full-length cDNA has an ORF of 1,320 bp corresponding to a deduced protein of 440 residues with a calculated molecular weight of 49,052, consistent with the size of the operationally soluble, monomeric, native acetyl transferase. The recombinant acetyl transferase has a pH optimum of 7.5, has Km values of 10 ?M and 8 ?M for 10-deacetylbaccatin III and acetyl CoA, respectively, and is apparently regiospecific toward the 10-hydroxyl group of the taxane ring. Amino acid sequence comparison of 10-deacetylbaccatin III-10-O-acetyl transferase with taxadienol-5-O-acetyl transferase and with other known acyl transferases of plant origin indicates a significant degree of similarity between these enzymes (80% and 6467%, respectively). PMID:10639122

  17. Thermodynamics of actinide hydrolysis

    SciTech Connect

    Hromadka, P.M.; Wong, P.J.; Sullivan, J.C.; Choppin, G.R.

    1996-10-01

    Thermodynamic parameters of Np(V), U(VI), and Pu(VI) hydrolysis have been measured at 5-45{degrees}C via calorimetric titration. These parameters are needed to evaluate actinide migration behavior in the near field of nuclear waste repositories which are expected to reach temperatures of 100{degrees}C. The enthalpies determined exhibit both a temperature as well as an ionic media dependence. Calorimetry provides a direct, quantitative method to observe the energetic consequences of hydrolysis and the solute-solvent interactions between actinide metals and their coordination spheres in aqueous solution. The different enthalpies of hydrolysis in 1M Me{sub 4}NCl and NaClO{sub 4} provide further insight concerning their second sphere salvation structures. Our results suggest {Delta}Cp is linear over the 40{degrees} temperature range. The linear correlation between {Delta}{Delta}H and {Delta}Cp reflects the dominance of solvent reorganization in the {Delta}Cp term. Based on our values of {Delta}Cp for Np(V), U(VI), and Pu(VI) hydrolysis, the Van`t Hoff equation (assumes {Delta}Cp=O) may not be used to estimate hydrolysis constants at elevated temperatures.

  18. Progressing batch hydrolysis process

    DOEpatents

    Wright, John D. (Denver, CO)

    1986-01-01

    A progressive batch hydrolysis process for producing sugar from a lignocellulosic feedstock, comprising passing a stream of dilute acid serially through a plurality of percolation hydrolysis reactors charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the cellulose component of the feedstock to glucose; cooling said dilute acid stream containing glucose, after exiting the last percolation hydrolysis reactor, then feeding said dilute acid stream serially through a plurality of prehydrolysis percolation reactors, charged with said feedstock, at a flow rate, temperature and pressure sufficient to substantially convert all the hemicellulose component of said feedstock to glucose; and cooling the dilute acid stream containing glucose after it exits the last prehydrolysis reactor.

  19. Riboflavin-responsive glutaryl CoA dehydrogenase deficiency.

    PubMed

    Chalmers, Ronald A; Bain, Murray D; Zschocke, Johannes

    2006-05-01

    We report here riboflavin responsiveness in a patient with glutaryl CoA dehydrogenase (GCDH) deficiency, compound heterozygous for the S139L and P248L mutations and with 20% residual GCDH enzyme activity in vitro. Our results suggest the mitochondrial GCDH homotetramer remains intact with one of these mutations associated with the binding site of the single FAD cofactor and that pharmacological doses of the cofactor precursor may be sufficient to induce an increase in activity in the mutant GCDH enzyme, although not sufficient to normalise urinary organic acid excretion. Serine139 is one of nine conserved amino acid residues that line the binding site of the protein and is in close proximity to both substrate and FAD cofactor. It is possible that steric alterations caused by substitution of serine with leucine at this position may be overcome with high cofactor concentrations. P248L is also associated with some residual GCDH activity in other patients and the unique combination of S139L with P248L may also explain the results in our patient. Responsiveness to riboflavin in our patient has been compared with two other patients with glutaric aciduria type 1 and minimal residual GCDH activity, one with homozygosity for the R257Q mutation and one with heterozygosity for the G354S mutation and a novel G156V mutation. A low lysine diet reduced glutaric acid excretion in our riboflavin-responsive GCDH-deficient patient almost to control values. She is now 21 years of age and clinically and neurologically normal. PMID:16377226

  20. Exome sequence reveals mutations in CoA synthase as a cause of neurodegeneration with brain iron accumulation.

    PubMed

    Dusi, Sabrina; Valletta, Lorella; Haack, Tobias B; Tsuchiya, Yugo; Venco, Paola; Pasqualato, Sebastiano; Goffrini, Paola; Tigano, Marco; Demchenko, Nikita; Wieland, Thomas; Schwarzmayr, Thomas; Strom, Tim M; Invernizzi, Federica; Garavaglia, Barbara; Gregory, Allison; Sanford, Lynn; Hamada, Jeffrey; Bettencourt, Conceio; Houlden, Henry; Chiapparini, Luisa; Zorzi, Giovanna; Kurian, Manju A; Nardocci, Nardo; Prokisch, Holger; Hayflick, Susan; Gout, Ivan; Tiranti, Valeria

    2014-01-01

    Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA. PMID:24360804

  1. Biochemical and kinetic characterization of the recombinant ADP-forming acetyl coenzyme A synthetase from the amitochondriate protozoan Entamoeba histolytica.

    PubMed

    Jones, Cheryl P; Ingram-Smith, Cheryl

    2014-12-01

    Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PPi-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PPi were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PPi displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed. PMID:25303954

  2. Characterization of O-acetylation of N-acetylglucosamine: a novel structural variation of bacterial peptidoglycan.

    PubMed

    Bernard, Elvis; Rolain, Thomas; Courtin, Pascal; Guillot, Alain; Langella, Philippe; Hols, Pascal; Chapot-Chartier, Marie-Pierre

    2011-07-01

    Peptidoglycan (PG) N-acetyl muramic acid (MurNAc) O-acetylation is widely spread in gram-positive bacteria and is generally associated with resistance against lysozyme and endogenous autolysins. We report here the presence of O-acetylation on N-acetylglucosamine (GlcNAc) in Lactobacillus plantarum PG. This modification of glycan strands was never described in bacteria. Fine structural characterization of acetylated muropeptides released from L. plantarum PG demonstrated that both MurNAc and GlcNAc are O-acetylated in this species. These two PG post-modifications rely on two dedicated O-acetyltransferase encoding genes, named oatA and oatB, respectively. By analyzing the resistance to cell wall hydrolysis of mutant strains, we showed that GlcNAc O-acetylation inhibits N-acetylglucosaminidase Acm2, the major L. plantarum autolysin. In this bacterial species, inactivation of oatA, encoding MurNAc O-acetyltransferase, resulted in marked sensitivity to lysozyme. Moreover, MurNAc over-O-acetylation was shown to activate autolysis through the putative N-acetylmuramoyl-L-alanine amidase LytH enzyme. Our data indicate that in L. plantarum, two different O-acetyltransferases play original and antagonistic roles in the modulation of the activity of endogenous autolysins. PMID:21586574

  3. Regulation of glycolysis and gluconeogenesis by acetylation of PKM and PEPCK.

    PubMed

    Xiong, Y; Lei, Q-Y; Zhao, S; Guan, K-L

    2011-01-01

    Glycolysis is a catabolic process of glucose hydrolysis needed for energy and biosynthetic intermediates, whereas gluconeogenesis is a glucose production process important for maintaining blood glucose levels during starvation. Although they share many enzymes, these two processes are not simply the reverse of each other and are instead reciprocally regulated. Two key enzymes that regulate irreversible steps in these two processes are pyruvate kinase (PK) and phosphoenolpyruvate carboxy kinase (PEPCK), which catalyze the last and first step of glycolysis and gluconeogenesis, respectively, and are both regulated by lysine acetylation. Acetylation at Lys305 of the PKM (muscle form of PK) decreases its activity and also targets it for chaperone-mediated autophagy and subsequent lysosome degradation. Acetylation of PEPCK, on the other hand, targets it for ubiquitylation by the HECT E3 ligase, UBR5/EDD1, and subsequent proteasomal degradation. These studies established a model in which acetylation regulates metabolic enzymes via different mechanisms and also revealed cross talk between acetylation and ubiquitination. Given that most metabolic enzymes are acetylated, we propose that acetylation is a major posttranslational modifier that regulates cellular metabolism. PMID:22096030

  4. Isomerization of 1-O-indol-3-ylacetyl-beta-D-glucose. Enzymatic hydrolysis of 1-O, 4-O, and 6-O-indol-3-ylacetyl-beta-D-glucose and the enzymatic synthesis of indole-3-acetyl glycerol by a hormone metabolizing complex

    NASA Technical Reports Server (NTRS)

    Kowalczyk, S.; Bandurski, R. S.

    1990-01-01

    The first compound in the series of reactions leading to the ester conjugates of indole-3-acetic acid (IAA) in kernels of Zea mays sweet corn is the acyl alkyl acetal, 1-O-indol-3-ylacetyl-beta-D-glucose (1-O-IAGlu). The enzyme catalyzing the synthesis of this compound is UDP-glucose:indol-3-ylacetate glucosyl-transferase (IAGlu synthase). The IAA moiety of the high energy compound 1-O-IAGlu may be enzymatically transferred to myo-inositol or to glycerol or the 1-O-IAGlu may be enzymatically hydrolyzed. Alternatively, nonenzymatic acyl migration may occur to yield the 2-O, 4-O, and 6-O esters of IAA and glucose. The 4-O and 6-O esters may then be enzymatically hydrolyzed to yield free IAA and glucose. This work reports new enzymatic activities, the transfer of IAA from 1-O-IAGlu to glycerol, and the enzyme-catalyzed hydrolysis of 4-O and 6-O-IAGlu. Data is also presented on the rate of non-enzymatic acyl migration of IAA from the 1-O to the 4-O and 6-O positions of glucose. We also report that enzymes catalyzing the synthesis of 1-O-IAGlu and the hydrolysis of 1-O, 4-O, and 6-O-IAGlu fractionate as a hormone metabolizing complex. The association of synthetic and hydrolytic capabilities in enzymes which cofractionate may have physiological significance.

  5. Effect of elevated total CoA levels on metabolic pathways in cultured hepatocytes

    SciTech Connect

    Steffen, C.A.; Smith, C.M.

    1987-05-01

    Livers from fasted rats have 30% higher total CoA levels than fed rats. To determine whether this increase of total CoA influences metabolism, the rates of gluconeogenesis, fatty acid oxidation and ketogenesis were measured in hepatocytes with cyanamide (CYM) or pantothenate (PA) deficient medium used to vary total CoA levels independently of hormonal status. Primary cultures of rat hepatocytes were incubated 14 hrs with Bt/sub 2/ cAMP, dexamethasone + theophylline in PA deficient medium or with CYM (500 ..mu..M) + PA, rinsed and preincubated 0.5 hr to remove the CYM. Hepatocytes treated with CYM had total CoA levels 10-24% higher than PA deficient cells and lower rates of glucose production from lactate + pyruvate (L/P) or from alanine (0.23 +/- 0.05 and 0.089 +/- 0.02 ..mu..m/mg protein, respectively in CYM treated cells compared to 0.33 +/- 0.06 and 0.130 +/- 0.006 in PA deficient cells). This decrease was not due to CYM per se, as the direct addition of CYM stimulated glucose production from L/P. CYM treated cells with 15-40% higher total CoA and 30% higher fatty acyl-CoA levels had the same rates of (/sup 14/C)-palmitate oxidation as PA deficient cells. However, rates of ketogenesis were lower in CYM treated cells (163 +/- 11 nm/mg compared to 217 +/- 14 nm/mg protein). These results suggest that physiological alterations of hepatic total CoA levels are not necessary for fasting rates of gluconeogenesis, fatty acid oxidation and ketogenesis.

  6. First principles study of structural, electronic and magnetic properties of Mn2CoAs

    NASA Astrophysics Data System (ADS)

    Berri, Saadi; Ibrir, M.; Maouche, D.; Bensalem, R.

    2014-06-01

    We have performed first-principle calculations of the structural, electronic and magnetic properties of Mn2CoAs Heusler alloy, using full-potential linearized augmented plane wave (FP-LAPW) scheme within the GGA. Features such as the lattice constant, the bulk modulus and its pressure derivative are reported. The electronic band structures and density of states of the Mn2CoAs compound show that the spin-up electrons are metallic, but the spin-down bands have a gap of 0.48 eV, resulting in stable half-metallic ferrimagnetic behavior with a magnetic moment of 4.00 ?B.

  7. Lysozyme catalysis: kinetics of the hydrolysis of cell wall oligosaccharides.

    PubMed

    Boekelheide, K; Patt, S L; Weisz, G; Baldo, J H; Sykes, B D

    1979-06-01

    The cleavage of cell wall tetrasaccharide, the beta(1 leads to 4)-linked dimer of the basic repeating disaccharide N-acetyl-D-glucosamine-beta(1 leads to 4)-N-acetyl-D-muramic acid, by lysozyme has been studied at various concentrations of lysozyme and over long time ranges. A theoretical analysis of the kinetic results indicates that direct hydrolysis of the tetrasaccharide by binding in subsities CDEF of the active site of lysozyme is significant at long times relative to the transglycosylation pathway. The binding constant for tetrasaccharide in CDEF is shown to be 10(3) times larger than that determined on the basis of an analysis of kinetic data over a more restricted range of times and concentrations. PMID:476521

  8. Binding of acyl CoA by fatty acid binding protein and the effect on fatty acid activation

    SciTech Connect

    Burrier, R.E.; Manson, C.R.; Brecher, P.

    1987-05-01

    The ability of purified rat liver and heart fatty acid binding proteins (FABPs) to bind oleoyl CoA and modulate acyl CoA synthesis by microsomal membranes was investigated. Using binding assays employing either Lipidex 1000 or multilamellar liposomes to sequester unbound ligand, rat liver but not rat heart FABP was shown to bind radiolabeled acyl CoA. Binding studies suggest that liver FABP has a single binding site for acyl CoA which is separate from the two binding sites for fatty acids. Experiments were then performed to determine how binding may influence acyl CoA metabolism by liver microsomes or heart sarcoplasmic reticulum. Using liposomes as fatty acid donors, liver FABP stimulated acyl CoA production whereas heart FABP did not stimulate production over control values. /sup 14/C-Fatty acid-FABP complexes were prepared, incubated with membranes and acyl CoA synthetase activity was determined. Up to 70% of the fatty acid could be converted to acyl CoA in the presence of liver FABP but in the presence of heart FABP, only 45% of the fatty acid was converted. The amount of product formed was not changed by additional membrane, enzyme cofactor, or incubation time. Liver but not heart FABP bound the acyl CoA formed and removed it from the membranes. These studies suggest that liver FABP can increase the amount of acyl CoA by binding this ligand thereby removing it from the membrane and possibly aiding transport within the cell.

  9. EXPRESSION OF TURKEY TRANSCRIPTION FACTORS AND ACYL COA OXIDASE IN DIFFERENT TISSUES AND GENETIC POPULATIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several transcription factors are involved in regulating lipid metabolism in various animal tissues. Peroxisome proliferator activated receptor (PPAR) gamma and PPAR alpha regulate both lipogenesis and fatty acid oxidation. Gene fragments for PPAR gamma, PPAR alpha, and acyl CoA oxidase (ACO) have b...

  10. Adenine recognition: a motif present in ATP-, CoA-, NAD-, NADP-, and FAD-dependent proteins.

    PubMed

    Denessiouk, K A; Rantanen, V V; Johnson, M S

    2001-08-15

    Adenosine triphosphate (ATP) plays an essential role in energy transfer within the cell. In the form of NAD, adenine participates in multiple redox reactions. Phosphorylation and ATP-hydrolysis reactions have key roles in signal transduction and regulation of many proteins, especially enzymes. In each cell, proteins with many different functions use adenine and its derivatives as ligands; adenine, of course, is present in DNA and RNA. We show that an adenine binding motif, which differs according to the backbone chain direction of a loop that binds adenine (and in one variant by the participation of an aspartate side-chain), is common to many proteins; it was found from an analysis of all adenylate-containing protein structures from the Protein Data Bank. Indeed, 224 protein-ligand complexes (86 different proteins) from a total of 645 protein structure files bind ATP, CoA, NAD, NADP, FAD, or other adenine-containing ligands, and use the same structural elements to recognize adenine, regardless of whether the ligand is a coenzyme, cofactor, substrate, or an allosteric effector. The common adenine-binding motif shown in this study is simple to construct. It uses only (1) backbone polar interactions that are not dependent on the protein sequence or particular properties of amino acid side-chains, and (2) nonspecific hydrophobic interactions. This is probably why so many different proteins with different functions use this motif to bind an adenylate-containing ligand. The adenylate-binding motif reported is present in "ancient proteins" common to all living organisms, suggesting that adenine-containing ligands and the common motif for binding them were exploited very early in evolution. The geometry of adenine binding by this motif mimics almost exactly the geometry of adenine base-pairing seen in DNA and RNA. PMID:11455601

  11. Acid hydrolysis of Jerusalem artichoke for ethanol fermentation

    SciTech Connect

    Kim, K.; Hamdy, M.K.

    1986-01-01

    An excellent substrate for ethanol production is the Jerusalem artichoke (JA) tuber (Helianthus tuberosus). This crop contains a high level of inulin that can be hydrolyzed mainly to D-fructose and has several distinct advantages as an energy source compared to others. The potential ethanol yield of ca. 4678 L/ha on good agricultural land is equivalent to that obtained from sugar beets and twice that of corn. When JA is to be used for ethanol fermentation by conventional yeast, it is first converted to fermentable sugars by enzymes or acids although various strains of yeast were used for the direct fermentation of JA extracts. Fleming and GrootWassink compared various acids (hydrochloric, sulfuric, citric, and phosphoric) and strong cation exchange resin for their effectiveness on inulin hydrolysis and reported that no differences were noted among the acids or resin in their influence on inulin hydrolysis. Undesirable side reactions were noted during acid hydrolysis leading to the formation of HMF and 2-(2-hydroxy acetyl) furan. The HMF at a level of 0.1% is known to inhibit growth and ethanol fermentation by yeast. In this study the authors established optimal conditions for complete acid-hydrolysis of JA with minimum side reactions and maximum sugar-ethanol production. A material balance for the ethanol production was also determined.

  12. Hydrolysis of biomass material

    DOEpatents

    Schmidt, Andrew J.; Orth, Rick J.; Franz, James A.; Alnajjar, Mikhail

    2004-02-17

    A method for selective hydrolysis of the hemicellulose component of a biomass material. The selective hydrolysis produces water-soluble small molecules, particularly monosaccharides. One embodiment includes solubilizing at least a portion of the hemicellulose and subsequently hydrolyzing the solubilized hemicellulose to produce at least one monosaccharide. A second embodiment includes solubilizing at least a portion of the hemicellulose and subsequently enzymatically hydrolyzing the solubilized hemicellulose to produce at least one monosaccharide. A third embodiment includes solubilizing at least a portion of the hemicellulose by heating the biomass material to greater than 110.degree. C. resulting in an aqueous portion that includes the solubilized hemicellulose and a water insoluble solids portion and subsequently separating the aqueous portion from the water insoluble solids portion. A fourth embodiment is a method for making a composition that includes cellulose, at least one protein and less than about 30 weight % hemicellulose, the method including solubilizing at least a portion of hemicellulose present in a biomass material that also includes cellulose and at least one protein and subsequently separating the solubilized hemicellulose from the cellulose and at least one protein.

  13. Acetyl-CoA Carboxylase Regulates Global Histone Acetylation*♦

    PubMed Central

    Galdieri, Luciano; Vancura, Ales

    2012-01-01

    Histone acetylation depends on intermediary metabolism for supplying acetyl-CoA in the nucleocytosolic compartment. However, because nucleocytosolic acetyl-CoA is also used for de novo synthesis of fatty acids, histone acetylation and synthesis of fatty acids compete for the same acetyl-CoA pool. The first and rate-limiting reaction in de novo synthesis of fatty acids is carboxylation of acetyl-CoA to form malonyl-CoA, catalyzed by acetyl-CoA carboxylase. In yeast Saccharomyces cerevisiae, acetyl-CoA carboxylase is encoded by the ACC1 gene. In this study, we show that attenuated expression of ACC1 results in increased acetylation of bulk histones, globally increased acetylation of chromatin histones, and altered transcriptional regulation. Together, our data indicate that Acc1p activity regulates the availability of acetyl-CoA for histone acetyltransferases, thus representing a link between intermediary metabolism and epigenetic mechanisms of transcriptional regulation. PMID:22580297

  14. Crystallographic trapping of the glutamyl-CoA thioester intermediate of family I CoA transferases

    SciTech Connect

    Rangarajan,E.; Li, Y.; Ajamian, E.; Iannuzzi, P.; Kernaghan, S.; Fraser, M.; Cygler, M.; Matte, A.

    2005-01-01

    Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 Angstrom resolution, respectively. YdiF is organized into tetramers, with each monomer having an open {alpha}/{beta} structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent {gamma}-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.

  15. Metabolic biology of 3-methylglutaconic acid-uria: a new perspective.

    PubMed

    Su, Betty; Ryan, Robert O

    2014-05-01

    Over the past 25 years a growing number of distinct syndromes/mutations associated with compromised mitochondrial function have been identified that share a common feature: urinary excretion of 3-methylglutaconic acid (3MGA). In the leucine degradation pathway, carboxylation of 3-methylcrotonyl CoA leads to formation of 3-methylglutaconyl CoA while 3-methylglutaconyl CoA hydratase converts this metabolite to 3-hydroxy-3-methylglutaryl CoA (HMG CoA). In "primary" 3MGA-uria, mutations in the hydratase are directly responsible for the accumulation of 3MGA. On the other hand, in all "secondary" 3MGA-urias, no defect in leucine catabolism exists and the metabolic origin of 3MGA is unknown. Herein, a path to 3MGA from mitochondrial acetyl CoA is proposed. The pathway is initiated when syndrome-associated mutations/DNA deletions result in decreased Krebs cycle flux. When this occurs, acetoacetyl CoA thiolase condenses two acetyl CoA into acetoacetyl CoA plus CoASH. Subsequently, HMG CoA synthase 2 converts acetoacetyl CoA and acetyl CoA to HMG CoA. Under syndrome-specific metabolic conditions, 3-methylglutaconyl CoA hydratase converts HMG CoA into 3-methylglutaconyl CoA in a reverse reaction of the leucine degradation pathway. This metabolite fails to proceed further up the leucine degradation pathway owing to the kinetic properties of 3-methylcrotonyl CoA carboxylase. Instead, hydrolysis of the CoA moiety of 3-methylglutaconyl CoA generates 3MGA, which appears in urine. If experimentally confirmed, this pathway provides an explanation for the occurrence of 3MGA in multiple disorders associated with compromised mitochondrial function. PMID:24407466

  16. Cellulose acetate from oil palm empty fruit bunch via a one step heterogeneous acetylation.

    PubMed

    Wan Daud, Wan Rosli; Djuned, Fauzi Muhammad

    2015-11-01

    Acetone soluble oil palm empty fruit bunch cellulose acetate (OPEFB-CA) of DS 2.52 has been successfully synthesized in a one-step heterogeneous acetylation of OPEFB cellulose without necessitating the hydrolysis stage. This has only been made possible by the mathematical modeling of the acetylation process by manipulating the variables of reaction time and acetic anhydride/cellulose ratio (RR). The obtained model was verified by experimental data with an error of less than 2.5%. NMR analysis showed that the distribution of the acetyl moiety among the three OH groups of cellulose indicates a preference at the C6 position, followed by C3 and C2. XRD revealed that OPEFB-CA is highly amorphous with a degree of crystallinity estimated to be ca. 6.41% as determined from DSC. The OPEFB-CA films exhibited good mechanical properties being their tensile strength and Young's modulus higher than those of the commercial CA. PMID:26256348

  17. Catalytic Depolymerization of Chitin with Retention of N-Acetyl Group.

    PubMed

    Yabushita, Mizuho; Kobayashi, Hirokazu; Kuroki, Kyoichi; Ito, Shogo; Fukuoka, Atsushi

    2015-11-01

    Chitin, a polymer of N-acetylglucosamine units with ?-1,4-glycosidic linkages, is the most abundant marine biomass. Chitin monomers containing N-acetyl groups are useful precursors to various fine chemicals and medicines. However, the selective conversion of robust chitin to N-acetylated monomers currently requires a large excess of acid or a long reaction time, which limits its application. We demonstrate a fast catalytic transformation of chitin to monomers with retention of N-acetyl groups by combining mechanochemistry and homogeneous catalysis. Mechanical-force-assisted depolymerization of chitin with a catalytic amount of H2 SO4 gave soluble short-chain oligomers. Subsequent hydrolysis of the ball-milled sample provided N-acetylglucosamine in 53?% yield, and methanolysis afforded 1-O-methyl-N-acetylglucosamine in yields of up to 70?%. Our process can greatly reduce the use of acid compared to the conventional process. PMID:26538108

  18. Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation.

    PubMed

    de Boor, Susanne; Knyphausen, Philipp; Kuhlmann, Nora; Wroblowski, Sarah; Brenig, Julian; Scislowski, Lukas; Baldus, Linda; Nolte, Hendrik; Krger, Marcus; Lammers, Michael

    2015-07-14

    Ran is a small GTP-binding protein of the Ras superfamily regulating fundamental cellular processes: nucleo-cytoplasmic transport, nuclear envelope formation and mitotic spindle assembly. An intracellular RanGTP/RanGDP gradient created by the distinct subcellular localization of its regulators RCC1 and RanGAP mediates many of its cellular effects. Recent proteomic screens identified five Ran lysine acetylation sites in human and eleven sites in mouse/rat tissues. Some of these sites are located in functionally highly important regions such as switch I and switch II. Here, we show that lysine acetylation interferes with essential aspects of Ran function: nucleotide exchange and hydrolysis, subcellular Ran localization, GTP hydrolysis, and the interaction with import and export receptors. Deacetylation activity of certain sirtuins was detected for two Ran acetylation sites in vitro. Moreover, Ran was acetylated by CBP/p300 and Tip60 in vitro and on transferase overexpression in vivo. Overall, this study addresses many important challenges of the acetylome field, which will be discussed. PMID:26124124

  19. Small GTP-binding protein Ran is regulated by posttranslational lysine acetylation

    PubMed Central

    de Boor, Susanne; Knyphausen, Philipp; Kuhlmann, Nora; Wroblowski, Sarah; Brenig, Julian; Scislowski, Lukas; Baldus, Linda; Nolte, Hendrik; Krger, Marcus; Lammers, Michael

    2015-01-01

    Ran is a small GTP-binding protein of the Ras superfamily regulating fundamental cellular processes: nucleo-cytoplasmic transport, nuclear envelope formation and mitotic spindle assembly. An intracellular RanGTP/RanGDP gradient created by the distinct subcellular localization of its regulators RCC1 and RanGAP mediates many of its cellular effects. Recent proteomic screens identified five Ran lysine acetylation sites in human and eleven sites in mouse/rat tissues. Some of these sites are located in functionally highly important regions such as switch I and switch II. Here, we show that lysine acetylation interferes with essential aspects of Ran function: nucleotide exchange and hydrolysis, subcellular Ran localization, GTP hydrolysis, and the interaction with import and export receptors. Deacetylation activity of certain sirtuins was detected for two Ran acetylation sites in vitro. Moreover, Ran was acetylated by CBP/p300 and Tip60 in vitro and on transferase overexpression in vivo. Overall, this study addresses many important challenges of the acetylome field, which will be discussed. PMID:26124124

  20. [Heroin. II. Preparation, hydrolysis, stability, pharmacokinetics].

    PubMed

    Hosztafi, S

    2001-10-01

    Heroin is prepared by treating morphine with acetyl chloride or acetic anhydride. It is a simple reaction and the yields are generally quantitative. Nowadays the whole process is illegal. Morphine is the major alkaloid present in the opium poppy. Opium is manufactured illicitly then morphine is extracted from it in clandestine laboratories. Numerous studies were carried out on heroin to investigate its rate of hydrolysis. It has been shown that heroin is rapidly deacylated in aqueous solution at alkaline or acidic pH to form 6-acethylmorphine and finally, to morphine. Heroin also rapidly decomposes in biological medium yielding first 6-acetylmorphine and then morphine. Hydrolysis can be performed in blood and in tissue homogenates. Heroin can be administered by several routes. Smoking and intravenous administration are preferred, but intranasal, intramuscular and subcutaneous administration are also common. Recently, there has been a shift in heroin use patterns from injection to sniffing and smoking. Sharing of the injection equipment can result in several severe infectious diseases, such as AIDS, hepatitis B and C. Soon after administration, heroin metabolizes to 6-acetylmorphine and morphine. Most of the pharmacological activities of heroin are due to these active metabolites. Therefore, knowledge of distribution of 6-acetylmorphine and morphine is essential to understand pharmacological properties of heroin. Heroin, which is relatively nonpolar compound compared with morphine, has high lipid solubility facilitating rapid absorption from the bloodstream and passage through the blood-brain barrier. When heroin is administered by intravenously the drug takes 10 s to reach the brain i.e. pharmacological effects appear quickly. PMID:11961908

  1. Specific interaction between S6K1 and CoA synthase: a potential link between the mTOR/S6K pathway, CoA biosynthesis and energy metabolism.

    PubMed

    Nemazanyy, Ivan; Panasyuk, Ganna; Zhyvoloup, Alexander; Panayotou, George; Gout, Ivan T; Filonenko, Valeriy

    2004-12-17

    Ribosomal protein S6 kinase (S6K) is a key regulator of cell size and growth. It is regulated via phosphoinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR) signaling pathways. We demonstrate for the first time that CoA synthase associates specifically with S6K1. The association was observed between native and transiently overexpressed proteins in vivo, as well as by BIAcore analysis in vitro. The sites of interaction were mapped to the C-terminal regions of both CoA synthase and S6K1. In vitro studies indicated that the interaction does not affect their enzymatic activities and that CoA synthase is not a substrate for S6 kinase. This study uncovers a potential link between mTor/S6K signaling pathway and energy metabolism through CoA and its thioester derivatives, but its physiological relevance should be further elucidated. PMID:15589845

  2. Regulation of schistosome egg production by HMG CoA reductase

    SciTech Connect

    VandeWaa, E.A.; Bennett, J.L.

    1986-03-05

    Hydroxymethylglutaryl coenzyme A reductase (HMG CoA reductase) catalyzes the conversion of HMG CoA to mevalonate in the synthesis of steroids, isoprenoids and terpenes. Mevinolin, an inhibitor of this enzyme, decreased egg production in Schistosoma mansoni during in vitro incubations. This was associated with a reduction in the incorporation of /sup 14/C-acetate into polyisoprenoids and a reduction in the formation of a lipid-linked oligosaccharide. In vivo, mevinolin in daily doses of 50 mg/kg (p.o., from days 30-48 post-infection) caused no change in gross liver pathology in S. mansoni infected mice. However, when parasites exposed to mevinolin or its vehicle in vivo were cultured in vitro, worms from mevinolin-treated mice produced six times more eggs than control parasites. When infected mice were dosed with 250 mg/kg mevinolin daily (p.o., from days 35-45 post-infection), liver pathology was reduced in comparison to control mice. Thus, during in vivo exposure to a high dose of the drug egg production is decreased, while at a lower dose it appears unaffected until the parasites are cultured in a drug-free in vitro system wherein egg production is stimulated to extraordinarily high levels. It may be that at low doses mevinolin, by inhibiting the enzyme, is blocking the formation of a product (such as an isoprenoid) which normally acts to down-regulate enzyme synthesis, resulting in enzyme induction. Induction of HMG CoA reductase is then expressed as increased egg production when the worms are removed from the drug. These data suggest that HMG CoA reductase plays a role in schistosome egg production.

  3. Fatty infiltration in the liver in medium chain acyl CoA dehydrogenase deficiency.

    PubMed

    Losty, H C; Lee, P; Alfaham, M; Gray, O P; Leonard, J V

    1991-06-01

    Fatty infiltration of the liver at postmortem examination has been recommended as a criterion for selection of infants who have died suddenly and unexpectedly for further biochemical investigation for disorders of fatty acid oxidation. We describe a boy with medium chain acyl CoA dehydrogenase deficiency who died four months after diagnosis and in whom only minimal hepatic fatty infiltration was found. PMID:2053798

  4. ESTIMATION OF PHOSPHATE ESTER HYDROLYSIS RATE CONSTANTS. I. ALKALINE HYDROLYSIS

    EPA Science Inventory

    SPARC (SPARC Performs Automated Reasoning in Chemistry) chemical reactivity models were extended to allow the calculation of alkaline hydrolysis rate constants of phosphate esters in water. The rate is calculated from the energy difference between the initial and transition state...

  5. ESTIMATION OF PHOSPHATE ESTER HYDROLYSIS RATE CONSTANTS - ALKALINE HYDROLYSIS

    EPA Science Inventory

    SPARC (SPARC Performs Automated Reasoning in Chemistry) chemical reactivity models were extended to allow the calculation of alkaline hydrolysis rate constants of phosphate esters in water. The rate is calculated from the energy difference between the initial and transition state...

  6. hCOA3 Stabilizes Cytochrome c Oxidase 1 (COX1) and Promotes Cytochrome c Oxidase Assembly in Human Mitochondria*

    PubMed Central

    Clemente, Paula; Peralta, Susana; Cruz-Bermudez, Alberto; Echevarra, Luca; Fontanesi, Flavia; Barrientos, Antoni; Fernandez-Moreno, Miguel A.; Garesse, Rafael

    2013-01-01

    Cytochrome c oxidase (COX) or complex IV of the mitochondrial respiratory chain plays a fundamental role in energy production of aerobic cells. In humans, COX deficiency is the most frequent cause of mitochondrial encephalomyopathies. Human COX is composed of 13 subunits of dual genetic origin, whose assembly requires an increasing number of nuclear-encoded accessory proteins known as assembly factors. Here, we have identified and characterized human CCDC56, an 11.7-kDa mitochondrial transmembrane protein, as a new factor essential for COX biogenesis. CCDC56 shares sequence similarity with the yeast COX assembly factor Coa3 and was termed hCOA3. hCOA3-silenced cells display a severe COX functional alteration owing to a decreased stability of newly synthesized COX1 and an impairment in the holoenzyme assembly process. We show that hCOA3 physically interacts with both the mitochondrial translation machinery and COX structural subunits. We conclude that hCOA3 stabilizes COX1 co-translationally and promotes its assembly with COX partner subunits. Finally, our results identify hCOA3 as a new candidate when screening for genes responsible for mitochondrial diseases associated with COX deficiency. PMID:23362268

  7. Hydrolysis reactor for hydrogen production

    SciTech Connect

    Davis, Thomas A.; Matthews, Michael A.

    2012-12-04

    In accordance with certain embodiments of the present disclosure, a method for hydrolysis of a chemical hydride is provided. The method includes adding a chemical hydride to a reaction chamber and exposing the chemical hydride in the reaction chamber to a temperature of at least about 100.degree. C. in the presence of water and in the absence of an acid or a heterogeneous catalyst, wherein the chemical hydride undergoes hydrolysis to form hydrogen gas and a byproduct material.

  8. Identification and Quantitative Analysis of Indole-3-Acetyl-l-Aspartate from Seeds of Glycine max L. 1

    PubMed Central

    Cohen, Jerry D.

    1982-01-01

    Indole-3-acetyl-l-aspartate (IAAsp) was isolated from seeds of Glycine max L. cv. Hark and its identity established by its chromatographic performance and its mass spectral fragmentation. Following acid hydrolysis, the aspartate moiety was shown to be the l-enantiomer by reverse phase high performance liquid chromatographic retention time of the bisethyl ester derivatized with 2,3,4,6-tetra-O-acetyl-?-d-glycopyranosyl isothiocyanate. Isotope dilution analysis using [14C]IAAsp as internal standard showed that soybean seed contained 10 ?mol/kg IAAsp and this accounted for one-half of the total indoleacetic acid of the seed. PMID:16662569

  9. Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions

    PubMed Central

    Weinert, Brian T; Moustafa, Tarek; Iesmantavicius, Vytautas; Zechner, Rudolf; Choudhary, Chunaram

    2015-01-01

    Acetylation is frequently detected on mitochondrial enzymes, and the sirtuin deacetylase SIRT3 is thought to regulate metabolism by deacetylating mitochondrial proteins. However, the stoichiometry of acetylation has not been studied and is important for understanding whether SIRT3 regulates or suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue of fasted mice, we found that SIRT3-targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3-dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation sites, and greater sensitivity of SIRT3-targeted sites to chemical acetylation in vitro and fasting-induced acetylation in vivo, suggest a nonenzymatic mechanism of acetylation. Our data indicate that most mitochondrial acetylation occurs as a low-level nonenzymatic protein lesion and that SIRT3 functions as a protein repair factor that removes acetylation lesions from lysine residues. PMID:26358839

  10. The Yeast AMPK Homolog SNF1 Regulates Acetyl Coenzyme A Homeostasis and Histone Acetylation

    PubMed Central

    Zhang, Man; Galdieri, Luciano

    2013-01-01

    Acetyl coenzyme A (acetyl-CoA) is a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Concentration of acetyl-CoA affects histone acetylation and links intermediary metabolism and transcriptional regulation. Here we show that SNF1, the budding yeast ortholog of the mammalian AMP-activated protein kinase (AMPK), plays a role in the regulation of acetyl-CoA homeostasis and global histone acetylation. SNF1 phosphorylates and inhibits acetyl-CoA carboxylase, which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting reaction in the de novo synthesis of fatty acids. Inactivation of SNF1 results in a reduced pool of cellular acetyl-CoA, globally decreased histone acetylation, and reduced fitness and stress resistance. The histone acetylation and transcriptional defects can be partially suppressed and the overall fitness improved in snf1Δ mutant cells by increasing the cellular concentration of acetyl-CoA, indicating that the regulation of acetyl-CoA homeostasis represents another mechanism in the SNF1 regulatory repertoire. PMID:24081331

  11. A Method to determine lysine acetylation stoichiometries

    SciTech Connect

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; Pasa-Tolic, Ljiljana; Qian, Weijun; Smith, Richard D.; Adkins, Joshua N.; Ansong, Charles

    2014-07-21

    A major bottleneck to fully understanding the functional aspects of lysine acetylation is the lack of stoichiometry information. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of lysine acetylation on proteins globally. Using this technique, we determined the modification occupancy on hundreds of acetylated peptides from cell lysates and cross-validated the measurements via immunoblotting.

  12. Characterization and prediction of lysine (K)-acetyl-transferase specific acetylation sites.

    PubMed

    Li, Tingting; Du, Yipeng; Wang, Likun; Huang, Lei; Li, Wenlin; Lu, Ming; Zhang, Xuegong; Zhu, Wei-Guo

    2012-01-01

    Lysine acetylation is a well-studied post-translational modification on both histone and nonhistone proteins. More than 2000 acetylated proteins and 4000 lysine acetylation sites have been identified by large scale mass spectrometry or traditional experimental methods. Although over 20 lysine (K)-acetyl-transferases (KATs) have been characterized, which KAT is responsible for a given protein or lysine site acetylation is mostly unknown. In this work, we collected KAT-specific acetylation sites manually and analyzed sequence features surrounding the acetylated lysine of substrates from three main KAT families (CBP/p300, GCN5/PCAF, and the MYST family). We found that each of the three KAT families acetylates lysines with different sequence features. Based on these differences, we developed a computer program, Acetylation Set Enrichment Based method to predict which KAT-families are responsible for acetylation of a given protein or lysine site. Finally, we evaluated the efficiency of our method, and experimentally detected four proteins that were predicted to be acetylated by two KAT families when one representative member of the KAT family is over expressed. We conclude that our approach, combined with more traditional experimental methods, may be useful for identifying KAT families responsible for acetylated substrates proteome-wide. PMID:21964354

  13. Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

    Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

    2011-08-22

    Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

  14. Rhabdomyolysis in a neonate due to very long chain acyl CoA dehydrogenase deficiency

    PubMed Central

    Scott Schwoerer, Jessica; Cooper, Gena; van Calcar, Sandra

    2015-01-01

    Very long chain acyl CoA dehydrogenase deficiency (VLCADD) is an inborn error in long chain fatty acid oxidation with significant variability in the severity and timing of its clinical presentation. Neonatal presentations of VLCADD have included hypoglycemia and cardiomyopathy while rhabdomyolysis is usually a later onset complication. We describe a neonate with VLCADD presenting with rhabdomyolysis prior to the return of an abnormal newborn screen. This report suggests that evaluating for rhabdomyolysis, in addition to a cardiac and hepatic work-up, is an important part of the initial evaluation of an infant with an abnormal newborn screen suggesting a diagnosis of VLCADD.

  15. Are long chain acyl CoAs responsible for suppression of mitochondrial metabolism in hibernating 13-lined ground squirrels?

    PubMed

    Cooper, Alex N; Brown, Jason C L; Staples, James F

    2014-04-01

    Hibernation in 13-lined ground squirrels (Ictidomys tridecemlineatus) is associated with a substantial suppression of whole-animal metabolism. We compared the metabolism of liver mitochondria isolated from torpid ground squirrels with those from interbout euthermic (IBE; recently aroused from torpor) and summer euthermic conspecifics. Succinate-fuelled state 3 respiration, calculated relative to mitochondrial protein, was suppressed in torpor by 48% and 44% compared with IBE and summer, respectively. This suppression remains when respiration is expressed relative to cytochrome c oxidase activity. We hypothesized that this suppression was caused by inhibition of succinate transport at the dicarboxylate transporter (DCT) by long-chain fatty acyl CoAs that may accumulate during torpor. We predicted, therefore, that exogenous palmitoyl CoA would inhibit respiration in IBE more than in torpor. Palmitoyl CoA inhibited respiration ~70%, in both torpor and IBE. The addition of carnitine, predicted to reverse palmitoyl CoA suppression by facilitating its transport into the mitochondrial matrix, did not rescue the respiration rates in IBE or torpor. Liver mitochondrial activities of carnitine palmitoyl transferase did not differ among IBE, torpor and summer animals. Although palmitoyl CoA inhibits succinate-fuelled respiration, this suppression is likely not related exclusively to inhibition of the DCT, and may involve additional mitochondrial transporters such as the adenine-nucleotide transporter. PMID:24561259

  16. COA6 is a mitochondrial complex IV assembly factor critical for biogenesis of mtDNA-encoded COX2.

    PubMed

    Stroud, David A; Maher, Megan J; Lindau, Caroline; Vgtle, F-Nora; Frazier, Ann E; Surgenor, Elliot; Mountford, Hayley; Singh, Abeer P; Bonas, Matteo; Oeljeklaus, Silke; Warscheid, Bettina; Meisinger, Chris; Thorburn, David R; Ryan, Michael T

    2015-10-01

    Biogenesis of complex IV of the mitochondrial respiratory chain requires assembly factors for subunit maturation, co-factor attachment and stabilization of intermediate assemblies. A pathogenic mutation in COA6, leading to substitution of a conserved tryptophan for a cysteine residue, results in a loss of complex IV activity and cardiomyopathy. Here, we demonstrate that the complex IV defect correlates with a severe loss in complex IV assembly in patient heart but not fibroblasts. Complete loss of COA6 activity using gene editing in HEK293T cells resulted in a profound growth defect due to complex IV deficiency, caused by impaired biogenesis of the copper-bound mitochondrial DNA-encoded subunit COX2 and subsequent accumulation of complex IV assembly intermediates. We show that the pathogenic mutation in COA6 does not affect its import into mitochondria but impairs its maturation and stability. Furthermore, we show that COA6 has the capacity to bind copper and can associate with newly translated COX2 and the mitochondrial copper chaperone SCO1. Our data reveal that COA6 is intricately involved in the copper-dependent biogenesis of COX2. PMID:26160915

  17. Proteomic profiling of lysine acetylation in Pseudomonas aeruginosa reveals the diversity of acetylated proteins.

    PubMed

    Ouidir, Tassadit; Cosette, Pascal; Jouenne, Thierry; Hardouin, Julie

    2015-07-01

    Protein lysine acetylation is a reversible and highly regulated post-translational modification with the well demonstrated physiological relevance in eukaryotes. Recently, its important role in the regulation of metabolic processes in bacteria was highlighted. Here, we reported the lysine acetylproteome of Pseudomonas aeruginosa using a proteomic approach. We identified 430 unique peptides corresponding to 320 acetylated proteins. In addition to the proteins involved in various metabolic pathways, several enzymes contributing to the lipopolysaccharides biosynthesis were characterized as acetylated. This data set illustrated the abundance and the diversity of acetylated lysine proteins in P. aeruginosa and opens opportunities to explore the role of the acetylation in the bacterial physiology. PMID:25900529

  18. A Method to Determine Lysine Acetylation Stoichiometries

    DOE PAGESBeta

    Nakayasu, Ernesto S.; Wu, Si; Sydor, Michael A.; Shukla, Anil K.; Weitz, Karl K.; Moore, Ronald J.; Hixson, Kim K.; Kim, Jong-Seo; Petyuk, Vladislav A.; Monroe, Matthew E.; et al

    2014-01-01

    Lysine acetylation is a common protein posttranslational modification that regulates a variety of biological processes. A major bottleneck to fully understanding the functional aspects of lysine acetylation is the difficulty in measuring the proportion of lysine residues that are acetylated. Here we describe a mass spectrometry method using a combination of isotope labeling and detection of a diagnostic fragment ion to determine the stoichiometry of protein lysine acetylation. Using this technique, we determined the modification occupancy for ~750 acetylated peptides from mammalian cell lysates. Furthermore, the acetylation on N-terminal tail of histone H4 was cross-validated by treating cells with sodiummore » butyrate, a potent deacetylase inhibitor, and comparing changes in stoichiometry levels measured by our method with immunoblotting measurements. Of note we observe that acetylation stoichiometry is high in nuclear proteins, but very low in mitochondrial and cytosolic proteins. In summary, our method opens new opportunities to study in detail the relationship of lysine acetylation levels of proteins with their biological functions.« less

  19. Enhanced acetyl-CoA production is associated with increased triglyceride accumulation in the green alga Chlorella desiccata

    PubMed Central

    Avidan, Omri; Brandis, Alexander; Rogachev, Ilana; Pick, Uri

    2015-01-01

    Triglycerides (TAGs) from microalgae can be utilized as food supplements and for biodiesel production, but little is known about the regulation of their biosynthesis. This work aimed to test the relationship between acetyl-CoA (Ac-CoA) levels and TAG biosynthesis in green algae under nitrogen deprivation. A novel, highly sensitive liquid chromatography mass spectrometry (LC-MS/MS) technique enabled us to determine the levels of Ac-CoA, malonyl-CoA, and unacetylated (free) CoA in green microalgae. A comparative study of three algal species that differ in TAG accumulation levels shows that during N starvation, Ac-CoA levels rapidly rise, preceding TAG accumulation in all tested species. The levels of Ac-CoA in the high TAG accumulator Chlorella desiccata exceed the levels in the moderate TAG accumulators Dunaliella tertiolecta and Chlamydomonas reinhardtii. Similarly, malonyl-CoA and free CoA levels also increase, but to lower extents. Calculated cellular concentrations of Ac-CoA are far lower than reported K mAc-CoA values of plastidic Ac-CoA carboxylase (ptACCase) in plants. Transcript level analysis of plastidic pyruvate dehydrogenase (ptPDH), the major chloroplastic Ac-CoA producer, revealed rapid induction in parallel with Ac-CoA accumulation in C. desiccata, but not in D. tertiolecta or C. reinhardtii. It is proposed that the capacity to accumulate high TAG levels in green algae critically depends on their ability to divert carbon flow towards Ac-CoA. This requires elevation of the chloroplastic CoA pool level and enhancement of Ac-CoA biosynthesis. These conclusions may have important implications for future genetic manipulation to enhance TAG biosynthesis in green algae. PMID:25922486

  20. HYDROLYSIS OF CHLOROSTILBENE OXIDE: I. HYDROLYSIS IN HOMOGENEOUS SYSTEMS

    EPA Science Inventory

    The hydrolysis kinetics of 4-chlorostilbene oxide (CSO) in buffered distilled water, in natural waters, and in sediment associated water are reported. he disappearance of CSO followed pseudo-first-order kinetics in buffered water over the experimental pH range of 3 to 11. elow pH...

  1. Unique response of lung acetyl-CoA carboxylase to inhibitors

    SciTech Connect

    Patterson, C.E.; Davis, K.S.; Rhoades, R.A.

    1986-05-01

    Fatty acid synthesis (FAS) in lung is not inhibited by c-AMP analogs or aminophylline although these agents inhibit FAS in other lipogenic tissues. To further characterize FAS in lung, the authors examined the response of cultured fetal lung explants to known inhibitors of FAS in liver: t-butyl benzoic acid (tBB-which binds CoA and inhibits acetyl-CoA carboxylase) and palmitate (an allosteric effector of acetyl-CoA carboxylase). Explants derived from d18 fetuses (term=22d) were cultured 2d in F12k media containing 10mM lactate, 2mM glucose, and 10mM Hepes. At 48h, FAS was determined by incubation with /sup 3/H/sub 2/O (control = 3892 +/- 755 nmoles C2 units/g/h) and surfactant lipid production estimated by incorporation of /sup 14/C-choline into DSPC (control = 35.8 +/- 9.0 nmoles/g/h). Addition of tBB (50uM) did not significantly alter FAS or choline incorporation. Addition of palmitate (0.15mM) in either ethanol (1% final conc.) or albumin (3% final conc.) did not result in diminished FAS. Palmitate did increase DSPC labeling 20%, indicating that in these cultures the rate of surfactant synthesis is partially dependent upon palmitate availability. These data show that lung is unique in its unresponsiveness to various inhibitors of FAS which act at the level acetyl-CoA carboxylase and suggest that FAS is maintained in order to insure a de novo palmitate supply for surfactant lipid synthesis.

  2. Purification and properties of acetyl-CoA synthetase from Bradyrhizobium japonicum bacteroids.

    PubMed Central

    Preston, G G; Wall, J D; Emerich, D W

    1990-01-01

    Acetyl-CoA synthetase was purified 800-fold from Bradyrhizobium japonicum bacteroids. A specific activity of 16 mumol/min per mg of protein was achieved, with a 30-40% yield. The purification scheme consisted of only three consecutive chromatography steps. The enzyme has a native Mr of 150,000, estimated by gel-permeation chromatography, and a subunit Mr of 72,000, determined by SDS/polyacrylamide-gel electrophoresis. The optimum pH and temperature are 8.5 and 50 degrees C respectively. The Km values for acetate, CoA and ATP were 146, 202 and 275 microM respectively. The reaction was specific for acetate, as propionate and oleate were used very poorly. Likewise, the enzyme used only ATP, ADP or dATP; AMP, GTP, XTP and UTP could not replace ATP. Acetyl-CoA synthetase showed a broad specificity for metals; MnCl2 could replace MgCl2. In addition, CaCl2 and CoCl2 were approx. 50% as effective as MgCl2, but FeCl3, NiCl2 or ZnCl2 could not effectively substitute for MgCl2. The enzyme may be regulated by NADP+ and pyruvate; no effect was seen of amino acids, glucose catabolites, reduced nicotinamide nucleotides or acetyl-CoA. Inhibition was seen with AMP, PPi, FMN and pyridoxal phosphate, with Ki values of 720, 222, 397 and 1050 microM respectively. Images Fig. 1. PMID:1970239

  3. Metabolic control of methylation and acetylation.

    PubMed

    Su, Xiaoyang; Wellen, Kathryn E; Rabinowitz, Joshua D

    2016-02-01

    Methylation and acetylation of DNA and histone proteins are the chemical basis for epigenetics. From bacteria to humans, methylation and acetylation are sensitive to cellular metabolic status. Modification rates depend on the availability of one-carbon and two-carbon substrates (S-adenosylmethionine, acetyl-CoA, and in bacteria also acetyl-phosphate). In addition, they are sensitive to demodification enzyme cofactors (?-ketoglutarate, NAD(+)) and structural analog metabolites that function as epigenetic enzyme inhibitors (e.g., S-adenosylhomocysteine, 2-hydroxyglutarate). Methylation and acetylation likely initially evolved to tailor protein activities in microbes to their metabolic milieu. While the extracellular environment of mammals is more tightly controlled, the combined impact of nutrient abundance and metabolic enzyme expression impacts epigenetics in mammals sufficiently to drive important biological outcomes such as stem cell fate and cancer. PMID:26629854

  4. The Cardiac Acetyl-Lysine Proteome

    PubMed Central

    Foster, D. Brian; Liu, Ting; Rucker, Jasma; OMeally, Robert N.; Devine, Lauren R.; Cole, Robert N.; ORourke, Brian

    2013-01-01

    In the heart, lysine acetylation has been implicated in processes ranging from transcriptional control of pathological remodeling, to cardioprotection arising from caloric restriction. Given the emerging importance of this post-translational modification, we used a proteomic approach to investigate the broader role of lysine acetylation in the heart using a guinea pig model. Briefly, hearts were fractionated into myofilament-, mitochondrial- and cytosol-enriched fractions prior to proteolysis and affinity-enrichment of acetylated peptides. LC-MS/MS analysis identified 1075 acetylated peptides, harboring 994 acetylation sites that map to 240 proteins with a global protein false discovery rate <0.8%. Mitochondrial targets account for 59% of identified proteins and 64% of sites. The majority of the acetyl-proteins are enzymes involved in fatty acid metabolism, oxidative phosphorylation or the TCA cycle. Within the cytosolic fraction, the enzymes of glycolysis, fatty acid synthesis and lipid binding are prominent. Nuclear targets included histones and the transcriptional regulators E1A(p300) and CREB binding protein. Comparison of our dataset with three previous global acetylomic studies uniquely revealed 53 lysine-acetylated proteins. Specifically, newly-identified acetyl-proteins include Ca2+-handling proteins, RyR2 and SERCA2, and the myofilament proteins, myosin heavy chain, myosin light chains and subunits of the Troponin complex, among others. These observations were confirmed by anti-acetyl-lysine immunoblotting. In summary, cardiac lysine acetylation may play a role in cardiac substrate selection, bioenergetic performance, and maintenance of redox balance. New sites suggest a host of potential mechanisms by which excitation-contraction coupling may also be modulated. PMID:23844019

  5. Acetylated glucopyranosyl esters of enkephalins.

    PubMed

    Skuri?, M; Horvat, J; Horvat, S; Chung, N N; Schiller, P W

    1994-04-01

    Acetylated D-glucopyranosyl esters of enkephalins were prepared by two different fragment condensation procedures involving direct participation of imidazole in the ester linkage formation. By both methods anomeric mixtures of D-glucosyl esters were obtained and resolved by column chromatography. Depending on coupling conditions, racemization of either the C-terminal or the penultimate amino acid residue of the enkephalin molecule occurred. The glucoconjugates with inverted stereochemistry were quantitated and separated from the main product by reversed-phase high-performance liquid chromatography. The opioid agonist potencies of the synthesized glucopyranosyl esters of enkephalins on electrically stimulated guinea pig ileum and mouse vas deferens preparations were determined in comparison with [Leu5]enkephalin. PMID:8045687

  6. Unsaturated Lipid Assimilation by Mycobacteria Requires Auxiliary cis-trans Enoyl CoA Isomerase.

    PubMed

    Srivastava, Sonali; Chaudhary, Sarika; Thukral, Lipi; Shi, Ce; Gupta, Rinkoo D; Gupta, Radhika; Priyadarshan, K; Vats, Archana; Haque, Asfarul S; Sankaranarayanan, Rajan; Natarajan, Vivek T; Sharma, Rakesh; Aldrich, Courtney C; Gokhale, Rajesh S

    2015-12-17

    Mycobacterium tuberculosis (Mtb) can survive in hypoxic necrotic tissue by assimilating energy from host-derived fatty acids. While the expanded repertoire of ?-oxidation auxiliary enzymes is considered crucial for Mtb adaptability, delineating their functional relevance has been challenging. Here, we show that the Mtb fatty acid degradation (FadAB) complex cannot selectively break down cis fatty acyl substrates. We demonstrate that the stereoselective binding of fatty acyl substrates in the Mtb FadB pocket is due to the steric hindrance from Phe287 residue. By developing a functional screen, we classify the family of Mtb Ech proteins as monofunctional or bifunctional enzymes, three of which complement the FadAB complex to degrade cis fatty acids. Crystal structure determination of two cis-trans enoyl coenzyme A (CoA) isomerases reveals distinct placement of active-site residue in Ech enzymes. Our studies thus reveal versatility of Mtb lipid-remodeling enzymes and identify an essential role of stand-alone cis-trans enoyl CoA isomerases in mycobacterial biology. PMID:26628360

  7. Flexible DAQ card for detector systems utilizing the CoaXPress communication standard

    NASA Astrophysics Data System (ADS)

    Neue, G.; Hejtmnek, M.; Mar?iovsk, M.; Vole, P.

    2015-04-01

    This work concerns the design and construction of a flexible FPGA based data acquisition system aimed for particle detectors. The interface card as presented was designed for large area detectors with millions of individual readout channels. Flexibility was achieved by partitioning the design into multiple PCBs, creating a set of modular blocks, allowing the creation of a wide variety of configurations by simply stacking functional PCBs together. This way the user can easily toggle the polarity of the high voltage bias supply or switch the downstream interface from CoaXPress to PCIe or stream directly HDMI. We addressed the issues of data throughput, data buffering, bias voltage generation, trigger timing and fine tuning of the whole readout chain enabling a smooth data transmission. On the current prototype, we have wire-bonded a MediPix2 MXR quad and connected it to a XILINX FPGA. For the downstream interface, we implemented the CoaXPress communication protocol, which enables us to stream data at 3.125 Gbps to a standard PC.

  8. A method of COA based on multi-agent evolutionary algorithm

    NASA Astrophysics Data System (ADS)

    Yu, Xin; Wang, Hui; Jiao, Licheng

    2009-10-01

    Planning detailed military course of action (COA) is very complex and time consuming. In this paper, a method based on multi-agent evolutionary algorithm was presented to solve COA' resource management and scheduling problems. Each individual can be seen as an agent, in order to realize the local perceptivity of agents, the environment is organized as a latticelike structure. Each agent is fixed on a lattice point and it can only interact with its neighbors .Two agent behaviors which are competition behavior and self-learning behavior are designed. In this work, constraint functions are considered as functions to be optimized like the objectives and then added in competition strategy to deal with the multi-objective aspect of resource-constrained project scheduling problems. This approach avoids the use of a penalty function to deal with constraints. At the same time, the added constraint functions could make the whole algorithm evolving feasible. The simulation results demonstrated that this approach could improve searching ability of this algorithm, and the precision of this method.

  9. Enzymatically hydrolysed, acetylated and dually modified corn starch: physico-chemical, rheological and nutritional properties and effects on cake quality.

    PubMed

    Sahnoun, Mouna; Ismail, Nouha; Kammoun, Radhouane

    2016-01-01

    Corn starch was treated by enzymatic hydrolysis with Aspergillus oryzae S2 α-amylase, acetylation with vinyl acetate, and dual modification. The dual modified starch displayed a higher substitution degree than the acetylated starch and lower reducing sugar content than the hydrolysed starch. The results revealed that the cooling viscosity and amylose content of those products decrease (P < 0.05). An increase in moisture, water, and oil absorption capacity was observed for the acetylated starch and, which was less pronounced for the enzymatically hydrolysed starch but more pronounced for the enzymatically hydrolysed acetylated product. The latter product underwent an increase in resistant starch content, which is induced by a rise in hydrolysis time to attain about 67 % after 1 h of reaction. The modified starch samples were added to cake formulations at 5 and 10 % concentrations on a wheat flour basis and compared to native starch. The results revealed that when applied at 5 % concentrations, the modified starches reduced the hardness, cohesion, adhesion and chewiness of baked cakes and enhanced their elasticity, volume, height, crust color, and appearance as compared to native starch. These effects were more pronounced for the cake incorporating the dually modified starch. PMID:26787967

  10. Enzymatic hydrolysis of organic phosphorus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Orthophosphate-releasing enzymatic hydrolysis is an alternative means for characterizing organic phosphorus (Po) in animal manure. The approach is not only simple and fast, but can also provide information difficult to obtain by other methods. Currently, commercially available phosphatases are mainl...

  11. Economics of enzymatic hydrolysis processes

    SciTech Connect

    Wright, J.D.

    1988-02-01

    Enzymatic hydrolysis processes have the ability to produce high yields of sugars for fermentation to fuel ethanol from lignocellulosic biomass. However, these systems have been plagued with yields, product concentrations, and reactions rates far below those that are theoretically possible. Engineering and economic analyses are presented on several fungal enzyme hydrolysis processes to illustrate the effects of the important process parameters, to quantify the progress that has been made to date, and to estimate the cost reductions that can be made through research improvements. All enzymatic hydrolysis processes require pretreatment, hydrolysis, fermentation, and enzyme production. The key effect of pretreatment is to allow access of the enzymes to the substrate. Pretreatments have been devised that make the biomass completely digestible that increase the xylose yield and concentration, and that integrate pretreatment with lignin utilization. Major improvements in enzyme activity and use of simultaneous saccharification and fermentation (SSF) have greatly reduced the inhibition of the enzymes. It now appears that ethanol inhibition of the yeast is the limiting factor. Enzyme production costs have been dramatically reduced because use of SSF has reduced enzyme loading. However, further improvements may be possible by using soluble carbon sources for production. Over the past decade, the predicted cost of ethanol from such processes has dropped from more than $4.00/gallon to approximately $1.60. Research is currently under way in the United States and has the potential to reduce the projected cost to less than $1.00/gallon. 65 refs., 16 figs., 1 tab.

  12. ABIOTIC HYDROLYSIS OF SORBED PESTICIDES

    EPA Science Inventory

    The hydrolysis of pesticides that are sorbed to sterilized natural sediments has been investigated in aqueous systems at acid, neutral and alkaline pH's. The results show that the rate constants of pH independent ('neutral') hydrolyses are the same within experimental uncertainti...

  13. Reverse genetic characterization of two paralogous acetoacetyl CoA thiolase genes in Arabidopsis reveals their importance in plant growth and development

    SciTech Connect

    Jin, Huanan; Song, Zhihong; Nikolau, Basil J.

    2012-03-31

    Acetoacetyl CoA thiolase (AACT, EC 2.3.1.9) catalyzes the condensation of two acetyl?CoA molecules to form acetoacetyl?CoA. Two AACT?encoding genes, At5g47720 (AACT1) and At5g48230 (AACT2), were functionally identified in the Arabidopsis genome by direct enzymological assays and functional expression in yeast. Promoter::GUS fusion experiments indicated that AACT1 is primarily expressed in the vascular system and AACT2 is highly expressed in root tips, young leaves, top stems and anthers. Characterization of T?DNA insertion mutant alleles at each AACT locus established that AACT2 function is required for embryogenesis and for normal male gamete transmission. In contrast, plants lacking AACT1 function are completely viable and show no apparent growth phenotypes, indicating that AACT1 is functionally redundant with respect to AACT2 function. RNAi lines that express reduced levels of AACT2 show pleiotropic phenotypes, including reduced apical dominance, elongated life span and flowering duration, sterility, dwarfing, reduced seed yield and shorter root length. Microscopic analysis reveals that the reduced stature is caused by a reduction in cell size and fewer cells, and male sterility is caused by loss of the pollen coat and premature degeneration of the tapetal cells. Biochemical analyses established that the roots of AACT2 RNAi plants show quantitative and qualitative alterations in phytosterol profiles. These phenotypes and biochemical alterations are reversed when AACT2 RNAi plants are grown in the presence of mevalonate, which is consistent with the role of AACT2 in generating the bulk of the acetoacetyl?CoA precursor required for the cytosol?localized, mevalonate?derived isoprenoid biosynthetic pathway.

  14. Acetylation of rice straw for thermoplastic applications.

    PubMed

    Zhang, Guangzhi; Huang, Kai; Jiang, Xue; Huang, Dan; Yang, Yiqi

    2013-07-01

    An inexpensive and biodegradable thermoplastic was developed through acetylation of rice straw (RS) with acetic anhydride. Acetylation conditions were optimized. The structure and properties of acetylated RS were characterized by fourier transform infrared (FTIR), solid-state (13)C NMR spectroscopy, X-ray diffractometer (XRD), scanning electron microscope (SEM), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The results showed that acetylation of RS has successfully taken place, and comparing with raw RS, the degree of crystallinity decreased and the decomposition rate was slow. The acetylated RS has got thermoplasticity when weight ratio of RS and acetic anhydride was 1:3, using sulphuric acid (9% to RS) as catalyst in glacial acetic acid 35C for 12h, and the dosage of solvent was 9 times RS, in which weight percent gain (WPG) of the modified RS powder was 35.5% and its percent acetyl content was 36.1%. The acetylated RS could be formed into transparent thin films with different amount of plasticizer diethyl phthalate (DEP) using tape casting technology. PMID:23688473

  15. Quantitative measurement of acetyl fentanyl and acetyl norfentanyl in human urine by LC-MS/MS.

    PubMed

    Patton, Amy L; Seely, Kathryn A; Pulla, Sharon; Rusch, Nancy J; Moran, Cindy L; Fantegrossi, William E; Knight, Laura D; Marraffa, Jeanna M; Kennedy, Paul D; James, Laura P; Endres, Gregory W; Moran, Jeffery H

    2014-02-01

    Opioid abuse involving emerging opioid compounds is a growing public health problem, which was highlighted recently by cases of human morbidity and mortality linked to acetyl fentanyl abuse. Unfortunately, the lack of information available on the toxicology and metabolism of acetyl fentanyl precludes its detection in human samples. The following study was conducted to test a new analytical procedure for the simultaneous quantification of acetyl fentanyl and its predicted metabolite, acetyl norfentanyl, in human urine. Metabolic reference standards and deuterium-labeled internal standards were synthesized for use in an assay that coupled solid-phase extraction (SPE) with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The accuracy (% Relative Error <5%) and inter- and intrarun precision (%CV <20%) of this new method resulted in low levels of quantification (∼1 ng/mL). Similar results were obtained using liquid chromatography columns manufactured with phenyl-hexyl and biphenyl stationary phases (r(2) > 0.98). Preliminary human liver microsomal and in vivo rodent studies demonstrated that acetyl fentanyl is metabolized by cytochrome P450s to acetyl norfentanyl. Urine samples from rats treated with a toxic dose of acetyl fentanyl contained high concentrations of acetyl fentanyl and acetyl norfentanyl. Further toxicokinetic studies are required to fully elucidate the metabolic pathways responsible for acetyl fentanyl detoxification and excretion. PMID:24354295

  16. Acetyl-phosphate is a critical determinant of lysine acetylation in E. coli.

    PubMed

    Weinert, Brian T; Iesmantavicius, Vytautas; Wagner, Sebastian A; Schölz, Christian; Gummesson, Bertil; Beli, Petra; Nyström, Thomas; Choudhary, Chunaram

    2013-07-25

    Lysine acetylation is a frequently occurring posttranslational modification in bacteria; however, little is known about its origin and regulation. Using the model bacterium Escherichia coli (E. coli), we found that most acetylation occurred at a low level and accumulated in growth-arrested cells in a manner that depended on the formation of acetyl-phosphate (AcP) through glycolysis. Mutant cells unable to produce AcP had significantly reduced acetylation levels, while mutant cells unable to convert AcP to acetate had significantly elevated acetylation levels. We showed that AcP can chemically acetylate lysine residues in vitro and that AcP levels are correlated with acetylation levels in vivo, suggesting that AcP may acetylate proteins nonenzymatically in cells. These results uncover a critical role for AcP in bacterial acetylation and indicate that most acetylation in E. coli occurs at a low level and is dynamically affected by metabolism and cell proliferation in a global, uniform manner. PMID:23830618

  17. GENES ENCODING PLASTID ACETYL-COA CARBOXYLASE AND 3-PHOSPHOGLYCERATE KINASE OF THE TRITICUM/AEGILOPS COMPLEX AND THE EVOLUTIONARY HISTORY OF POLYPLOID WHEAT.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The classic wheat evolutionary history is one of adaptive radiation of the diploid Triticum/Aegilops species (A, S, D) , genome convergence and divergence of the tetraploid (T. turgidum AABB, and T. timopheevii AAAGG) and hexaploid (T. aestivum, AABBDD) species. The objective of this study was to a...

  18. Akt-dependent metabolic reprogramming regulates tumor cell histone acetylation

    PubMed Central

    Snyder, Nathaniel W.; Wei, Shuanzeng; Venneti, Sriram; Worth, Andrew J.; Yuan, Zuo-Fei; Lim, Hee-Woong; Liu, Shichong; Jackson, Ellen; Aiello, Nicole M.; Haas, Naomi B.; Rebbeck, Timothy R.; Judkins, Alexander; Won, Kyoung-Jae; Chodosh, Lewis A.; Garcia, Benjamin A.; Stanger, Ben Z.; Feldman, Michael D.; Blair, Ian A.; Wellen, Kathryn E.

    2014-01-01

    SUMMARY Histone acetylation plays important roles in gene regulation, DNA replication, and the response to DNA damage, and it is frequently deregulated in tumors. We postulated that tumor cell histone acetylation levels are determined in part by changes in acetyl-CoA availability mediated by oncogenic metabolic reprogramming. Here, we demonstrate that acetyl-CoA is dynamically regulated by glucose availability in cancer cells and that the ratio of acetyl-CoA: coenzyme A within the nucleus modulates global histone acetylation levels. In vivo, expression of oncogenic Kras or Akt stimulates histone acetylation changes that precede tumor development. Furthermore, we show that Akt's effects on histone acetylation are mediated through the metabolic enzyme ATP-citrate lyase (ACLY), and that pAkt(Ser473) levels correlate significantly with histone acetylation marks in human gliomas and prostate tumors. The data implicate acetyl-CoA metabolism as a key determinant of histone acetylation levels in cancer cells. PMID:24998913

  19. Succinyl CoA: 3-oxoacid CoA transferase (SCOT): Human cDNA cloning, human chromosomal mapping to 5p13, and mutation detection in a SCOT-deficient patient

    SciTech Connect

    Kassovska-Bratinova, S.; Robert, M.F.; Mitchell, G.A.

    1996-09-01

    Succinyl CoA: 3-oxoacid CoA transferase (SCOT; E.C.2.8.3.5) mediates the rate-determining step of ketolysis in extrahepatic tissues, the esterification of acetoacetate to CoA for use in energy production. Hereditary SCOT deficiency in humans causes episodes of severe ketoacidosis. We obtained human-heart SCOT cDNA clones spanning the entire 1,560-nt coding sequence. Sequence alignment of the human SCOT peptides with other known CoA transferases revealed several conserved regions of potential functional importance. A single {approximately}3.2-kb SCOT mRNA is present in human tissues (heart > leukocytes {much_gt} fibroblasts), but no signal is detectable in the human hepatoma cell line HepG2. We mapped the human SCOT locus (OXCT) to the cytogenetic band 5p13 by in situ hybridization. From fibroblasts of a patient with hereditary SCOT deficiency, we amplified and cloned cDNA fragments containing the entire SCOT coding sequence. We found a homozygous C-to-G transversion at nt 848, which changes the Ser 283 codon to a stop codon. This mutation (S283X) is incompatible with normal enzyme function and represents the first documentation of a pathogenic mutation in SCOT deficiency. 45 refs., 6 figs.

  20. Succinyl CoA: 3-oxoacid CoA transferase (SCOT): human cDNA cloning, human chromosomal mapping to 5p13, and mutation detection in a SCOT-deficient patient.

    PubMed Central

    Kassovska-Bratinova, S.; Fukao, T.; Song, X. Q.; Duncan, A. M.; Chen, H. S.; Robert, M. F.; Prez-Cerd, C.; Ugarte, M.; Chartrand, C.; Vobecky, S.; Kondo, N.; Mitchell, G. A.

    1996-01-01

    Succinyl CoA: 3-oxoacid CoA transferase (SCOT; E.C.2.8.3.5) mediates the rate-determining step of ketolysis in extrahepatic tissues, the esterification of acetoacetate to CoA for use in energy production. Hereditary SCOT deficiency in humans causes episodes of severe ketoacidosis. We obtained human-heart SCOT cDNA clones spanning the entire 1,560-nt coding sequence. Sequence alignment of the human SCOT peptides with other known CoA transferases revealed several conserved regions of potential functional importance. A single approximately 3.2-kb SCOT mRNA is present in human tissues (heart > leukocytes >> fibroblasts), but no signal is detectable in the human hepatoma cell line HepG2. We mapped the human SCOT locus (OXCT) to the cytogenetic band 5p13 by in situ hybridization. From fibroblasts of a patient with hereditary SCOT deficiency, we amplified and cloned cDNA fragments containing the entire SCOT coding sequence. We found a homozygous C-to-G transversion at nt 848, which changes the Ser 283 codon to a stop codon. This mutation (S283X) is incompatible with normal enzyme function and represents the first documentation of a pathogenic mutation in SCOT deficiency. Images Figure 2 Figure 6 PMID:8751852

  1. Structure of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase in complex with the feedback inhibitor CoA reveals only one active-site conformation

    SciTech Connect

    Wubben, T.; Mesecar, A.D.

    2014-10-02

    Phosphopantetheine adenylyltransferase (PPAT) catalyzes the penultimate step in the coenzyme A (CoA) biosynthetic pathway, reversibly transferring an adenylyl group from ATP to 4'-phosphopantetheine to form dephosphocoenzyme A (dPCoA). To complement recent biochemical and structural studies on Mycobacterium tuberculosis PPAT (MtPPAT) and to provide further insight into the feedback regulation of MtPPAT by CoA, the X-ray crystal structure of the MtPPAT enzyme in complex with CoA was determined to 2.11 {angstrom} resolution. Unlike previous X-ray crystal structures of PPAT-CoA complexes from other bacteria, which showed two distinct CoA conformations bound to the active site, only one conformation of CoA is observed in the MtPPAT-CoA complex.

  2. OUTCROP-BASED HIGH RESOLUTION GAMMA-RAY CHARACTERIZATION OF ARSENIC-BEARING LITHOFACIES IN THE PERMIAN GARBER SANDSTONE AND WELLINGTON FORMATION, CENTRAL OKLAHOMA AQUIFER (COA). CLEVELAND COUNTY, OKLAHOMA

    EPA Science Inventory

    The COA supplies drinking water to a number of municipalities in central Oklahoma. Two major stratigraphic units in the COA, the Garber Sandstone and Wellington Formation, contain naturally occurring arsenic that exceeds government mandated drinking-water standards (EPA, 2001). ...

  3. Unraveling Cholesterol Catabolism in Mycobacterium tuberculosis: ChsE4-ChsE5 α2β2 Acyl-CoA Dehydrogenase Initiates β-Oxidation of 3-Oxo-cholest-4-en-26-oyl CoA

    PubMed Central

    2016-01-01

    The metabolism of host cholesterol by Mycobacterium tuberculosis (Mtb) is an important factor for both its virulence and pathogenesis, although how and why cholesterol metabolism is required is not fully understood. Mtb uses a unique set of catabolic enzymes that are homologous to those required for classical β-oxidation of fatty acids but are specific for steroid-derived substrates. Here, we identify and assign the substrate specificities of two of these enzymes, ChsE4-ChsE5 (Rv3504-Rv3505) and ChsE3 (Rv3573c), that carry out cholesterol side chain oxidation in Mtb. Steady-state assays demonstrate that ChsE4-ChsE5 preferentially catalyzes the oxidation of 3-oxo-cholest-4-en-26-oyl CoA in the first cycle of cholesterol side chain β-oxidation that ultimately yields propionyl-CoA, whereas ChsE3 specifically catalyzes the oxidation of 3-oxo-chol-4-en-24-oyl CoA in the second cycle of β-oxidation that generates acetyl-CoA. However, ChsE4-ChsE5 can catalyze the oxidation of 3-oxo-chol-4-en-24-oyl CoA as well as 3-oxo-4-pregnene-20-carboxyl-CoA. The functional redundancy of ChsE4-ChsE5 explains the in vivo phenotype of the igr knockout strain of Mycobacterium tuberculosis; the loss of ChsE1-ChsE2 can be compensated for by ChsE4-ChsE5 during the chronic phase of infection. The X-ray crystallographic structure of ChsE4-ChsE5 was determined to a resolution of 2.0 Å and represents the first high-resolution structure of a heterotetrameric acyl-CoA dehydrogenase (ACAD). Unlike typical homotetrameric ACADs that bind four flavin adenine dinucleotide (FAD) cofactors, ChsE4-ChsE5 binds one FAD at each dimer interface, resulting in only two substrate-binding sites rather than the classical four active sites. A comparison of the ChsE4-ChsE5 substrate-binding site to those of known mammalian ACADs reveals an enlarged binding cavity that accommodates steroid substrates and highlights novel prospects for designing inhibitors against the committed β-oxidation step in the first cycle of cholesterol side chain degradation by Mtb. PMID:26161441

  4. Hydrolysis and transesterification of platelet-activating factor by lecithin-cholesterol acyltransferase.

    PubMed Central

    Liu, M; Subbaiah, P V

    1994-01-01

    Purified lecithin-cholesterol acyltransferase (LCAT, EC 2.3.1.43) from human plasma was found to hydrolyze platelet-activating factor (PAF) to lyso-PAF and acetate. In addition, it catalyzed the transfer of the acetate group from PAF to lysophosphatidylcholine, forming lyso-PAF and a 1-acyl analog of PAF. In contrast to the cholesterol-esterification reaction carried out by the enzyme, the hydrolysis and transacetylation of PAF by LCAT did not require an apoprotein activator and were not inhibited by sulfhydryl inhibitors but were inhibited by serum albumin. When added to a proteoliposome substrate of LCAT or to whole plasma, PAF inhibited cholesterol esterification by LCAT competitively. PAF acetylhydrolase (EC 3.1.1.47), purified from human plasma, also catalyzed the transfer of acetate from PAF to lysophosphatidylcholine. However, the LCAT-catalyzed reactions of PAF were not due to contamination with PAF acetylhydrolase, since the ratio of acetyl transfer to acetyl hydrolysis was 3 times greater for LCAT, when compared with PAF acetylhydrolase under identical conditions. Furthermore, recombinant human LCAT secreted by baby hamster kidney cells also catalyzed the hydrolysis and transacetylation of PAF. These results demonstrate that LCAT can inactivate PAF in plasma by transacetylation and suggest that it may have a role in the metabolism of PAF, and possibly of oxidized phospholipids, in plasma. PMID:8016111

  5. Structural insights into rice straw pretreated by hot-compressed water in relation to enzymatic hydrolysis.

    PubMed

    Yu, Guoce; Yano, Shinichi; Inoue, Hiroyuki; Inoue, Seiichi; Wang, Jianlong; Endo, Takashi

    2014-11-01

    Pretreatment-induced structural alteration is critical in influencing the rate and extent of enzymatic saccharification of lignocellulosic biomass. The present work has investigated structural features of rice straw pretreated by hot-compressed water (HCW) from 140 to 240 °C for 10 or 30 min and enzymatic hydrolysis profiles of pretreated rice straw. Compositional profiles of pretreated rice straw were examined to offer the basis for structural changes. The wide-angle X-ray diffraction analysis revealed possible modification in crystalline microstructure of cellulose and the severity-dependent variation of crystallinity. The specific surface area (SSA) of pretreated samples was able to achieve more than 10-fold of that of the raw material and was in linear relationship with the removal of acetyl groups and xylan. The glucose yield by enzymatic hydrolysis of pretreated materials correlated linearly with the SSA increase and the dissolution of acetyl and xylan. A quantitatively intrinsic relationship was suggested to exist between enzymatic hydrolysis and the extraction of hemicellulose components in hydrothermally treated rice straw, and SSA was considered one important structural parameter signaling the efficiency of enzymatic digestibility in HCW-treated materials in which hemicellulose removal and lignin redistribution happened. PMID:25178420

  6. Development and validation of an LC-MS/MS method for the toxicokinetic study of deoxynivalenol and its acetylated derivatives in chicken and pig plasma.

    PubMed

    Broekaert, N; Devreese, M; De Mil, T; Fraeyman, S; De Baere, S; De Saeger, S; De Backer, P; Croubels, S

    2014-11-15

    This study aims to develop an LC-MS/MS method allowing the determination of 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, deoxynivalenol and its main in vivo metabolite, deepoxy-deoxynivalenol, in broiler chickens and pigs. These species have a high exposure to these toxins, given their mainly cereal based diet. Several sample cleanup strategies were tested and further optimized by means of fractional factorial designs. A simple and straightforward sample preparation method was developed consisting out of a deproteinisation step with acetonitrile, followed by evaporation of the supernatant and reconstitution in water. The method was single laboratory validated according to European guidelines and found to be applicable for the intended purpose, with a linear response up to 200ngml(-1) and limits of quantification of 0.1-2ngml(-1). As a proof of concept, biological samples from a broiler chicken that received either deoxynivalenol, 3- or 15-acetyl-deoxynivalenol were analyzed. Preliminary results indicate nearly complete hydrolysis of 3-acetyl-deoxynivalenol to deoxynivalenol; and to a lesser extent of 15-acetyl-deoxynivalenol to deoxynivalenol. No deepoxy-deoxynivalenol was detected in any of the plasma samples. The method will be applied to study full toxicokinetic properties of deoxynivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol in broiler chickens and pigs. PMID:25264912

  7. 4-Acetylpiperazinium picrate

    PubMed Central

    Kavitha, Channappa N.; Kaur, Manpreet; Jasinski, Jerry P.; Yathirajan, Hemmige S.

    2014-01-01

    In the title salt, C6H13N2O+C6H2N3O7 ? (systematic name: 4-acetylpiperazin-1-ium 2,4,6-trinitrophenolate), the piperazin-1-ium ring has a slightly distorted chair conformation. In the picrate anion, the mean planes of the two o-NO2 and p-NO2 groups are twisted with respect to the benzene ring by 15.0?(2), 68.9?(4) and 4.4?(3), respectively. In the crystal, NH?O hydrogen bonds are observed, linking the ions into an infinite chain along [010]. In addition, weak cationanion CH?O intermolecular interactions and a weak ?? stacking interaction between the benzene rings of the anions, with an inter-centroid distance of 3.771?(8)?, help to stabilize the crystal packing, giving an overall sheet structure lying parallel to (100). Disorder was modelled for one of the O atoms in one of the o-NO2 groups over two sites with an occupancy ratio of 0.57?(6):0.43?(6). PMID:24940287

  8. DOWN-REGULATION OF CINNAMOYL-COA REDUCTASE (CCR) IN POPLAR INVESTIGATED WITH CHEMOMETRICS AND 2D-NMR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An understanding of the lignification process is of vital importance, especially for the pulp and paper industry. Cinnamoyl-coa reductase (CCR) is an enzyme that plays a central role in the lignification process. Previous results have shown that down-regulation of CCR decreases the lignin content. B...

  9. Histone deacetylase 3 indirectly modulates tubulin acetylation.

    PubMed

    Bacon, Travis; Seiler, Caroline; Wolny, Marcin; Hughes, Ruth; Watson, Peter; Schwabe, John; Grigg, Ronald; Peckham, Michelle

    2015-12-15

    Histone deacetylase 3 (HDAC3), a member of the Class I subfamily of HDACs, is found in both the nucleus and the cytoplasm. Its roles in the nucleus have been well characterized, but its cytoplasmic roles are still not elucidated fully. We found that blocking HDAC3 activity using MI192, a compound specific for HDAC3, modulated tubulin acetylation in the human prostate cancer cell line PC3. A brief 1 h treatment of PC3 cells with MI192 significantly increased levels of tubulin acetylation and ablated the dynamic behaviour of microtubules in live cells. siRNA-mediated knockdown (KD) of HDAC3 in PC3 cells, significantly increased levels of tubulin acetylation, and overexpression reduced it. However, the active HDAC3-silencing mediator of retinoic and thyroid receptors (SMRT)-deacetylase-activating domain (DAD) complex did not directly deacetylate tubulin in vitro. These data suggest that HDAC3 indirectly modulates tubulin acetylation. PMID:26450925

  10. Acetylator phenotypes in Papua New Guinea

    PubMed Central

    Penketh, R J A; Gibney, S F A; Nurse, G T; Hopkinson, D A

    1983-01-01

    Acetylator phenotypes have been determined in 139 unrelated subjects from the hitherto untested populations of Papua New Guinea, and their relevance to current antituberculous isoniazid chemotherapy is discussed. PMID:6842533

  11. Acetyl-L-carnitine increases mitochondrial protein acetylation in the aged rat heart

    PubMed Central

    Kerner, Janos; Yohannes, Elizabeth; Lee, Kwangwon; Virmani, Ashraf; Koverech, Aleardo; Cavazza, Claudio; Chance, Mark R.; Hoppel, Charles

    2015-01-01

    Previously we showed that in vivo treatment of elderly Fisher 344 rats with acetylcarnitine abolished the age-associated defect in respiratory chain complex III in interfibrillar mitochondria and improved the functional recovery of the ischemic/reperfused heart. Herein we explored mitochondrial protein acetylation as a possible mechanism for acetylcarnitines effect. In vivo treatment of elderly rats with acetylcarnitine restored cardiac acetylcarnitine content and increased mitochondrial protein lysine acetylation and increased the number of lysine-acetylated proteins in cardiac subsarcolemmal and interfibrillar mitochondria. Enzymes of the tricarboxylic acid cycle, mitochondrial ?-oxidation, and ATP synthase of the respiratory chain showed the greatest acetylation. Acetylation of isocitrate dehydrogenase, long-chain acyl-CoA dehydrogenase, complex V, and aspartate aminotransferase was accompanied by decreased catalytic activity. Several proteins were found to be acetylated only after treatment with acetylcarnitine, suggesting that exogenous acetylcarnitine served as the acetyl-donor. 2-D fluorescence difference gel electrophoresis analysis revealed that acetylcarnitine treatment also induced changes in mitochondrial protein amount; a twofold or greater increase/decrease in abundance was observed for thirty one proteins. Collectively, our data provide evidence for the first time that in the aged rat heart in vivo administration of acetylcarnitine provides acetyl groups for protein acetylation and affects the amount of mitochondrial proteins. PMID:25660059

  12. Purification, characterization, and mode of action of a rhamnogalacturonan hydrolase from Irpex lacteus, tolerant to an acetylated substrate.

    PubMed

    Normand, Jessica; Ralet, Marie-Christine; Thibault, Jean-Franois; Rogniaux, Hlne; Delavault, Philippe; Bonnin, Estelle

    2010-03-01

    A novel rhamnogalacturonase (RGase) acting on an acetylated substrate was detected in the commercial preparation Driselase, an enzymatic mixture derived from the basidiomycete Irpex lacteus. The activity was isolated by hydrophobic interaction chromatography, gel filtration, and preparative isoelectric focusing, resulting in the isolation of five different rhamnogalacturonan hydrolases exhibiting various isoelectric points from 6.2 to 7.7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry analyses after trypsin cleavage of the five fractions revealed that the five rhamnogalacturonases have a molar mass of 55 kDa without any divergences in the identified peptides. The RGase with a pI of 7.2 exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40 degrees C and 50 degrees C. Its mode of action was analyzed by mass spectrometry of the oligosaccharides produced after hydrolysis of acetylated and nonacetylated rhamnogalacturonan. Oligomers esterified by an acetyl group on the reducing galacturonic acid residue or fully acetylated were detected in the hydrolysate showing that the novel enzyme is able to bind acetylated galacturonic acid in its active site. PMID:19862512

  13. Protein acetylation in prokaryotes increases stress resistance.

    PubMed

    Ma, Qun; Wood, Thomas K

    2011-07-15

    Acetylation of lysine residues is conserved in all three kingdoms; however, its role in prokaryotes is unknown. Here we demonstrate that acetylation enables the reference bacterium Escherichia coli to withstand environmental stress. Specifically, the bacterium reaches higher cell densities and becomes more resistant to heat and oxidative stress when its proteins are acetylated as shown by deletion of the gene encoding acetyltransferase YfiQ and the gene encoding deacetylase CobB as well as by overproducing YfiQ and CobB. Furthermore, we show that the increase in oxidative stress resistance with acetylation is due to the induction of catalase activity through enhanced katG expression. We also found that two-component system proteins CpxA, PhoP, UvrY, and BasR are associated with cell catalase activity and may be responsible as the connection between bacterial acetylation and the stress response. This is the first demonstration of a specific environmental role of acetylation in prokaryotes. PMID:21703240

  14. Protein acetylation in metabolism - metabolites and cofactors.

    PubMed

    Menzies, Keir J; Zhang, Hongbo; Katsyuba, Elena; Auwerx, Johan

    2016-01-01

    Reversible acetylation was initially described as an epigenetic mechanism regulating DNA accessibility. Since then, this process has emerged as a controller of histone and nonhistone acetylation that integrates key physiological processes such as metabolism, circadian rhythm and cell cycle, along with gene regulation in various organisms. The widespread and reversible nature of acetylation also revitalized interest in the mechanisms that regulate lysine acetyltransferases (KATs) and deacetylases (KDACs) in health and disease. Changes in protein or histone acetylation are especially relevant for many common diseases including obesity, diabetes mellitus, neurodegenerative diseases and cancer, as well as for some rare diseases such as mitochondrial diseases and lipodystrophies. In this Review, we examine the role of reversible acetylation in metabolic control and how changes in levels of metabolites or cofactors, including nicotinamide adenine dinucleotide, nicotinamide, coenzyme A, acetyl coenzyme A, zinc and butyrate and/or β-hydroxybutyrate, directly alter KAT or KDAC activity to link energy status to adaptive cellular and organismal homeostasis. PMID:26503676

  15. p53 Acetylation: Regulation and Consequences

    PubMed Central

    Reed, Sara M.; Quelle, Dawn E.

    2014-01-01

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer. PMID:25545885

  16. HYDROLYSIS

    EPA Science Inventory

    Hydrolytic processes provide the baseline loss rate for any chemical in an aqueous envi- ronment. Although various hydrolytic pathways account for significant degradation of certain classes of organic chemicals, other organic structures are completely inert. Strictly speaking, hy...

  17. Photosensitized [2 + 2] cycloaddition of N-acetylated cytosine affords stereoselective formation of cyclobutane pyrimidine dimer

    PubMed Central

    Yamamoto, Junpei; Nishiguchi, Kosuke; Manabe, Koichiro; Masutani, Chikahide; Hanaoka, Fumio; Iwai, Shigenori

    2011-01-01

    Photocycloaddition between two adjacent bases in DNA produces a cyclobutane pyrimidine dimer (CPD), which is one of the major UV-induced DNA lesions, with either the cis-syn or trans-syn structure. In this study, we investigated the photosensitized intramolecular cycloaddition of partially-protected thymidylyl-(3??5?)-N4-acetyl-2?-deoxy-5-methylcytidine, to clarify the effect of the base modification on the cycloaddition reaction. The reaction resulted in the stereoselective formation of the trans-syn CPD, followed by hydrolysis of the acetylamino group. The same result was obtained for the photocycloaddition of thymidylyl-(3??5?)-N4-acetyl-2?-deoxycytidine, whereas both the cis-syn and trans-syn CPDs were formed from thymidylyl-(3??5?)-thymidine. Kinetic analyses revealed that the activation energy of the acid-catalyzed hydrolysis is comparable to that reported for the thymine-cytosine CPD. These findings provided a new strategy for the synthesis of oligonucleotides containing the trans-syn CPD. Using the synthesized oligonucleotide, translesion synthesis by human DNA polymerase ? was analyzed. PMID:20880992

  18. Yeast Phospholipase C Is Required for Normal Acetyl-CoA Homeostasis and Global Histone Acetylation*

    PubMed Central

    Galdieri, Luciano; Chang, Jennifer; Mehrotra, Swati; Vancura, Ales

    2013-01-01

    Phospholipase C (Plc1p) is required for the initial step of inositol polyphosphate (InsP) synthesis, and yeast cells with deletion of the PLC1 gene are completely devoid of any InsPs and display aberrations in transcriptional regulation. Here we show that Plc1p is required for a normal level of histone acetylation; plc1? cells that do not synthesize any InsPs display decreased acetylation of bulk histones and global hypoacetylation of chromatin histones. In accordance with the role of Plc1p in supporting histone acetylation, plc1? mutation is synthetically lethal with mutations in several subunits of SAGA and NuA4 histone acetyltransferase (HAT) complexes. Conversely, the growth rate, sensitivity to multiple stresses, and the transcriptional defects of plc1? cells are partially suppressed by deletion of histone deacetylase HDA1. The histone hypoacetylation in plc1? cells is due to the defect in degradation of repressor Mth1p, and consequently lower expression of HXT genes and reduced conversion of glucose to acetyl-CoA, a substrate for HATs. The histone acetylation and transcriptional defects can be partially suppressed and the overall fitness improved in plc1? cells by increasing the cellular concentration of acetyl-CoA. Together, our data indicate that Plc1p and InsPs are required for normal acetyl-CoA homeostasis, which, in turn, regulates global histone acetylation. PMID:23913687

  19. Vibrational and ab initio studies of 3-acetyl-6-bromocoumarin and 3-acetyl-6-methylcoumarin

    NASA Astrophysics Data System (ADS)

    Ramoji, Anuradha; Yenagi, Jayashree; Tonannavar, J.; Jadhav, V. B.; Kulkarni, M. V.

    2010-12-01

    Infrared absorption and Raman spectra (3500-50 cm -1) of 3-acetyl-6-bromocoumarin and 3-acetyl-6-methylcoumarin have been measured and interpreted, aided by electronic structure calculations at RHF and B3LYP using 6-31(d, p) basis set. It has been determined that the rotation of the acetyl group with respect to the coumarin ring results in three conformers - two trans and one cis - for each molecule, with one trans conformer being the most stable in both cases. There are significant changes in the vibrational structure as characterized by positions and intensities of certain modes in going from 3-acetyl-6-bromocoumarin to 3-acetyl-6-methylcoumarin. The carbonyl stretching mode of the pyrone ring is stable in both molecules whereas the same mode in acetyl groups is not. Ring stretching vibrations are coupled to C-H in-plane bending vibrations. Down-shifting of frequencies of methyl vibrations in acetyl group occurs vis-à-vis methyl vibrations in 3-acetyl-6-methylcoumarin. A strong Raman band at 126 cm -1 in both molecules is structure-independent non-genuine mode, correlated to lattice vibrations in the solid phase.

  20. Vibrational and ab initio studies of 3-acetyl-6-bromocoumarin and 3-acetyl-6-methylcoumarin.

    PubMed

    Ramoji, Anuradha; Yenagi, Jayashree; Tonannavar, J; Jadhav, V B; Kulkarni, M V

    2010-12-01

    Infrared absorption and Raman spectra (3500-50 cm(-1)) of 3-acetyl-6-bromocoumarin and 3-acetyl-6-methylcoumarin have been measured and interpreted, aided by electronic structure calculations at RHF and B3LYP using 6-31(d, p) basis set. It has been determined that the rotation of the acetyl group with respect to the coumarin ring results in three conformers--two trans and one cis--for each molecule, with one trans conformer being the most stable in both cases. There are significant changes in the vibrational structure as characterized by positions and intensities of certain modes in going from 3-acetyl-6-bromocoumarin to 3-acetyl-6-methylcoumarin. The carbonyl stretching mode of the pyrone ring is stable in both molecules whereas the same mode in acetyl groups is not. Ring stretching vibrations are coupled to C-H in-plane bending vibrations. Down-shifting of frequencies of methyl vibrations in acetyl group occurs vis--vis methyl vibrations in 3-acetyl-6-methylcoumarin. A strong Raman band at 126 cm(-1) in both molecules is structure-independent non-genuine mode, correlated to lattice vibrations in the solid phase. PMID:20855229

  1. Molecular Characterization of a Heteromeric ATP-Citrate Lyase That Generates Cytosolic Acetyl-Coenzyme A in Arabidopsis1[w

    PubMed Central

    Fatland, Beth L.; Ke, Jinshan; Anderson, Marc D.; Mentzen, Wieslawa I.; Cui, Li Wei; Allred, C. Christy; Johnston, Jerry L.; Nikolau, Basil J.; Wurtele, Eve Syrkin

    2002-01-01

    Acetyl-coenzyme A (CoA) is used in the cytosol of plant cells for the synthesis of a diverse set of phytochemicals including waxes, isoprenoids, stilbenes, and flavonoids. The source of cytosolic acetyl-CoA is unclear. We identified two Arabidopsis cDNAs that encode proteins similar to the amino and carboxy portions of human ATP-citrate lyase (ACL). Coexpression of these cDNAs in yeast (Saccharomyces cerevisiae) confers ACL activity, indicating that both the Arabidopsis genes are required for ACL activity. Arabidopsis ACL is a heteromeric enzyme composed of two distinct subunits, ACLA (45 kD) and ACLB (65 kD). The holoprotein has a molecular mass of 500 kD, which corresponds to a heterooctomer with an A4B4 configuration. ACL activity and the ACLA and ACLB polypeptides are located in the cytosol, consistent with the lack of targeting peptides in the ACLA and ACLB sequences. In the Arabidopsis genome, three genes encode for the ACLA subunit (ACLA-1, At1g10670; ACLA-2, At1g60810; and ACLA-3, At1g09430), and two genes encode the ACLB subunit (ACLB-1, At3g06650 and ACLB-2, At5g49460). The ACLA and ACLB mRNAs accumulate in coordinated spatial and temporal patterns during plant development. This complex accumulation pattern is consistent with the predicted physiological needs for cytosolic acetyl-CoA, and is closely coordinated with the accumulation pattern of cytosolic acetyl-CoA carboxylase, an enzyme using cytosolic acetyl-CoA as a substrate. Taken together, these results indicate that ACL, encoded by the ACLA and ACLB genes of Arabidopsis, generates cytosolic acetyl-CoA. The heteromeric organization of this enzyme is common to green plants (including Chlorophyceae, Marchantimorpha, Bryopsida, Pinaceae, monocotyledons, and eudicots), species of fungi, Glaucophytes, Chlamydomonas, and prokaryotes. In contrast, all known animal ACL enzymes have a homomeric structure, indicating that a evolutionary fusion of the ACLA and ACLB genes probably occurred early in the evolutionary history of this kingdom. PMID:12376641

  2. QSAR for cholinesterase inhibition by organophosphorus esters and CNDO/2 calculations for organophosphorus ester hydrolysis

    NASA Technical Reports Server (NTRS)

    Johnson, H.; Kenley, R. A.; Rynard, C.; Golub, M. A.

    1985-01-01

    Quantitative structure-activity relationships were derived for acetyl- and butyrylcholinesterase inhibition by various organophosphorus esters. Bimolecular inhibition rate constants correlate well with hydrophobic substituent constants, and with the presence or absence of catonic groups on the inhibitor, but not with steric substituent constants. CNDO/2 calculations were performed on a separate set of organophosphorus esters, RR'P(O)X, where R and R' are alkyl and/or alkoxy groups and X is fluorine, chlorine or a phenoxy group. For each subset with the same X, the CNDO-derived net atomic charge at the central phosphorus atom in the ester correlates well with the alkaline hydrolysis rate constant. For the whole set of esters with different X, two equations were derived that relate either charge and leaving group steric bulk, or orbital energy and bond order to the hydrogen hydrolysis rate constant.

  3. Structural, Kinetic and Proteomic Characterization of Acetyl Phosphate-Dependent Bacterial Protein Acetylation

    PubMed Central

    Sahu, Alexandria; Sorensen, Dylan; Minasov, George; Lima, Bruno P.; Scholle, Michael; Mrksich, Milan; Anderson, Wayne F.; Gibson, Bradford W.; Schilling, Birgit; Wolfe, Alan J.

    2014-01-01

    The emerging view of N?-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ?-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that N?-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial N?-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase. PMID:24756028

  4. Sulfonamide acetylation in isolated rat liver cells.

    PubMed

    Olsen, H; Mørland, J

    1981-08-01

    Primary suspension of isolated liver cells, prepared from rat livers perfused with Ca++ free buffer and 0.05% collagenase, were used for studies of sulfadimidine uptake and metabolism at various temperatures (29 degrees -41 degrees) and pH (6.4 - 7.8). The intracellular: extracellular ratio for sulfanilamide was found to be insensitive to both temperature and pH variations, while the corresponding ratio for sulfadimidine was pH dependent but insensitive to temperature variation. Decreasing pH increased the cell content of sulfadimidine. Sulfanilamide was metabolized by acetylation only, while sulfadimidine gave rise to several metabolites. The rate of sulfanilamide acetylation in primary cell suspensions was of the same order of magnitude as the acetylation rate of sulfanilamide published previously for the intact organ. Sulfanilamide was acetylated at the highest rate in the pH-region from 7.0 to 7.5, while sulfadimidine was metabolized most rapidly between pH 6.7 and 7.3. At pH 7.5 sulfadimidine was metabolized at a rate only 35% of the rate at pH 7.3. The acetylation rate of both drugs increased with increasing temperature, approximately 5% per degree celcius, and exhibited a Q10 of 1.5. PMID:7336968

  5. Histone acetylation regulates intracellular pH.

    PubMed

    McBrian, Matthew A; Behbahan, Iman Saramipoor; Ferrari, Roberto; Su, Trent; Huang, Ta-Wei; Li, Kunwu; Hong, Candice S; Christofk, Heather R; Vogelauer, Maria; Seligson, David B; Kurdistani, Siavash K

    2013-01-24

    Differences in global levels of histone acetylation occur in normal and cancer cells, although the reason why cells regulate these levels has been unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pH(i)). As pH(i) decreases, histones are globally deacetylated by histone deacetylases (HDACs), and the released acetate anions are coexported with protons out of the cell by monocarboxylate transporters (MCTs), preventing further reductions in pH(i). Conversely, global histone acetylation increases as pH(i) rises, such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pH(i), particularly compromising pH(i) maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation throughout the genome. Thus, acetylation of chromatin functions as a rheostat to regulate pH(i) with important implications for mechanism of action and therapeutic use of HDAC inhibitors. PMID:23201122

  6. Proteomic analysis of acetylation in thermophilic Geobacillus kaustophilus.

    PubMed

    Lee, Dong-Woo; Kim, Dooil; Lee, Yong-Jik; Kim, Jung-Ae; Choi, Ji Young; Kang, Sunghyun; Pan, Jae-Gu

    2013-08-01

    Recent analysis of prokaryotic N(ε)-lysine-acetylated proteins highlights the posttranslational regulation of a broad spectrum of cellular proteins. However, the exact role of acetylation remains unclear due to a lack of acetylated proteome data in prokaryotes. Here, we present the N(ε)-lysine-acetylated proteome of gram-positive thermophilic Geobacillus kaustophilus. Affinity enrichment using acetyl-lysine-specific antibodies followed by LC-MS/MS analysis revealed 253 acetylated peptides representing 114 proteins. These acetylated proteins include not only common orthologs from mesophilic Bacillus counterparts, but also unique G. kaustophilus proteins, indicating that lysine acetylation is pronounced in thermophilic bacteria. These data complement current knowledge of the bacterial acetylproteome and provide an expanded platform for better understanding of the function of acetylation in cellular metabolism. PMID:23696451

  7. Acid Hydrolysis of Trioxalatocobaltate (III) Ion

    ERIC Educational Resources Information Center

    Wiggans, P. W.

    1975-01-01

    Describes an investigation involving acid hydrolysis and using both volumetric and kinetic techniques. Presents examples of the determination of the rate constant and its variation with temperature. (GS)

  8. Beating the acetyl coenzyme A-pathway to the origin of life.

    PubMed

    Nitschke, Wolfgang; Russell, Michael J

    2013-07-19

    Attempts to draft plausible scenarios for the origin of life have in the past mainly built upon palaeogeochemical boundary conditions while, as detailed in a companion article in this issue, frequently neglecting to comply with fundamental thermodynamic laws. Even if demands from both palaeogeochemistry and thermodynamics are respected, then a plethora of strongly differing models are still conceivable. Although we have no guarantee that life at its origin necessarily resembled biology in extant organisms, we consider that the only empirical way to deduce how life may have emerged is by taking the stance of assuming continuity of biology from its inception to the present day. Building upon this conviction, we have assessed extant types of energy and carbon metabolism for their appropriateness to conditions probably pertaining in those settings of the Hadean planet that fulfil the thermodynamic requirements for life to come into being. Wood-Ljungdahl (WL) pathways leading to acetyl CoA formation are excellent candidates for such primordial metabolism. Based on a review of our present understanding of the biochemistry and biophysics of acetogenic, methanogenic and methanotrophic pathways and on a phylogenetic analysis of involved enzymes, we propose that a variant of modern methanotrophy is more likely than traditional WL systems to date back to the origin of life. The proposed model furthermore better fits basic thermodynamic demands and palaeogeochemical conditions suggested by recent results from extant alkaline hydrothermal seeps. PMID:23754811

  9. Beating the acetyl coenzyme A-pathway to the origin of life

    PubMed Central

    Nitschke, Wolfgang; Russell, Michael J.

    2013-01-01

    Attempts to draft plausible scenarios for the origin of life have in the past mainly built upon palaeogeochemical boundary conditions while, as detailed in a companion article in this issue, frequently neglecting to comply with fundamental thermodynamic laws. Even if demands from both palaeogeochemistry and thermodynamics are respected, then a plethora of strongly differing models are still conceivable. Although we have no guarantee that life at its origin necessarily resembled biology in extant organisms, we consider that the only empirical way to deduce how life may have emerged is by taking the stance of assuming continuity of biology from its inception to the present day. Building upon this conviction, we have assessed extant types of energy and carbon metabolism for their appropriateness to conditions probably pertaining in those settings of the Hadean planet that fulfil the thermodynamic requirements for life to come into being. Wood–Ljungdahl (WL) pathways leading to acetyl CoA formation are excellent candidates for such primordial metabolism. Based on a review of our present understanding of the biochemistry and biophysics of acetogenic, methanogenic and methanotrophic pathways and on a phylogenetic analysis of involved enzymes, we propose that a variant of modern methanotrophy is more likely than traditional WL systems to date back to the origin of life. The proposed model furthermore better fits basic thermodynamic demands and palaeogeochemical conditions suggested by recent results from extant alkaline hydrothermal seeps. PMID:23754811

  10. A Novel Functional Site in the PB2 Subunit of Influenza A Virus Essential for Acetyl-CoA Interaction, RNA Polymerase Activity, and Viral Replication*

    PubMed Central

    Hatakeyama, Dai; Shoji, Masaki; Yamayoshi, Seiya; Hirota, Takenori; Nagae, Monami; Yanagisawa, Shin; Nakano, Masahiro; Ohmi, Naho; Noda, Takeshi; Kawaoka, Yoshihiro; Kuzuhara, Takashi

    2014-01-01

    The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, are essential for viral transcription and replication. The PB2 subunit binds to the host RNA cap (7-methylguanosine triphosphate (m7GTP)) and supports the endonuclease activity of PA to snatch the cap from host pre-mRNAs. However, the structure of PB2 is not fully understood, and the functional sites remain unknown. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is involved in interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m7GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m7GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced the binding ability of PB2 to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication. PMID:25063805

  11. [Transferase activity of horse blood serum cholinesterase at hydrolysis of 1-methyl-8-acetoxychinolium iodide in the presence of aliphatic alcohols].

    PubMed

    Basova, N E; Kormilitsyn, B N; Perchenok, A Yu; Rozengart, E V; Saakov, V S; Suvorov, A A

    2014-01-01

    To check whether the horse blood serum butyrylcholinesterase expresses transferase activity at the complex ester hydrolysis in the presense of several low-molecular aliphatic alcohols, a study was performed with aid of the chromogenic substrate 1-methyl-8-acetoxychinolium whose phenolic hydrolysis product absorbs intensively at 445 nm, whereas the initial ester in this specter area practically does not absorb. This allowed measuring simultaneously the products of accumulation of both products of enzymatic hydrolysis: of acetic acid by the potentiometric, while of phenol--by the photometric method. Rates of formation of both products of enzymatic hydrolysis are practically equal in experiments with all studied alcohols. This indicates that horse blood serum butyrylcholinesterase under these experimental conditions does not catalize transfer of acetyl residue to the studied aliphatic alcohols, i. e. does not have transefase activity. PMID:25486801

  12. Molecular biology of acetyl-CoA metabolism.

    PubMed

    Nikolau, B J; Oliver, D J; Schnable, P S; Wurtele, E S

    2000-12-01

    We have characterized the expression of potential acetyl-CoA-generating genes (acetyl-CoA synthetase, pyruvate decarboxylase, acetaldehyde dehydrogenase, plastidic pyruvate dehydrogenase complex and ATP-citrate lyase), and compared these with the expression of acetyl-CoA-metabolizing genes (heteromeric and homomeric acetyl-CoA carboxylase). These comparisons have led to the development of testable hypotheses as to how distinct pools of acetyl-CoA are generated and metabolized. These hypotheses are being tested by combined biochemical, genetic and molecular biological experiments, which is providing insights into how acetyl-CoA metabolism is regulated. PMID:11171136

  13. Acetylation of bleached Kraft pulp: effect of xylan content on properties of acetylated compounds.

    PubMed

    Peredo, Karol; Reyes, Herna; Escobar, Danilo; Vega-Lara, Johana; Berg, Alex; Pereira, Miguel

    2015-03-01

    Bleached Kraft pulp (BKP) from Eucalyptus globulus and cotton xylan blends (CXB) was acetylated. The effects of xylan content on cellulose acetylation and the properties of the acetylated material were studied. An increase in xylan content caused a slight decrease in the degree of substitution (2.98 to 2.68 for CXB; 2.93 to 2.84 for BKP). Thermal analysis showed that the melting temperature also decreases from 268.0 to 188.8 °C for CXB and from 221.4 to 212.8 °C for BKP. Moreover, the solubility decreased due to the partial dissolution of acetylated xylans. The presence of xylans during Kraft pulp acetylation does not have a significant negative effect on the physical properties of the acetylated material, but the decrease in melting temperature was beneficial for the application of acetylated polymer as a natural internal plasticizer. This is considered to be an important argument for BKP utilization in the cellulose acetate manufacturing process. PMID:25498729

  14. Preliminary toxicological study of ferric acetyl acetonate

    SciTech Connect

    London, J.E.; Smith, D.M.

    1983-01-01

    The calculated acute oral LD/sub 50//sup 30/ (lethal does for 50% of the animals occuring with 30 days after compound administration) values for ferric acetyl acetonate were 584 mg/kg in mice and 995 mg/kg in rats. According to classical guidelines, this compound would be considered slightly toxic in both species. Skin application studies in the rabbit demonstrated the compound to be irritating. The eye irritation study disclosed the compound to be a severe irritant causing permanent damage to the cornea (inflammation and scarring resulting in blindness). The sensitization study in the guinea pig did not show ferric acetyl acetonate to be deleterious in this regard.

  15. SUBSURFACE WELL-LOG CORRELATION OF ARSENIC-BEARING LITHOFACIES IN THE PERMIAN GARBER SANDSTONE AND WELLINGTON FORMATION, CENTRAL OKLAHOMA AQUIFER (COA), CLEVELAND COUNTY, OKLAHOMA

    EPA Science Inventory

    The fluvial Garber Sandstone and the underlying Wellington Formation are important sources of drinking water in central Oklahoma. These formations, which make up much of the COA, consist of amalgamated sandstones with some interbedded mudstones, siltstones, and local mudstone- a...

  16. ?-Lapachone Ameliorates Lipotoxic Cardiomyopathy in Acyl CoA Synthase Transgenic Mice

    PubMed Central

    Jeong, Moon Hee; Tran, Nguyen Khoi Song; Kwak, Tae Hwan; Park, Byung Keon; Lee, Chul Soon; Park, Tae-Sik; Lee, Young-Hoon; Park, Woo Jin; Yang, Dong Kwon

    2014-01-01

    Lipotoxic cardiomyopathy is caused by myocardial lipid accumulation and often occurs in patients with diabetes and obesity. This study investigated the effects of ?-lapachone (?-lap), a natural compound that activates Sirt1 through elevation of the intracellular NAD+ level, on acyl CoA synthase (ACS) transgenic (Tg) mice, which have lipotoxic cardiomyopathy. Oral administration of ?-lap to ACS Tg mice significantly attenuated heart failure and inhibited myocardial accumulation of triacylglycerol. Electron microscopy and measurement of mitochondrial complex II protein and mitochondrial DNA revealed that administration of ?-lap restored mitochondrial integrity and biogenesis in ACS Tg hearts. Accordingly, ?-lap administration significantly increased the expression of genes associated with mitochondrial biogenesis and fatty acid metabolism that were down-regulated in ACS Tg hearts. ?-lap also restored the activities of Sirt1 and AMP-activated protein kinase (AMPK), the two key regulators of metabolism, which were suppressed in ACS Tg hearts. In H9C2 cells, ?-lap-mediated elevation of AMPK activity was retarded when the level of Sirt1 was reduced by transfection of siRNA against Sirt1. Taken together, these results indicate that ?-lap exerts cardioprotective effects against cardiac lipotoxicity through the activation of Sirt1 and AMPK. ?-lap may be a novel therapeutic agent for the treatment of lipotoxic cardiomyopathy. PMID:24614171

  17. Expression and Characterization of ?-Methylacyl CoA Racemase from Anisakis simplex Larvae

    PubMed Central

    Kim, Bong Jin; Kim, Sun Mi; Cho, Min Kyung; Yu, Hak Sun; Lee, Yong Seok; Cha, Hee Jae

    2012-01-01

    Larval excretory-secretory products of Anisakis simplex are known to cause allergic reactions in humans. A cDNA library of A. simplex 3rd-stage larvae (L3) was immunoscreened with polyclonal rabbit serum raised against A. simplex L3 excretory-secretory products to identify an antigen that elicits the immune response. One cDNA clone, designated as ?-methylacyl CoA racemase (Amacr) contained a 1,412 bp cDNA transcript with a single open reading frame that encoded 418 amino acids. A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species. Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40?) and time (24-48 hr). Immunohistochemical analysis revealed that Amacr was expressed mainly in the ventriculus of A. simplex larvae. The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae. PMID:22711931

  18. Expression and characterization of ?-methylacyl CoA racemase from Anisakis simplex larvae.

    PubMed

    Kim, Bong Jin; Kim, Sun Mi; Cho, Min Kyung; Yu, Hak Sun; Lee, Yong Seok; Cha, Hee Jae; Ock, Meesun

    2012-06-01

    Larval excretory-secretory products of Anisakis simplex are known to cause allergic reactions in humans. A cDNA library of A. simplex 3rd-stage larvae (L3) was immunoscreened with polyclonal rabbit serum raised against A. simplex L3 excretory-secretory products to identify an antigen that elicits the immune response. One cDNA clone, designated as ?-methylacyl CoA racemase (Amacr) contained a 1,412 bp cDNA transcript with a single open reading frame that encoded 418 amino acids. A. simplex Amacr showed a high degree of homology compared to Amacr orthologs from other species. Amacr mRNA was highly and constitutively expressed regardless of temperature (10-40?) and time (24-48 hr). Immunohistochemical analysis revealed that Amacr was expressed mainly in the ventriculus of A. simplex larvae. The Amacr protein produced in large quantities from the ventriculus is probably responsible for many functions in the development and growth of A. simplex larvae. PMID:22711931

  19. Nonenzymatic Protein Acetylation Detected by NAPPA Protein Arrays.

    PubMed

    Olia, Adam S; Barker, Kristi; McCullough, Cheryl E; Tang, Hsin-Yao; Speicher, David W; Qiu, Ji; LaBaer, Joshua; Marmorstein, Ronen

    2015-09-18

    Acetylation is a post-translational modification that occurs on thousands of proteins located in many cellular organelles. This process mediates many protein functions and modulates diverse biological processes. In mammalian cells, where acetyl-CoA is the primary acetyl donor, acetylation in the mitochondria is thought to occur by chemical means due to the relatively high concentration of acetyl-CoA located in this organelle. In contrast, acetylation outside of the mitochondria is thought to be mediated predominantly by acetyltransferase enzymes. Here, we address the possibility that nonenzymatic chemical acetylation outside of the mitochondria may be more common than previously appreciated. We employed the Nucleic Acid Programmable Protein Array platform to perform an unbiased screen for human proteins that undergo chemical acetylation, which resulted in the identification of a multitude of proteins with diverse functions and cellular localization. Mass spectrometry analysis revealed that basic residues typically precede the acetylated lysine in the -7 to -3 position, and we show by mutagenesis that these basic residues contribute to chemical acetylation capacity. We propose that these basic residues lower the pKa of the substrate lysine for efficient chemical acetylation. Many of the identified proteins reside outside of the mitochondria and have been previously demonstrated to be acetylated in vivo. As such, our studies demonstrate that chemical acetylation occurs more broadly throughout the eukaryotic cell than previously appreciated and suggests that this post-translational protein modification may have more diverse roles in protein function and pathway regulation. PMID:26083674

  20. Non-enzymatic protein acetylation detected by NAPPA protein arrays*

    PubMed Central

    Olia, Adam S.; Barker, Kristi; McCullough, Cheryl E.; Tang, Hsin-Yao; Speicher, David W.; Qiu, Ji; LaBaer, Joshua; Marmorstein, Ronen

    2015-01-01

    Acetylation is a post-translational modification that occurs on thousands of proteins located in many cellular organelles. This process mediates many protein functions and modulates diverse biological processes. In mammalian cells, where acetyl-CoA is the primary acetyl donor, acetylation in the mitochondria is thought to occur by chemical means due to the relatively high concentration of acetyl-CoA located in this organelle. In contrast, acetylation outside of the mitochondria is thought to be mediated predominantly by acetyltransferase enzymes. Here we address the possibility that non-enzymatic chemical acetylation outside of the mitochondria may be more common than previously appreciated. We employed the Nucleic Acid Programmable Protein Array platform to perform an unbiased screen for human proteins that undergo chemical acetylation, which resulted in the identification of a multitude of proteins with diverse functions and cellular localization. Mass spectrometry analysis revealed that basic residues typically precede the acetylated lysine in the ?7 to ?3 position, and we show by mutagenesis that these basic residues contribute to chemical acetylation capacity. We propose that these basic residues lower the pKa of the substrate lysine for efficient chemical acetylation. Many of the identified proteins reside outside of the mitochondria, and have been previously demonstrated to be acetylated in vivo. As such, our studies demonstrate that chemical acetylation occurs more broadly throughout the eukaryotic cell than previously appreciated, and suggests that this post-translational protein modification may have more diverse roles in protein function and pathway regulation. PMID:26083674

  1. CoA Synthase is phosphorylated on tyrosines in mammalian cells, interacts with and is dephosphorylated by Shp2PTP.

    PubMed

    Breus, Oksana; Panasyuk, Ganna; Gout, Ivan T; Filonenko, Valeriy; Nemazanyy, Ivan

    2010-02-01

    CoA Synthase (CoASy, 4'-phosphopantetheine adenylyltransferase/dephospho-CoA kinase) mediates two final stages of de novo coenzyme A (CoA) biosynthesis in higher eukaryotes. Unfortunately very little is known about regulation of this important metabolic pathway. In this study, we demonstrate that CoASy interacts in vitro with Src homology-2 (SH2) domains of a number of signaling proteins, including Src homology-2 domains containing protein tyrosine phosphatase (Shp2PTP). Complexes between CoASy and Shp2PTP exist in vivo in mammalian cells and this interaction is regulated in a growth-factor-dependent manner. We have also demonstrated that endogenous CoASy is phosphorylated on tyrosine residues in vivo, and that cytoplasmic protein tyrosine kinases can mediate this phosphorylation in vitro and in vivo. Importantly, Shp2PTP-mediated CoASy in vitro dephosphorylation leads to an increase in CoASy enzymatic phosphopantetheine adenylyltransferase (PPAT) activity. We therefore argue that CoASy is a novel potential substrate of Shp2PTP and phosphorylation of CoASy at tyrosine residue(s) could represent unrecognized before mechanism of modulation intracellular CoA level in response to hormonal and (or) other extracellular stimuli. PMID:19763791

  2. Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of Saccharomyces cerevisiae

    PubMed Central

    Kozak, Barbara U.; van Rossum, Harmen M.; Luttik, Marijke A. H.; Akeroyd, Michiel; Benjamin, Kirsten R.; Wu, Liang; de Vries, Simon; Daran, Jean-Marc; Pronk, Jack T.

    2014-01-01

    ABSTRACT The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E.faecalis PDH required simultaneous expression of E.faecalis genes encoding its E1?, E1?, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E.faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E.faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E.faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. PMID:25336454

  3. Rate of Hydrolysis of Tertiary Halogeno Alkanes

    ERIC Educational Resources Information Center

    Pritchard, D. R.

    1978-01-01

    Describes an experiment to measure the relative rate of hydrolysis of the 2-x-2 methylpropanes, where x is bromo, chloro or iodo. The results are plotted on a graph from which the relative rate of hydrolysis can be deduced. (Author/GA)

  4. Microwave Pretreatment For Hydrolysis Of Cellulose

    NASA Technical Reports Server (NTRS)

    Cullingford, Hatice S.; George, Clifford E.; Lightsey, George R.

    1993-01-01

    Microwave pretreatment enhances enzymatic hydrolysis of cellulosic wastes into soluble saccharides used as feedstocks for foods, fuels, and other products. Low consumption of energy, high yield, and low risk of proposed hydrolysis process incorporating microwave pretreatment makes process viable alternative to composting.

  5. PHTHALATE ESTER HYDROLYSIS: LINEAR FREE ENERGY RELATIONSHIPS

    EPA Science Inventory

    Alkaline hydrolysis rate constants were measured for dimethyl, diethyl, di-n-butyl, di-iso-butyl, and di-(2-ethylhexyl) phthalate esters in water. A linear free energy relationship (LFER) was established for estimating alkaline hydrolysis rate constants for other phthalate esters...

  6. Identification of 9?-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid in steroid degradation by Comamonas testosteroni TA441 and its conversion to the corresponding 6-en-5-oyl coenzyme A (CoA) involving open reading frame 28 (ORF28)- and ORF30-encoded acyl-CoA dehydrogenases.

    PubMed

    Horinouchi, Masae; Hayashi, Toshiaki; Koshino, Hiroyuki; Malon, Michal; Hirota, Hiroshi; Kudo, Toshiaki

    2014-10-01

    Comamonas testosteroni TA441 degrades steroids via aromatization and meta-cleavage of the A ring, followed by hydrolysis, and produces 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid as an intermediate compound. Herein, we identify a new intermediate compound, 9?-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. Open reading frame 28 (ORF28)- and ORF30-encoded acyl coenzyme A (acyl-CoA) dehydrogenase was shown to convert the CoA ester of 9?-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid to the CoA ester of 9?-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid. A homology search of the deduced amino acid sequences suggested that the ORF30-encoded protein is a member of the acyl-CoA dehydrogenase_fadE6_17_26 family, whereas the deduced amino acid sequence of ORF28 showed no significant similarity to specific acyl-CoA dehydrogenase family proteins. Possible steroid degradation gene clusters similar to the cluster of TA441 appear in bacterial genome analysis data. In these clusters, ORFs similar to ORFs 28 and 30 are often found side by side and ordered in the same manner as ORFs 28 and 30. PMID:25092028

  7. Identification of 9?-Hydroxy-17-Oxo-1,2,3,4,10,19-Hexanorandrostan-5-Oic Acid in Steroid Degradation by Comamonas testosteroni TA441 and Its Conversion to the Corresponding 6-En-5-Oyl Coenzyme A (CoA) Involving Open Reading Frame 28 (ORF28)- and ORF30-Encoded Acyl-CoA Dehydrogenases

    PubMed Central

    Hayashi, Toshiaki; Koshino, Hiroyuki; Malon, Michal; Hirota, Hiroshi; Kudo, Toshiaki

    2014-01-01

    Comamonas testosteroni TA441 degrades steroids via aromatization and meta-cleavage of the A ring, followed by hydrolysis, and produces 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid as an intermediate compound. Herein, we identify a new intermediate compound, 9?-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. Open reading frame 28 (ORF28)- and ORF30-encoded acyl coenzyme A (acyl-CoA) dehydrogenase was shown to convert the CoA ester of 9?-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid to the CoA ester of 9?-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid. A homology search of the deduced amino acid sequences suggested that the ORF30-encoded protein is a member of the acyl-CoA dehydrogenase_fadE6_17_26 family, whereas the deduced amino acid sequence of ORF28 showed no significant similarity to specific acyl-CoA dehydrogenase family proteins. Possible steroid degradation gene clusters similar to the cluster of TA441 appear in bacterial genome analysis data. In these clusters, ORFs similar to ORFs 28 and 30 are often found side by side and ordered in the same manner as ORFs 28 and 30. PMID:25092028

  8. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...) The direct acetylation of edible monoglycerides with acetic anhydride without the use of catalyst or molecular distillation, and with the removal by vacuum distillation, if necessary, of the acetic acid, acetic anhydride, and triacetin. (b) The food additive has a Reichert-Meissl value of 75-200 and an...

  9. Palladium-Catalyzed Acetylation of Arenes

    PubMed Central

    2015-01-01

    A simple method for the preparation of aryl methyl ketones is reported. The transformation involves the Pd-catalyzed coupling of an acyl anion equivalent, acetyltrimethylsilane, with aryl bromides to afford the corresponding acetylated arenes in synthetically useful yields. The methodology is tolerant of heterocycles and provides a new method for arene functionalization. PMID:24405108

  10. Gene encoding acetyl-coenzyme A carboxylase

    SciTech Connect

    Roessler, Paul G.; Ohlrogge, John B.

    1996-01-01

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides.

  11. Histone deacetylase 3 indirectly modulates tubulin acetylation

    PubMed Central

    Bacon, Travis; Seiler, Caroline; Wolny, Marcin; Hughes, Ruth; Watson, Peter; Schwabe, John; Grigg, Ronald; Peckham, Michelle

    2015-01-01

    Histone deacetylase 3 (HDAC3), a member of the Class I subfamily of HDACs, is found in both the nucleus and the cytoplasm. Its roles in the nucleus have been well characterized, but its cytoplasmic roles are still not elucidated fully. We found that blocking HDAC3 activity using MI192, a compound specific for HDAC3, modulated tubulin acetylation in the human prostate cancer cell line PC3. A brief 1 h treatment of PC3 cells with MI192 significantly increased levels of tubulin acetylation and ablated the dynamic behaviour of microtubules in live cells. siRNA-mediated knockdown (KD) of HDAC3 in PC3 cells, significantly increased levels of tubulin acetylation, and overexpression reduced it. However, the active HDAC3–silencing mediator of retinoic and thyroid receptors (SMRT)–deacetylase-activating domain (DAD) complex did not directly deacetylate tubulin in vitro. These data suggest that HDAC3 indirectly modulates tubulin acetylation. PMID:26450925

  12. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, P.G.; Ohlrogge, J.B.

    1996-09-24

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

  13. Peroxisomal Delta(3),Delta(2)-enoyl CoA isomerases and evolution of cytosolic paralogues in embryophytes.

    PubMed

    Goepfert, Simon; Vidoudez, Charles; Tellgren-Roth, Christian; Delessert, Syndie; Hiltunen, J Kalervo; Poirier, Yves

    2008-12-01

    Delta(3),Delta(2)-enoyl CoA isomerase (ECI) is an enzyme that participates in the degradation of unsaturated fatty acids through the beta-oxidation cycle. Three genes encoding Delta(3),Delta(2)-enoyl CoA isomerases and named AtECI1, AtECI2 and AtECI3 have been identified in Arabidopsis thaliana. When expressed heterologously in Saccharomyces cerevisiae, all three ECI proteins were targeted to the peroxisomes and enabled the yeast Deltaeci1 mutant to degrade 10Z-heptadecenoic acid, demonstrating Delta(3),Delta(2)-enoyl CoA isomerase activity in vivo. Fusion proteins between yellow fluorescent protein and AtECI1 or AtECI2 were targeted to the peroxisomes in onion epidermal cells and Arabidopsis root cells, but a similar fusion protein with AtECI3 remained in the cytosol for both tissues. AtECI3 targeting to peroxisomes in S. cerevisiae was dependent on yeast PEX5, while expression of Arabidopsis PEX5 in yeast failed to target AtECI3 to peroxisomes. AtECI2 and AtECI3 are tandem duplicated genes and show a high level of amino acid conservation, except at the C-terminus; AtECI2 ends with the well conserved peroxisome targeting signal 1 (PTS1) terminal tripeptide PKL, while AtECI3 possesses a divergent HNL terminal tripeptide. Evolutionary analysis of ECI genes in plants revealed several independent duplication events, with duplications occurring in rice and Medicago truncatula, generating homologues with divergent C-termini and no recognizable PTS1. All plant ECI genes analyzed, including AtECI3, are under negative purifying selection, implying functionality of the cytosolic AtECI3. Analysis of the mammalian and fungal genomes failed to identify cytosolic variants of the Delta(3),Delta(2)-enoyl CoA isomerase, indicating that evolution of cytosolic Delta(3),Delta(2)-enoyl CoA isomerases is restricted to the plant kingdom. PMID:18657232

  14. Acetylation of blasticidin S by its producing actinomycetes.

    PubMed

    Sugiyama, M; Takeda, A; Paik, S Y; Nimi, O; Nomi, R

    1986-06-01

    A blasticidin S-producing actinomycetes, Streptoverticillium sp. JCM 4673 possesses an enzyme activity which acetylates the drug in the presence of acetyl coenzyme A. The modified drug was biologically inactive when tested against protein synthesis in vivo and in vitro. Production of the enzyme which acetylates blasticidin S increases with formation of the antibiotic during cell growth. PMID:3733531

  15. 40 CFR 721.10520 - Acetylated fatty acid glycerides (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Acetylated fatty acid glycerides... Specific Chemical Substances 721.10520 Acetylated fatty acid glycerides (generic). (a) Chemical substance... acetylated fatty acid glycerides (PMN P-11-160) is subject to reporting under this section for...

  16. 40 CFR 721.10520 - Acetylated fatty acid glycerides (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Acetylated fatty acid glycerides... Specific Chemical Substances 721.10520 Acetylated fatty acid glycerides (generic). (a) Chemical substance... acetylated fatty acid glycerides (PMN P-11-160) is subject to reporting under this section for...

  17. Retarded hydrolysis-condensing reactivity of tetrabutyl titanate by acetylacetone and the application in dye-sensitized solar cells

    SciTech Connect

    Zhou, Conghua Ouyang, Jun; Yang, Bingchu

    2013-10-15

    Graphical abstract: - Highlights: • Effect of acetone acetyl on coarsening rate of TiO{sub 2} nanocrystallites was studied. • Hydrolysis reactivity of alkoxide was retarded with addition of acetone acetyl. • Coarsening rate of TiO{sub 2} nanocrystallites is retarded with addition of acetone acetyl. • The synthesized TiO{sub 2} sols were utilized in dye sensitized solar cells. • Small particles formed by Ti-complexes were beneficial for device performance. - Abstract: TiO{sub 2} nanocrystallites have been synthesized by hydrothermal reaction using tetrabutyl titanate as source material. Acetylacetone was utilized to modify hydrolysis-condensation behavior of the alkoxide and thus coarsening dynamics of TiO{sub 2} nanocrystallites in the reaction. With assistance of Fourier transformation infrared spectrum, transmission electron microscopy, selected area electron diffraction and X-ray diffraction, interaction between acetylacetone and tetrabutyltitanate was explored, crystallographic and morphological properties of TiO{sub 2} nanocrystallites were monitored. Less hydrolysable complex was formed by “method of chelating” as tetrabutyltitanate was mixed with acetylacetone, leading to retarded coarsening rate of nanocrystallites. The obtained TiO{sub 2} nanocrystallites were applied to fabricate nanoporous photoanode of dye sensitized solar cells. Improvement of 18% has been achieved for photo-to-electric energy conversion efficiency of the devices due to both upgraded open circuit voltage and photocurrent density.

  18. HMG CoA Reductase Inhibitors, NSAIDs and Risk of Glioma

    PubMed Central

    Ferris, Jennifer; McCoy, Lucie; Neugut, Alfred I; Wrensch, Margaret; Lai, Rose

    2012-01-01

    HMG Co-A reductase inhibitors (statins) have shown inverse associations with cancer risks, but the results have been inconsistent. Since there is no previous published data in brain tumors, we conducted a case-control study to investigate statin therapy and risk of glioma. We also further evaluated the use of nonsteriodal anti-inflammatory drugs (NSAIDs) in the risk of these tumors. We recruited newly diagnosed glioma cases and frequency matched controls at Columbia University and the University of California San Francisco. Standardized questions on statins and NSAIDs were used at both institutions. Intakes of these drugs were defined as > 6 months of at least twice weekly use versus less than this amount or never use. From July 2007 to January 2010, we recruited a total of 517 cases and 400 controls. Simvastatin and lovastatin showed significant inverse associations with glioma (OR = 0.49, 95% CI 0.30, 0.81 and OR = 0.47, 95% CI 0.24, 0.93, respectively). In NSAIDs, aspirin use was also inversely related to glioma risk (OR = 0.68, 95% CI 0.49, 0.96). Both statins and NSAIDs showed significant inverse trends between duration of drug use and glioma risks (trend tests p = 0.03 and p = 0.02, respectively), and drug intake > 120 months demonstrated the most significant associations for both types of medication. The inverse association between statin therapy and risk of glioma supports the roles of Ras/Rho GTPases or inflammatory cytokines in gliomagenesis, and similar relationship between NSAIDs and glioma highlights the importance of cyclo-oxygenase-2 in glioma pathogenesis. PMID:22419506

  19. Depolymerization of ?-1,6-N-Acetyl-d-Glucosamine Disrupts the Integrity of Diverse Bacterial Biofilms

    PubMed Central

    Itoh, Yoshikane; Wang, Xin; Hinnebusch, B. Joseph; Preston, James F.; Romeo, Tony

    2005-01-01

    Polymeric ?-1,6-N-acetyl-d-glucosamine (poly-?-1,6-GlcNAc) has been implicated as an Escherichia coli and Staphylococcus epidermidis biofilm adhesin, the formation of which requires the pgaABCD and icaABCD loci, respectively. Enzymatic hydrolysis of poly-?-1,6-GlcNAc, demonstrated for the first time by chromatography and mass spectrometry, disrupts biofilm formation by these species and by Yersinia pestis and Pseudomonas fluorescens, which possess pgaABCD homologues. PMID:15601723

  20. Isolation, purification and structural characterization of an acetylated heteroglycan from the unripe fruits of Manilkara zapota L.

    PubMed

    Mondal, Subhas; Das, Debsankar; Roy, Sadhan K; Islam, Syed S

    2012-06-01

    A water soluble polysaccharide isolated from the hot water extract of the unripe fruits of Manilkara zapota L. was found to consist of 3-O-acyl-L-rhamnose, L-arabinose, 3-O-acetyl-D-methyl galacturonate in a molar proportion of nearly 1:1:1. Structural investigation of the polysaccharide was carried out using total hydrolysis, methylation analysis; periodate oxidation followed by GLC-MS, and NMR experiments. On the basis of the above experiments it is concluded that the following repeating unit is present in the polysaccharide. PMID:22560630

  1. Hydrolysis of cellulose with superconcentrated hydrochloric acid

    SciTech Connect

    Goldstein, I.S.; Pereira, H.; Pittman, J.L.; Strouse, B.A.; Scaringelli, F.P.

    1983-01-01

    Batch hydrolysis of pure cellulose and cellulose in prehydrolyzed sweetgum wood with superconcentrated hydrochloric acid (15-16N) has been studied at moderate temperatures (20-50/sup 0/C). In the absence of agitation the hydrolysis does not go to completion at low liquid-to-solid ratios, but does appear to follow diffusion-controlled first-order kinetics. With agitation, hydrolysis goes to completion with glucose yields of 85-90%, and the higher activation energy associated with the homogeneous hydrolysis of glycosidic bonds is observed. The first-order rate constants are about 5-20 times greater than without agitation. Complete hydrolysis can be effected in 10 min at 50/sup 0/C and 60 min at 30/sup 0/C. Cellulose hydrolysis in the absence of agitation can be increased from less than 60% to over 90% by the addition of lithium or zinc ions. Even with agitation the hydrolysis of wood chips is diffusion controlled. 14 references, 4 figures, 3 tables.

  2. Degradation of Aromatics and Chloroaromatics by Pseudomonas sp. Strain B13: Purification and Characterization of 3-Oxoadipate:Succinyl-Coenzyme A (CoA) Transferase and 3-Oxoadipyl-CoA Thiolase

    PubMed Central

    Kaschabek, Stefan R.; Kuhn, Bernd; Mller, Dagmar; Schmidt, Eberhard; Reineke, Walter

    2002-01-01

    The degradation of 3-oxoadipate in Pseudomonas sp. strain B13 was investigated and was shown to proceed through 3-oxoadipyl-coenzyme A (CoA) to give acetyl-CoA and succinyl-CoA. 3-Oxoadipate:succinyl-CoA transferase of strain B13 was purified by heat treatment and chromatography on phenyl-Sepharose, Mono-Q, and Superose 6 gels. Estimation of the native molecular mass gave a value of 115,000 5,000 Da with a Superose 12 column. Polyacrylamide gel electrophoresis under denaturing conditions resulted in two distinct bands of equal intensities. The subunit A and B values were 32,900 and 27,000 Da. Therefore it can be assumed that the enzyme is a heterotetramer of the type A2B2 with a molecular mass of 120,000 Da. The N-terminal amino acid sequences of both subunits are as follows: subunit A, AELLTLREAVERFVNDGTVALEGFTHLIPT; subunit B, SAYSTNEMMTVAAARRLKNGAVVFV. The pH optimum was 8.4. Km values were 0.4 and 0.2 mM for 3-oxoadipate and succinyl-CoA, respectively. Reversibility of the reaction with succinate was shown. The transferase of strain B13 failed to convert 2-chloro- and 2-methyl-3-oxoadipate. Some activity was observed with 4-methyl-3-oxoadipate. Even 2-oxoadipate and 3-oxoglutarate were shown to function as poor substrates of the transferase. 3-Oxoadipyl-CoA thiolase was purified by chromatography on DEAE-Sepharose, blue 3GA, and reactive brown-agarose. Estimation of the native molecular mass gave 162,000 5,000 Da with a Superose 6 column. The molecular mass of the subunit of the denatured protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42 kDa. On the basis of these results, 3-oxoadipyl-CoA thiolase should be a tetramer of the type A4. The N-terminal amino acid sequence of 3-oxoadipyl-CoA thiolase was determined to be SREVYI-DAVRTPIGRFG. The pH optimum was 7.8. Km values were 0.15 and 0.01 mM for 3-oxoadipyl-CoA and CoA, respectively. Sequence analysis of the thiolase terminus revealed high percentages of identity (70 to 85%) with thiolases of different functions. The N termini of the transferase subunits showed about 30 to 35% identical amino acids with the glutaconate-CoA transferase of an anaerobic bacterium but only an identity of 25% with the respective transferases of aromatic compound-degrading organisms was found. PMID:11741862

  3. Acetyl coenzyme A: a central metabolite and second messenger.

    PubMed

    Pietrocola, Federico; Galluzzi, Lorenzo; Bravo-San Pedro, Jos Manuel; Madeo, Frank; Kroemer, Guido

    2015-06-01

    Acetyl-coenzyme A (acetyl-CoA) is a central metabolic intermediate. The abundance of acetyl-CoA in distinct subcellular compartments reflects the general energetic state of the cell. Moreover, acetyl-CoA concentrations influence the activity or specificity of multiple enzymes, either in an allosteric manner or by altering substrate availability. Finally, by influencing the acetylation profile of several proteins, including histones, acetyl-CoA controls key cellular processes, including energy metabolism, mitosis, and autophagy, both directly and via the epigenetic regulation of gene expression. Thus, acetyl-CoA determines the balance between cellular catabolism and anabolism by simultaneously operating as a metabolic intermediate and as a second messenger. PMID:26039447

  4. Histone Acetylation Enzymes Coordinate Metabolism and Gene Expression.

    PubMed

    Shen, Yuan; Wei, Wei; Zhou, Dao-Xiu

    2015-10-01

    Histone lysine acetylation is well known for being important in the epigenetic regulation of gene expression in eukaryotic cells. Recent studies have uncovered a plethora of acetylated proteins involved in important metabolic pathways, such as photosynthesis and respiration in plants. Enzymes involved in histone acetylation and deacetylation are being identified as regulators of acetylation of metabolic enzymes. Importantly, key metabolites, such as acetyl-CoA and NAD(+), are involved in protein acetylation and deacetylation processes, and their cellular levels may regulate the activity of histone acetyltransferases (HAT) and deacetylases (HDAC). Further research is required to determine whether and how HATs and HDACs sense cellular metabolite signals to control gene expression and metabolic enzyme activity through lysine acetylation and deacetylation. PMID:26440431

  5. Engineering acetyl coenzyme A supply: functional expression of a bacterial pyruvate dehydrogenase complex in the cytosol of Saccharomyces cerevisiae.

    PubMed

    Kozak, Barbara U; van Rossum, Harmen M; Luttik, Marijke A H; Akeroyd, Michiel; Benjamin, Kirsten R; Wu, Liang; de Vries, Simon; Daran, Jean-Marc; Pronk, Jack T; van Maris, Antonius J A

    2014-01-01

    The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1?, E1?, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs(+) reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. Importance: Genetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy (ATP) costs for product formation from sugar must be minimized. Here, we focus on an important ATP-requiring process in baker's yeast (Saccharomyces cerevisiae): synthesis of cytosolic acetyl coenzyme A, a key precursor for many industrially important products, ranging from biofuels to fragrances. We demonstrate that pyruvate dehydrogenase from the bacterium Enterococcus faecalis, a huge enzyme complex with a size similar to that of a ribosome, can be functionally expressed and assembled in the cytosol of baker's yeast. Moreover, we show that this ATP-independent mechanism for cytosolic acetyl-CoA synthesis can entirely replace the ATP-costly native yeast pathway. This work provides metabolic engineers with a new option to optimize the performance of baker's yeast as a "cell factory" for sustainable production of fuels and chemicals. PMID:25336454

  6. Aspergillus oryzae CsyB Catalyzes the Condensation of Two β-Ketoacyl-CoAs to Form 3-Acetyl-4-hydroxy-6-alkyl-α-pyrone*

    PubMed Central

    Hashimoto, Makoto; Koen, Tsukasa; Takahashi, Hiroaki; Suda, Chihiro; Kitamoto, Katsuhiko; Fujii, Isao

    2014-01-01

    The type III polyketide synthases from fungi produce a variety of secondary metabolites including pyrones, resorcinols, and resorcylic acids. We previously reported that CsyB from Aspergillus oryzae forms α-pyrone csypyrone B compounds when expressed in A. oryzae. Feeding experiments of labeled acetates indicated that a fatty acyl starter is involved in the reaction catalyzed by CsyB. Here we report the in vivo and in vitro reconstitution analysis of CsyB. When CsyB was expressed in Escherichia coli, we observed the production of 3-acetyl-4-hydroxy-α-pyrones with saturated or unsaturated straight aliphatic chains of C9–C17 in length at the 6 position. Subsequent in vitro analysis using recombinant CsyB revealed that CsyB could accept butyryl-CoA as a starter substrate and malonyl-CoA and acetoacetyl-CoA as extender substrates to form 3-acetyl-4-hydroxy-6-propyl-α-pyrone. CsyB also afforded dehydroacetic acid from two molecules of acetoacetyl-CoA. Furthermore, synthetic N-acetylcysteamine thioester of β-ketohexanoic acid was converted to 3-butanoyl-4-hydroxy-6-propyl-α-pyrone by CsyB. These results therefore confirmed that CsyB catalyzed the synthesis of β-ketoacyl-CoA from the reaction of the starter fatty acyl CoA thioesters with malonyl-CoA as the extender through decarboxylative condensation and further coupling with acetoacetyl-CoA to form 3-acetyl-4-hydroxy-6-alkyl-α-pyrone. CsyB is the first type III polyketide synthase that synthesizes 3-acetyl-4-hydroxy-6-alkyl-α-pyrone by catalyzed the coupling of two β-ketoacyl-CoAs. PMID:24895122

  7. Aspergillus oryzae CsyB catalyzes the condensation of two β-ketoacyl-CoAs to form 3-acetyl-4-hydroxy-6-alkyl-α-pyrone.

    PubMed

    Hashimoto, Makoto; Koen, Tsukasa; Takahashi, Hiroaki; Suda, Chihiro; Kitamoto, Katsuhiko; Fujii, Isao

    2014-07-18

    The type III polyketide synthases from fungi produce a variety of secondary metabolites including pyrones, resorcinols, and resorcylic acids. We previously reported that CsyB from Aspergillus oryzae forms α-pyrone csypyrone B compounds when expressed in A. oryzae. Feeding experiments of labeled acetates indicated that a fatty acyl starter is involved in the reaction catalyzed by CsyB. Here we report the in vivo and in vitro reconstitution analysis of CsyB. When CsyB was expressed in Escherichia coli, we observed the production of 3-acetyl-4-hydroxy-α-pyrones with saturated or unsaturated straight aliphatic chains of C9-C17 in length at the 6 position. Subsequent in vitro analysis using recombinant CsyB revealed that CsyB could accept butyryl-CoA as a starter substrate and malonyl-CoA and acetoacetyl-CoA as extender substrates to form 3-acetyl-4-hydroxy-6-propyl-α-pyrone. CsyB also afforded dehydroacetic acid from two molecules of acetoacetyl-CoA. Furthermore, synthetic N-acetylcysteamine thioester of β-ketohexanoic acid was converted to 3-butanoyl-4-hydroxy-6-propyl-α-pyrone by CsyB. These results therefore confirmed that CsyB catalyzed the synthesis of β-ketoacyl-CoA from the reaction of the starter fatty acyl CoA thioesters with malonyl-CoA as the extender through decarboxylative condensation and further coupling with acetoacetyl-CoA to form 3-acetyl-4-hydroxy-6-alkyl-α-pyrone. CsyB is the first type III polyketide synthase that synthesizes 3-acetyl-4-hydroxy-6-alkyl-α-pyrone by catalyzed the coupling of two β-ketoacyl-CoAs. PMID:24895122

  8. Comprehensive profiling of protein lysine acetylation in Escherichia coli.

    PubMed

    Zhang, Kai; Zheng, Shuzhen; Yang, Jeong Soo; Chen, Yue; Cheng, Zhongyi

    2013-02-01

    Protein lysine acetylation plays a key role in regulating chromatin dynamics, gene expression and metabolic pathways in eukaryotes, and, thus, contributes to diverse cellular processes like transcription, cell cycle regulation, and apoptosis. Although recent evidence suggests that acetylated proteins impact broadly cellular functions in prokaryotes, the substrates and localization of this modification remain widely unknown due to the limitations of analytical methods. Comprehensive identification of protein acetylation is a major bottleneck due to its dynamic property and pretty low abundance. A complete atlas of acetylome will significantly advance our understanding of this modification functions in prokaryotes. To achieve this goal, we have developed an intergraded approach to identifying lysine acetylation. Combining immunoaffinity enrichment with high sensitive mass spectrometry, we identified 349 acetylated proteins and addressed 1070 acetylation sites in Escherichia coli. To our knowledge, the acetylated proteins and acetylated sites were increased to 3 times and 8 times, respectively, compared to that in previous report. To further characterize this modification, we classified acetylated proteins into several groups according to cell components, molecular functions and biological process. Additionally, interaction networks and high confident domains architectures of acetylated proteins were investigated with the aid of bioinformatics tools. Finally, the acetylated metabolic enzymes were analyzed on the basis of acetylated proteins identified by proteomic survey in E. coli. Our study has demonstrated that the combined approach is powerful for identification and characterization of protein lysine acetylation on a large scale. These results not only greatly expand the number of acetylated proteins, but also provide a series of important information including localization, networks and characterization of acetylome. PMID:23294111

  9. Hydrolysis of enaminoamides in an alkaline medium

    SciTech Connect

    Kurbatova, T.Yu.; Kiselev, S.S.; Rubtsov, N.M.; Granik, V.G.; Sheinker, u.N.

    1988-12-20

    The hydrolysis of the derivatives of /alpha/-cyano-/beta/-aminoacrylamide and /alpha/cyano-/beta/-aminocrotonamide in an alkaline medium was studied. First order with respect to the hydroxide ion is observed for the /beta/-dimethylamino derivatives, whereas the hydrolysis rate of the /beta/-arylamino derivatives depends very little on the concentration of hydroxide ions. Derivatives of /alpha/-cyano-/beta/-hydroxyacrylamide and /alpha/-cyano-/beta/-hydroxycrotonamide were obtained as reaction products. The hydrolysis was conducted at 60/degree/C in a 0.1 N solution of sodium hydroxide. A decrease in the intensity of the absorption maxima for initial enamines with time and an increase in the absorption corresponding to the hydrolysis products were observed.

  10. Modeling the mechanisms of biological GTP hydrolysis.

    PubMed

    Carvalho, Alexandra T P; Szeler, Klaudia; Vavitsas, Konstantinos; Åqvist, Johan; Kamerlin, Shina C L

    2015-09-15

    Enzymes that hydrolyze GTP are currently in the spotlight, due to their molecular switch mechanism that controls many cellular processes. One of the best-known classes of these enzymes are small GTPases such as members of the Ras superfamily, which catalyze the hydrolysis of the γ-phosphate bond in GTP. In addition, the availability of an increasing number of crystal structures of translational GTPases such as EF-Tu and EF-G have made it possible to probe the molecular details of GTP hydrolysis on the ribosome. However, despite a wealth of biochemical, structural and computational data, the way in which GTP hydrolysis is activated and regulated is still a controversial topic and well-designed simulations can play an important role in resolving and rationalizing the experimental data. In this review, we discuss the contributions of computational biology to our understanding of GTP hydrolysis on the ribosome and in small GTPases. PMID:25731854

  11. Continuous steam hydrolysis of tulip poplar

    SciTech Connect

    Fieber, C.A.; Roberts, R.S.; Faass, G.S.; Muzzy, J.D.; Colcord, A.R.; Bery, M.K.

    1982-01-01

    The continuous hydrolysis of poplar chips by steam at 300-350 psi resulted in the separation of hemicellulose (I) cellulose and lignin components. The I fraction was readily depolymerised by steam to acetic acid, furfural, methanol, and xylose.

  12. Fragrance material review on acetyl cedrene.

    PubMed

    Scognamiglio, J; Letizia, C S; Politano, V T; Api, A M

    2013-12-01

    A toxicologic and dermatologic review of acetyl cedrene when used as a fragrance ingredient is presented. Acetyl cedrene is a member of the fragrance structural group Alkyl Cyclic Ketones. The generic formula for this group can be represented as (R1)(R2)CO. These fragrances can be described as being composed of an alkyl, R1, and various substituted and bicyclic saturated or unsaturated cyclic hydrocarbons, R2, in which one of the rings may include up to 12 carbons. Alternatively, R2 may be a carbon bridge of C2-C4 carbon chain length between the ketone and cyclic hydrocarbon. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for acetyl cedrene were evaluated then summarized and includes physical properties, acute toxicity, skin irritation, mucous membrane (eye) irritation, skin sensitization, elicitation, phototoxicity, photoallergy, toxicokinetics, repeated dose, reproductive toxicity, and genotoxicity data. A safety assessment of the entire Alkyl Cyclic Ketones will be published simultaneously with this document; please refer to Belsito et al. (2013) (Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M., Fryer, A.D., Greim, H., Miyachi, Y., Saurat, J.H., Sipes, I.G., 2013. A Toxicologic and Dermatologic Assessment of Alkyl Cyclic Ketones When Used as Fragrance Ingredients. Submitted with this manuscript.) for an overall assessment of the safe use of this material and all Alkyl Cyclic Ketones in fragrances. PMID:23907023

  13. Fragrance material review on acetyl carene.

    PubMed

    Scognamiglio, J; Letizia, C S; Api, A M

    2013-12-01

    A toxicologic and dermatologic review of acetyl carene when used as a fragrance ingredient is presented. Acetyl carene is a member of the fragrance structural group Alkyl Cyclic Ketones. These fragrances can be described as being composed of an alkyl, R1, and various substituted and bicyclic saturated or unsaturated cyclic hydrocarbons, R2, in which one of the rings may include up to 12 carbons. Alternatively, R2 may be a carbon bridge of C2-C4 carbon chain length between the ketone and cyclic hydrocarbon. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for acetyl carene were evaluated then summarized and includes physical properties, acute toxicity, skin irritation, and skin sensitization data. A safety assessment of the entire Alkyl Cyclic Ketones will be published simultaneously with this document; please refer to Belsito et al. (Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M., Fryer, A.D., Greim, H., Miyachi, Y., Saurat, J.H., Sipes, I.G., 2013A Toxicologic and dermatologic assessment of alkyl cyclic ketones when used as fragrance ingredients. (submitted for publication).) for an overall assessment of the safe use of this material and all Alkyl Cyclic Ketones in fragrances. PMID:23911801

  14. [Biotechnological production of acetylated thymosin beta4].

    PubMed

    Be?rakhova, K A; Stepanenko, V N; Miroshnikov, A I; Esipov, R S

    2011-01-01

    Thymosin beta4 (43 aa) is a highly conserved acidic peptide which regulates actin polymerization in mammalian cells by sequestering globular actin. Thymosin beta4 is undergoing clinical trials as a drug for the treatment of venous stasis ulcers, corneal wounds and injuries, as well as acute myocardial infarction. Currently, thymosin beta4 is produced with solid-phase chemical synthesis. Biotechnological synthesis of this peptide presents difficulties because N-terminal amino acid residue of thymosin beta4 is acetylated. In this study we propose a method for producing the recombinant precursor of thymosin beta4 and its subsequent targeted chemical acetylation. Desacetylthymosin beta4 was synthesized as a part of a hybrid protein with thioredoxin and a specific TEV (tobacco etch virus) protease cleavage site. The following scheme was developed for the purification of desacetylthymosin beta4: (i) the biosynthesis of a soluble hybrid protein (HP) in Escherichia coli; (ii) isolation of the HP by ion exchange chromatography; (iii) cleavage of the HP with TEVprotease; (iv) purification of desacetylthymosin beta4 by ultra-filtration. N-terminal acetylation of desacetylthymosin beta4 was performed with acetic anhydride under acidic conditions (pH 3). The reaction yield was 55%. Thymosin beta4 was then purified by reverse-phase high performance liquid chromatography. The proposed synthetic approach to recombinant thymosin beta4 is suitable for scale-up and can provide for the medical use of highly purified preparation with a yield of 20 mg from 1 L of culture. PMID:21721255

  15. Software interface for high-speed readout of particle detectors based on the CoaXPress communication standard

    NASA Astrophysics Data System (ADS)

    Hejtmánek, M.; Neue, G.; Voleš, P.

    2015-06-01

    This article is devoted to the software design and development of a high-speed readout application used for interfacing particle detectors via the CoaXPress communication standard. The CoaXPress provides an asymmetric high-speed serial connection over a single coaxial cable. It uses a widely available 75 Ω BNC standard and can operate in various modes with a data throughput ranging from 1.25 Gbps up to 25 Gbps. Moreover, it supports a low speed uplink with a fixed bit rate of 20.833 Mbps, which can be used to control and upload configuration data to the particle detector. The CoaXPress interface is an upcoming standard in medical imaging, therefore its usage promises long-term compatibility and versatility. This work presents an example of how to develop DAQ system for a pixel detector. For this purpose, a flexible DAQ card was developed using the XILINX Spartan 6 FPGA. The DAQ card is connected to the framegrabber FireBird CXP6 Quad, which is plugged in the PCI Express bus of the standard PC. The data transmission was performed between the FPGA and framegrabber card via the standard coaxial cable in communication mode with a bit rate of 3.125 Gbps. Using the Medipix2 Quad pixel detector, the framerate of 100 fps was achieved. The front-end application makes use of the FireBird framegrabber software development kit and is suitable for data acquisition as well as control of the detector through the registers implemented in the FPGA.

  16. Modeling of percolation process in hemicellulose hydrolysis.

    PubMed

    Cahela, D R; Lee, Y Y; Chambers, R P

    1983-01-01

    A mathematical model was developed for a percolation reactor in connection with consecutive first-order reactions. The model was designed to simulated acid-catalyzed cellulose or hemicellulose hydrolysis. The modeling process resulted in an analytically derived reactor equation, including mass-transfer effects, which was found to be useful in process desing and reactor optimization. The modedl was verified by experimental data obtained from hemicellulose hydrolysis. PMID:18548535

  17. Discovery of tumor-specific irreversible inhibitors of stearoyl CoA desaturase | Office of Cancer Genomics

    Cancer.gov

    A hallmark of targeted cancer therapies is selective toxicity among cancer cell lines. We evaluated results from a viability screen of over 200,000 small molecules to identify two chemical series, oxalamides and benzothiazoles, that were selectively toxic at low nanomolar concentrations to the same 4 of 12 human lung cancer cell lines. Sensitive cell lines expressed cytochrome P450 (CYP) 4F11, which metabolized the compounds into irreversible inhibitors of stearoyl CoA desaturase (SCD). SCD is recognized as a promising biological target in cancer and metabolic disease.

  18. Biosynthesis and turnover of O-acetyl and N-acetyl groups in the gangliosides of human melanoma cells

    SciTech Connect

    Manzi, A.E.; Sjoberg, E.R.; Diaz, S.; Varki, A.

    1990-08-05

    We and others previously described the melanoma-associated oncofetal glycosphingolipid antigen 9-O-acetyl-GD3, a disialoganglioside O-acetylated at the 9-position of the outer sialic acid residue. We have now developed methods to examine the biosynthesis and turnover of disialogangliosides in cultured melanoma cells and in Golgi-enriched vesicles from these cells. O-Acetylation was selectively expressed on di- and trisialogangliosides, but not on monosialogangliosides, nor on glycoprotein-bound sialic acids. Double-labeling of cells with (3H)acetate and (14C)glucosamine introduced easily detectable labels into each of the components of the ganglioside molecules. Pulse-chase studies of such doubly labeled molecules indicated that the O-acetyl groups turn over faster than the parent molecule. When Golgi-enriched vesicles from these cells were incubated with (acetyl-3H)acetyl-coenzyme A, the major labeled products were disialogangliosides. (Acetyl-3H)O-acetyl groups were found at both the 7- and the 9-positions, indicating that both 7-O-acetyl GD3 and 9-O-acetyl GD3 were synthesized by the action of O-acetyltransferase(s) on endogenous GD3. Analysis of the metabolically labeled molecules confirmed the existence of both 7- and 9-O-acetylated GD3 in the intact cells. Surprisingly, the major 3H-labeled product of the in vitro labeling reaction was not O-acetyl-GD3, but GD3, with the label exclusively in the sialic acid residues. Fragmentation of the labeled sialic acids by enzymatic and chemical methods showed that the 3H-label was exclusively in (3H)N-acetyl groups. Analyses of the double-labeled sialic acids from intact cells also showed that the 3H-label from (3H)acetate was exclusively in the form of (3H)N-acetyl groups, whereas the 14C-label was at the 4-position.

  19. O-Acetylation of Plant Cell Wall Polysaccharides

    PubMed Central

    Gille, Sascha; Pauly, Markus

    2011-01-01

    Plant cell walls are composed of structurally diverse polymers, many of which are O-acetylated. How plants O-acetylate wall polymers and what its function is remained elusive until recently, when two protein families were identified in the model plant Arabidopsis that are involved in the O-acetylation of wall polysaccharides the reduced wall acetylation (RWA) and the trichome birefringence-like (TBL) proteins. This review discusses the role of these two protein families in polysaccharide O-acetylation and outlines the differences and similarities of polymer acetylation mechanisms in plants, fungi, bacteria, and mammals. Members of the TBL protein family had been shown to impact pathogen resistance, freezing tolerance, and cellulose biosynthesis. The connection of TBLs to polysaccharide O-acetylation thus gives crucial leads into the biological function of wall polymer O-acetylation. From a biotechnological point understanding the O-acetylation mechanism is important as acetyl-substituents inhibit the enzymatic degradation of wall polymers and released acetate can be a potent inhibitor in microbial fermentations, thus impacting the economic viability of, e.g., lignocellulosic based biofuel production. PMID:22639638

  20. Acetylation of the chemotaxis response regulator CheY by acetyl-CoA synthetase purified from Escherichia coli.

    PubMed

    Barak, Rina; Prasad, Krishna; Shainskaya, Alla; Wolfe, Alan J; Eisenbach, Michael

    2004-09-10

    Acetylation of CheY, the excitatory response regulator of bacterial chemotaxis, by the enzyme acetyl-CoA synthetase (Acs) is involved in Escherichia coli chemotaxis, but its function is obscure. Here, we overproduced Acs from E.coli, purified it in quantities sufficient for biochemical work, and characterized both the enzyme and the CheY acetylation reaction that it catalyzes. Such characterization is essential for revealing the function of CheY acetylation in chemotaxis. The enzyme exhibited characteristics typical of prokaryotic Acs enzymes, and it could use either acetate or AcCoA as an acetyl donor for CheY acetylation. The Acs-catalyzed acetylation of CheY was reversible, an essential property for a regulatory process, and cooperative (Hill coefficient approximately 3). By Western blotting with specific anti-acetyl-lysine antibody we demonstrated that Acs undergoes autoacetylation, that CheY is acetylated to a small extent when isolated, and that the extent is elevated following in vitro acetylation. Exposing the intact protein to matrix-assisted laser desorption ionization time-of-flight mass spectrometry and electro-spray mass spectrometry, we found that, in most cases, purified CheY is a mixture of species having zero to six acetyl groups per molecule, with non-acetylated CheY being the most abundant species. By proteolytic in-gel digestion of non-treated CheY followed by peptide fingerprinting, precursor ion scan, and tandem mass spectrometry, we found that the acetylation sites of CheY are clustered at the C terminus of the protein, with lysine residues 91, 92, 109, 119, 122 and 126 being the main acetylation sites. Following in vitro acetylation, the main change that seemed to occur was an incremental increase in the extent of acetylation of the same lysine residues. Thus, CheY is similar to many eukaryotic proteins involved in signaling, which undergo both phosphorylation and multiple acetylation, and in which the acetylation sites are restricted to a particular region. PMID:15327942

  1. N-acetyl-l-histidine, a Prominent Biomolecule in Brain and Eye of Poikilothermic Vertebrates

    PubMed Central

    Baslow, Morris H.; Guilfoyle, David N.

    2015-01-01

    N-acetyl-l-histidine (NAH) is a prominent biomolecule in brain, retina and lens of poikilothermic vertebrates. In fish lens, NAH exhibits an unusual compartmentalized metabolism. It is synthesized from l-histidine (His) and acetyl Co-enzyme A. However, NAH cannot be catabolized by lens cells. For its hydrolysis, NAH is exported to ocular fluid where a specific acylase cleaves His which is then actively taken up by lens and re-synthesized into NAH. This energy-dependent cycling suggested a pump mechanism operating at the lens/ocular fluid interface. Additional studies led to the hypothesis that NAH functioned as a molecular water pump (MWP) to maintain a highly dehydrated lens and avoid cataract formation. In this process, each NAH molecule released to ocular fluid down its gradient carries with it 33 molecules of bound water, effectively transporting the water against a water gradient. In ocular fluid the bound water is released for removal from the eye by the action of NAH acylase. In this paper, we demonstrate for the first time the identification of NAH in fish brain using proton magnetic resonance spectroscopy (MRS) and describe recent evidence supporting the NAH MWP hypothesis. Using MRS, we also document a phylogenetic transition in brain metabolism between poikilothermic and homeothermic vertebrates. PMID:25919898

  2. Spectrophotometric assay for urinary N-acetyl-. beta. -d-glucosaminidase activity

    SciTech Connect

    Horak, E.; Hopfer, S.M.; Sunderman, F.W. Jr.

    1981-07-01

    An improved assay for N-acetyl-..beta..-D-glucosaminidase activity in urine is described that involves (a) gel filtration to separate the enzyme from inhibitors in urine, (b) enzymic hydrolysis of p-nitrophenyl-N-acetyl-..beta..-D-glucosaminide at pH 4.4, and (c) spectrophotometry of the liberated p-nitrophenylate. Measurements of activity of the enzyme in 58 urine specimens correlated closely (r = 0.9998) with results by an established procedure. The within-run coefficient of variation (CV) was 3.7%; the between-run CV averaged 6.8%. Reference values for the activity were established by assays of urine specimens from 135 healthy persons, age two weeks to 52 years. Efficacy of the assay for detection of nephrotoxicity was demonstrated in rats after experimental induction of reversible renal insufficiency by intraperitoneal injection of nickel chloride. Clinical application of the assay in approximately 1000 patients corroborated its utility for detection and monitoring of renal disorders.

  3. Selective detection of O(1 S) and CO(a 3 Pi) following electron impact on CO using solid xenon

    NASA Astrophysics Data System (ADS)

    Leclair, Lance R.; Brown, Michael D.; McConkey, J. William

    1994-12-01

    The neutral metastable species O(1 S) and CO(a 3 Pi) produce distinctly different red and UV emissions respectively within a few microseconds of their impact on a surface of solid Xe. This effect has been employed as a novel means of detection in a crossed beam experiment to obtain time-of-flight (TOF) spectra and relative cross sections of both species following electron impact on carbon monoxide. TOF spectra of O(1 S) show five possible dissociation channels. Semi-quantitative partial potential curves were constructed for the parent molecular states of two of those channels. The relative cross section for O(1 S) production was made absolute by comparison with production of the same from O2. The cross section reaches a maximum of 5 times 10(exp -19)/sq cm at 100 eV. The relative cross section for CO(a 3 Pi) production includes cascade from the higher triplet states of CO and is uncontaminated by the 10 eV metastable state of CO.

  4. Preparation, physicochemical characterization and application of acetylated lotus rhizome starches.

    PubMed

    Sun, Suling; Zhang, Ganwei; Ma, Chaoyang

    2016-01-01

    Acetylated lotus rhizome starches were prepared, physicochemically characterized and used as food additives in puddings. The percentage content of the acetyl groups and degree of substitution increased linearly with the amount of acetic anhydride used. The introduction of acetyl groups was confirmed via Fourier transform infrared (FT-IR) spectroscopy. The values of the pasting parameters were lower for acetylated starch than for native starch. Acetylation was found to increase the light transmittance (%), the freeze-thaw stability, the swelling power and the solubility of the starch. Sensorial scores for puddings prepared using native and acetylated lotus rhizome starches as food additives indicated that puddings produced from the modified starches with superior properties over those prepared from native starch. PMID:26453845

  5. Molecular Bases for Sensitivity to Acetyl-Coenzyme A Carboxylase Inhibitors in Black-Grass1

    PubMed Central

    Dlye, Christophe; Zhang, Xiao-Qi; Michel, Sverine; Matjicek, Annick; Powles, Stephen B.

    2005-01-01

    In grasses, residues homologous to residues Ile-1,781 and Ile-2,041 in the carboxyl-transferase (CT) domain of the chloroplastic acetyl-coenzyme A (CoA) carboxylase (ACCase) from the grass weed black-grass (Alopecurus myosuroides [Huds.]) are critical determinants for sensitivity to two classes of ACCase inhibitors, aryloxyphenoxypropionates (APPs) and cyclohexanediones. Using natural mutants of black-grass, we demonstrated through a molecular, biological, and biochemical approach that residues Trp-2,027, Asp-2,078, and Gly-2,096 are also involved in sensitivity to ACCase inhibitors. In addition, residues Trp-2,027 and Asp-2,078 are very likely involved in CT activity. Using three-dimensional modeling, we found that the side chains of the five residues are adjacent, located at the surface of the inside of the cavity of the CT active site, in the vicinity of the binding site for APPs. Residues 1,781 and 2,078 are involved in sensitivity to both APPs and cyclohexanediones, whereas residues 2,027, 2,041, and 2,096 are involved in sensitivity to APPs only. This suggests that the binding sites for these two classes of compounds are overlapping, although distinct. Comparison of three-dimensional models for black-grass wild-type and mutant CTs and for CTs from organisms with contrasted sensitivity to ACCase inhibitors suggested that inhibitors fitting into the cavity of the CT active site of the chloroplastic ACCase from grasses to reach their active sites may be tight. The three-dimensional shape of this cavity is thus likely of high importance for the efficacy of ACCase inhibitors. PMID:15579665

  6. Biochemical and Crystallographic Analysis of Substrate Binding and Conformational Changes in Acetyl-CoA Synthetase

    SciTech Connect

    Reger,A.; Carney, J.; Gulick, A.

    2007-01-01

    The adenylate-forming enzymes, including acyl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPS), and firefly luciferase, perform two half-reactions in a ping-pong mechanism. We have proposed a domain alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain of these enzymes undergoes a 140{sup o} rotation to perform the second thioester-forming half-reaction. Structural and kinetic data of mutant enzymes support this hypothesis. We present here mutations to Salmonella enterica acetyl-CoA synthetase (Acs) and test the ability of the enzymes to catalyze the complete reaction and the adenylation half-reaction. Substitution of Lys609 with alanine results in an enzyme that is unable to catalyze the adenylate reaction, while the Gly524 to leucine substitution is unable to catalyze the complete reaction yet catalyzes the adenylation half-reaction with activity comparable to the wild-type enzyme. The positions of these two residues, which are located on the mobile C-terminal domain, strongly support the domain alternation hypothesis. We also present steady-state kinetic data of putative substrate-binding residues and demonstrate that no single residue plays a dominant role in dictating CoA binding. We have also created two mutations in the active site to alter the acyl substrate specificity. Finally, the crystallographic structures of wild-type Acs and mutants R194A, R584A, R584E, K609A, and V386A are presented to support the biochemical analysis.

  7. Selective Acetylation of per-O-TMS-Protected Monosaccharides

    PubMed Central

    Witschi, Mark A.

    2010-01-01

    Selective acetylation of various per-O-TMS-protected carbohydrates has been accomplished. Using a protecting group exchange strategy and microwave assistance, monosaccharides (glucose, galactose and mannose) can be selectively acetylated producing either the 6-O-monoacetate or 1,6-O-diacetylated species. This new class of molecules can be deprotected without migration of the acetyl groups providing useful synthetic intermediates. To demonstrate the scope of the reaction, the methodology was successfully extended to TMS-protected ceramide. PMID:20799705

  8. Review: Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

    Yang, Bin; Dai, Ziyu; Ding, Shi-You; Wyman, Charles E.

    2011-07-16

    Biological conversion of cellulosic biomass to fuels and chemicals offers the high yields to products vital to economic success and the potential for very low costs. Enzymatic hydrolysis that converts lignocellulosic biomass to fermentable sugars may be the most complex step in this process due to substrate-related and enzyme-related effects and their interactions. Although enzymatic hydrolysis offers the potential for higher yields, higher selectivity, lower energy costs, and milder operating conditions than chemical processes, the mechanism of enzymatic hydrolysis and the relationship between the substrate structure and function of various glycosyl hydrolase components are not well understood. Consequently, limited success has been realized in maximizing sugar yields at very low cost. This review highlights literature on the impact of key substrate and enzyme features that influence performance to better understand fundamental strategies to advance enzymatic hydrolysis of cellulosic biomass for biological conversion to fuels and chemicals. Topics are summarized from a practical point of view including characteristics of cellulose (e.g., crystallinity, degree of polymerization, and accessible surface area) and soluble and insoluble biomass components (e.g., oligomeric xylan, lignin, etc.) released in pretreatment, and their effects on the effectiveness of enzymatic hydrolysis. We further discuss the diversity, stability, and activity of individual enzymes and their synergistic effects in deconstructing complex lignocellulosic biomass. Advanced technologies to discover and characterize novel enzymes and to improve enzyme characteristics by mutagenesis, post-translational modification, and over-expression of selected enzymes and modifications in lignocellulosic biomass are also discussed.

  9. Cross sections for production of the CO(A 1 Pi)-(X 1 Sigma) fourth positive band system and O(3 S) by photodissociation of CO2

    NASA Technical Reports Server (NTRS)

    Gentieu, E. P.; Mentall, J. E.

    1972-01-01

    The CO(A 1 Pi) cross sections reported here, along with previously determined electron impact results, establish the basis for calculating CO fourth positive system volume emission rates in the Martian dayglow. Calculated volume emission rates in turn determine relative distribution of photon vs. electron impact as mechanisms for producing CO(A 1 Pi) in the Mars atmosphere. The smallness of the O(1304) cross section confirms previous indirect evidence that photodissociative excitation of CO2 is not an important source of O(3 S) in the upper atmosphere of Mars.

  10. Intramuscular triacylglycerol, glycogen and acetyl group metabolism during 4 h of moderate exercise in man

    PubMed Central

    Watt, Matthew J; Heigenhauser, George J F; Dyck, David J; Spriet, Lawrence L

    2002-01-01

    This study investigated intramuscular triacylglycerol (IMTG) and glycogen utilisation, pyruvate dehydrogenase activation (PDHa) and acetyl group accumulation during prolonged moderate intensity exercise. Seven endurance-trained men cycled for 240 min at 57 % maximal oxygen consumption (V?O2,max) and duplicate muscle samples were obtained at rest and at 10, 120 and 240 min of exercise. We hypothesised that IMTG utilisation would be augmented during 2-4 h of exercise, while PDHa would be decreased secondary to reduced glycogen metabolism. IMTG was measured on both muscle samples at each time point and the coefficient of variation was 12.3 9.4 %. Whole body respiratory exchange ratio (RER) decreased from 0.89 0.01 at 30 min to 0.83 0.01 at 150 min and remained low throughout exercise. Plasma glycerol and free fatty acids (FFAs) had increased compared with rest at 90 min and progressively increased until exercise cessation. Although plasma glucose tended to decrease with exercise, this was not significant. IMTG was reduced at 120 min compared with rest (0 min, 15.6 0.8 mmol kg?1 d.m.; 120 min, 12.8 0.7 mmol kg?1 d.m.) but no further reduction in IMTG was observed at 240 min. Muscle glycogen was 468 49 mmol kg?1 d.m. at rest and decreased at 120 min and again at 240 min (217 48 and 144 + 47 mmol kg?1 d.m.). PDHa increased above rest at 10 and 120 min, but decreased at 240 min, which coincided with reduced whole body carbohydrate oxidation. Muscle pyruvate and ATP were unchanged with exercise. Acetyl CoA increased at 10 min and remained elevated throughout exercise. Acetylcarnitine increased at exercise onset but returned to resting values by 240 min. Contrary to our first hypothesis, significant utilisation of IMTG occurred during the first 2 h of moderate exercise but not during hours 2-4. The reduced utilisation of intramuscular fuels during the last 120 min was offset by greater FFA delivery and oxidation. Consistent with the second hypothesis, PDHa decreased late in moderate exercise and closely matched the estimates of lower carbohydrate flux. Although the factor underlying the PDHa decrease was not apparent, reduced pyruvate provision secondary to diminished glycolytic flux is the most likely mechanism. PMID:12068055

  11. Growth rate regulation of Escherichia coli acetyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis.

    PubMed Central

    Li, S J; Cronan, J E

    1993-01-01

    Acetyl coenzyme A (CoA) carboxylase catalyzes the synthesis of malonyl-CoA, the first intermediate of fatty acid synthesis. The Escherichia coli enzyme is encoded by four subunits located at three different positions on the E. coli chromosome. The accBC genes lie in a small operon at min 72, whereas accA and accD are located at min 4.3 and 50, respectively. We examined the expression of the genes that encode the E. coli acetyl-CoA carboxylase subunits (accA, accBC, and accD) under a variety of growth conditions by quantitative Northern (RNA) blot analysis. We found a direct correlation between the levels of transcription of the acc genes and the rate of cellular growth. Consistent results were also obtained upon nutritional upshift and downshift experiments and upon dilution of stationary-phase cultures into fresh media. We also determined the 5' end of the accA and accD mRNAs by primer extension and did transcriptional fusion analysis of the previously reported accBC promoter. Several interesting features were found in the promoter regions of these genes, including a bent DNA sequence and an open reading frame within the unusually long leader mRNA of the accBC operon, potential stem-loop structures in the accA and accD mRNA leader regions, and a stretch of GC-rich sequences followed by AT-rich sequences common to all three promoters. In addition, both accA and accD are located in complex gene clusters. For example, the accA promoter was localized within the upstream polC gene (which encodes the DNA polymerase III catalytic subunit), suggesting that additional regulatory mechanisms exist. Images PMID:7678242

  12. Nickel-dependent oligomerization of the alpha subunit of acetyl-coenzyme a synthase/carbon monoxide dehydrogenase.

    PubMed

    Tan, Xiangshi; Kagiampakis, Ioannis; Surovtsev, Ivan V; Demeler, Borries; Lindahl, Paul A

    2007-10-16

    After activation with NiCl2, the recombinant alpha subunit of the Ni-containing alpha2beta2 acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH) catalyzes the synthesis of acetyl-CoA from CO, CoA, and a methyl group donated from the corrinoid-iron-sulfur protein (CoFeSP). The alpha subunit has two conformations (open and closed), and contains a novel [Fe4S4]-[Nip Nid] active site in which the proximal Nip ion is labile. Prior to Ni activation, recombinant apo-alpha contain only an Fe4S4 cluster. Ni-activated alpha subunits exhibit catalytic, spectroscopic and heterogeneity properties typical of alpha subunits contained in ACS/CODH. Evidence presented here indicates that apo-alpha is a monomer whereas Ni-treated alpha oligomerizes, forming dimers and higher molecular weight species including tetramers. No oligomerization occurred when apo-alpha was treated with Cu(II), Zn(II), or Co(II) ions, but oligomerization occurred when apo-alpha was treated with Pt(II) and Pd(II) ions. The dimer accepted only 0.5 methyl group/alpha and exhibited, upon treatment with CO and under reducing conditions, the NiFeC EPR signal quantifying to 0.4 spin/alpha. Dimers appear to consist of two types of alpha subunits, including one responsible for catalytic activity and one that provides a structural scaffold. Higher molecular weight species may be similarly constituted. It is concluded that Ni binding to the A-cluster induces a conformational change in the alpha subunit, possibly to the open conformation, that promotes oligomerization. These interrelated events demonstrate previously unrealized connections between (a) the conformation of the alpha subunit; (b) the metal which occupies the proximal/distal sites of the A-cluster; and (c) catalytic activity. PMID:17887777

  13. Protein lysine acetylation in bacteria: Current state of the art.

    PubMed

    Ouidir, Tassadit; Kentache, Takfarinas; Hardouin, Julie

    2016-01-01

    Post-translational modifications of proteins are key events in cellular metabolism and physiology regulation. Lysine acetylation is one of the best studied protein modifications in eukaryotes, but, until recently, ignored in bacteria. However, proteomic advances have highlighted the diversity of bacterial lysine-acetylated proteins. The current data support the implication of lysine acetylation in various metabolic pathways, adaptation and virulence. In this review, we present a broad overview of the current knowledge of lysine acetylation in bacteria. We emphasize particularly the significant contribution of proteomics in this field. PMID:26390373

  14. Role of transcription factor acetylation in diabetic kidney disease.

    PubMed

    Liu, Ruijie; Zhong, Yifei; Li, Xuezhu; Chen, Haibing; Jim, Belinda; Zhou, Ming-Ming; Chuang, Peter Y; He, John Cijiang

    2014-07-01

    Nuclear factor (NF)-?B and signal transducer and activator of transcription 3 (STAT3) play a critical role in diabetic nephropathy (DN). Sirtuin-1 (SIRT1) regulates transcriptional activation of target genes through protein deacetylation. Here, we determined the roles of Sirt1 and the effect of NF-?B (p65) and STAT3 acetylation in DN. We found that acetylation of p65 and STAT3 was increased in both mouse and human diabetic kidneys. In human podocytes, advanced glycation end products (AGEs) induced p65 and STAT3 acetylation and overexpression of acetylation-incompetent mutants of p65 and STAT3 abrogated AGE-induced expression of NF-?B and STAT3 target genes. Inhibition of AGE formation in db/db mice by pyridoxamine treatment attenuated proteinuria and podocyte injury, restored SIRT1 expression, and reduced p65 and STAT3 acetylation. Diabetic db/db mice with conditional deletion of SIRT1 in podocytes developed more proteinuria, kidney injury, and acetylation of p65 and STAT3 compared with db/db mice without SIRT1 deletion. Treatment of db/db mice with a bromodomain and extraterminal (BET)-specific bromodomain inhibitor (MS417) which blocks acetylation-mediated association of p65 and STAT3 with BET proteins, attenuated proteinuria, and kidney injury. Our findings strongly support a critical role for p65 and STAT3 acetylation in DN. Targeting protein acetylation could be a potential new therapy for DN. PMID:24608443

  15. Differential patterns of histone acetylation in inflammatory bowel diseases

    PubMed Central

    2011-01-01

    Post-translational modifications of histones, particularly acetylation, are associated with the regulation of inflammatory gene expression. We used two animal models of inflammation of the bowel and biopsy samples from patients with Crohn's disease (CD) to study the expression of acetylated histones (H) 3 and 4 in inflamed mucosa. Acetylation of histone H4 was significantly elevated in the inflamed mucosa in the trinitrobenzene sulfonic acid model of colitis particularly on lysine residues (K) 8 and 12 in contrast to non-inflamed tissue. In addition, acetylated H4 was localised to inflamed tissue and to Peyer's patches (PP) in dextran sulfate sodium (DSS)-treated rat models. Within the PP, H3 acetylation was detected in the mantle zone whereas H4 acetylation was seen in both the periphery and the germinal centre. Finally, acetylation of H4 was significantly upregulated in inflamed biopsies and PP from patients with CD. Enhanced acetylation of H4K5 and K16 was seen in the PP. These results demonstrate that histone acetylation is associated with inflammation and may provide a novel therapeutic target for mucosal inflammation. PMID:21272292

  16. Structure, morphology and functionality of acetylated and oxidised barley starches.

    PubMed

    El Halal, Shanise Lisie Mello; Colussi, Rosana; Pinto, Vânia Zanella; Bartz, Josiane; Radunz, Marjana; Carreño, Neftali Lenin Villarreal; Dias, Alvaro Renato Guerra; Zavareze, Elessandra da Rosa

    2015-02-01

    Acetylation and oxidation are chemical modifications which alter the properties of starch. The degree of modification of acetylated and oxidized starches is dependent on the catalyst and active chlorine concentrations, respectively. The objective of this study was to evaluate the effect of acetylation and oxidation on the structural, morphological, physical-chemical, thermal and pasting properties of barley starch. Barley starches were acetylated at different catalyst levels (11%, 17%, and 23% of NaOH solution) and oxidized at different sodium hypochlorite concentrations (1.0%, 1.5%, and 2.0% of active chlorine). Fourier-transformed infrared spectroscopy (FTIR), X-ray diffractograms, thermal, morphological, and pasting properties, swelling power and solubility of starches were evaluated. The degree of substitution (DS) of the acetylated starches increased with the rise in catalyst concentration. The percentage of carbonyl (CO) and carboxyl (COOH) groups in oxidized starches also increased with the rise of active chlorine level. The presence of hydrophobic acetyl groups, carbonyl and carboxyl groups caused a partial disorganization and depolymerization of starch granules. The structural, morphological and functional changes in acetylated and oxidized starches varied according to reaction conditions. Acetylation makes barley starch more hydrophobic by the insertion of acetyl groups. Also the oxidation promotes low retrogradation and viscosity. All these characteristics are important for biodegradable film production. PMID:25172707

  17. Lipase-catalyzed synthesis of acetylated EGCG and antioxidant properties of the acetylated derivatives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    (-)-Epigallocatechin-3-O-gallate (EGCG) acetylated derivatives were prepared by lipase catalyzed acylation of EGCG with vinyl acetate to improve its lipophilicity and expand its application in lipophilic media. The immobilized lipase, Lipozyme RM IM, was found to be the optimum catalyst. The optimiz...

  18. Lysine Acetylation Activates Mitochondrial Aconitase in the Heart.

    PubMed

    Fernandes, Jolyn; Weddle, Alexis; Kinter, Caroline S; Humphries, Kenneth M; Mather, Timothy; Szweda, Luke I; Kinter, Michael

    2015-06-30

    High-throughput proteomics studies have identified several thousand acetylation sites on more than 1000 proteins. Mitochondrial aconitase, the Krebs cycle enzyme that converts citrate to isocitrate, has been identified in many of these reports. Acetylated mitochondrial aconitase has also been identified as a target for sirtuin 3 (SIRT3)-catalyzed deacetylation. However, the functional significance of mitochondrial aconitase acetylation has not been determined. Using in vitro strategies, mass spectrometric analyses, and an in vivo mouse model of obesity, we found a significant acetylation-dependent activation of aconitase. Isolated heart mitochondria subjected to in vitro chemical acetylation with either acetic anhydride or acetyl-coenzyme A resulted in increased aconitase activity that was reversed with SIRT3 treatment. Quantitative mass spectrometry was used to measure acetylation at 21 lysine residues and revealed significant increases with both in vitro treatments. A high-fat diet (60% of kilocalories from fat) was used as an in vivo model and also showed significantly increased mitochondrial aconitase activity without changes in protein level. The high-fat diet also produced an increased level of aconitase acetylation at multiple sites as measured by the quantitative mass spectrometry assays. Treatment of isolated mitochondria from these mice with SIRT3 abolished the high-fat diet-induced activation of aconitase and reduced acetylation. Finally, kinetic analyses found that the increase in activity was a result of increased maximal velocity, and molecular modeling suggests the potential for acetylation at K144 to perturb the tertiary structure of the enzyme. The results of this study reveal a novel activation of mitochondrial aconitase by acetylation. PMID:26061789

  19. Site-Specific Reactivity of Nonenzymatic Lysine Acetylation

    PubMed Central

    2016-01-01

    Protein acetylation of lysine ?-amino groups is abundant in cells, particularly within mitochondria. The contribution of enzyme-catalyzed and nonenzymatic acetylation in mitochondria remains unresolved. Here, we utilize a newly developed approach to measure site-specific, nonenzymatic acetylation rates for 90 sites in eight native purified proteins. Lysine reactivity (as second-order rate constants) with acetyl-phosphate and acetyl-CoA ranged over 3 orders of magnitude, and higher chemical reactivity tracked with likelihood of dynamic modification in vivo, providing evidence that enzyme-catalyzed acylation might not be necessary to explain the prevalence of acetylation in mitochondria. Structural analysis revealed that many highly reactive sites exist within clusters of basic residues, whereas lysines that show low reactivity are engaged in strong attractive electrostatic interactions with acidic residues. Lysine clusters are predicted to be high-affinity substrates of mitochondrial deacetylase SIRT3 both in vitro and in vivo. Our analysis describing rate determination of lysine acetylation is directly applicable to investigate targeted and proteome-wide acetylation, whether or not the reaction is enzyme catalyzed. PMID:25555129

  20. Acetylation regulates Cyclophilin A catalysis, immunosuppression and HIV isomerisation

    PubMed Central

    Lammers, Michael; Neumann, Heinz; Chin, Jason W.; James, Leo C.

    2013-01-01

    Cyclophilin A (CypA) is a ubiquitous cis-trans-prolyl isomerase with key roles in immunity and viral infection. CypA suppresses T-cell activation through cyclosporine (Cs) complexation and is required for effective HIV-1 replication in host cells. We show that CypA is acetylated in diverse human cell lines and use a synthetically evolved acetyl-lysyl-tRNA synthetase/tRNACUA pair to produce recombinant acetylated CypA in E. coli. We determine atomic resolution structures of acetylated CypA and its complexes with Cs and HIV-1 capsid. Acetylation dramatically inhibits CypA catalysis of cis to trans isomerisation and stabilises cis rather than trans forms of the HIV-1 capsid. Furthermore, CypA acetylation antagonizes the immunosuppressive effects of Cs, by inhibiting the sequential steps of Cs binding and calcineurin inhibition. Our results reveal that acetylation regulates key functions of CypA in immunity and viral infection and provide a general set of mechanisms by which acetylation modulates interactions to regulate cell function. PMID:20364129

  1. Metobromuron: acetylation of the aniline moiety as a detoxification mechanism.

    PubMed

    Tweedy, B G; Loeppky, C; Ross, J A

    1970-04-24

    p-Bromoaniline is rapidly acetylated by four soil microorganisms. Two fungal species convert metobromuron to p-bromoacetanilide, but a bacterial and an algal species do not metabolize metobromuron. Acetylation may serve as a detoxification mechanism by competing with azobenzene formation in utilizing the aniline formed by metabolism of substituted urea herbicides. PMID:5436083

  2. Effect of acetaminophen on sulfamethazine acetylation in male volunteers.

    PubMed

    Tahir, I M; Iqbal, T; Saleem, S; Mehboob, H; Akhter, N; Riaz, M

    2016-03-01

    The effect of acetaminophen on sulfamethazine N-acetylation by human N-acetyltrasferase-2 (NAT2) was studied in 19 (n = 19) healthy male volunteers in two different phases. In the first phase of the study the volunteers were given an oral dose of sulfamethazine 500 mg alone and blood and urine samples were collected. After the 10-day washout period the same selected volunteers were again administered sulfamethazine 500 mg along with 1000 mg acetaminophen. The acetylation of sulfamethazine by human NAT2 in both phases with and without acetaminophen was determined by HPLC to establish their respective phenotypes. In conclusion obtained statistics of present study revealed that acetaminophen significantly (P <0.0001) decreased sulfamethazine acetylation in plasma of both slow and fast acetylator male volunteers. A highly significant (P <0.0001) decrease in plasma-free and total sulfamethazine concentration was also observed when acetaminophen was co-administered. Urine acetylation status in both phases of the study was found not to be in complete concordance with that of plasma. Acetaminophen significantly (P <0.0001) increased the acetyl, free and total sulfamethazine concentration in urine of both slow and fast acetylators. Urine acetylation analysis has not been found to be a suitable approach for phenotypic studies. PMID:26519524

  3. Global analysis of lysine acetylation in strawberry leaves

    PubMed Central

    Fang, Xianping; Chen, Wenyue; Zhao, Yun; Ruan, Songlin; Zhang, Hengmu; Yan, Chengqi; Jin, Liang; Cao, Lingling; Zhu, Jun; Ma, Huasheng; Cheng, Zhongyi

    2015-01-01

    Protein lysine acetylation is a reversible and dynamic post-translational modification. It plays an important role in regulating diverse cellular processes including chromatin dynamic, metabolic pathways, and transcription in both prokaryotes and eukaryotes. Although studies of lysine acetylome in plants have been reported, the throughput was not high enough, hindering the deep understanding of lysine acetylation in plant physiology and pathology. In this study, taking advantages of anti-acetyllysine-based enrichment and high-sensitive-mass spectrometer, we applied an integrated proteomic approach to comprehensively investigate lysine acetylome in strawberry. In total, we identified 1392 acetylation sites in 684 proteins, representing the largest dataset of acetylome in plants to date. To reveal the functional impacts of lysine acetylation in strawberry, intensive bioinformatic analysis was performed. The results significantly expanded our current understanding of plant acetylome and demonstrated that lysine acetylation is involved in multiple cellular metabolism and cellular processes. More interestingly, nearly 50% of all acetylated proteins identified in this work were localized in chloroplast and the vital role of lysine acetylation in photosynthesis was also revealed. Taken together, this study not only established the most extensive lysine acetylome in plants to date, but also systematically suggests the significant and unique roles of lysine acetylation in plants. PMID:26442052

  4. Thermochemical characteristics of cellulose acetates with different degrees of acetylation

    NASA Astrophysics Data System (ADS)

    Larina, V. N.; Ur'yash, V. F.; Kushch, D. S.

    2012-12-01

    The standard enthalpies of combustion and formation of cellulose acetates with different degrees of acetylation are determined. It is established that there is a proportional dependence of these thermochemical characteristics vs. the degree of acetylation, weight fraction of bonded acetic acid, and molar mass of the repeating unit of cellulose acetates.

  5. Study on Dendrobium officinale O-acetyl-glucomannan (Dendronan): part II. Fine structures of O-acetylated residues.

    PubMed

    Xing, Xiaohui; Cui, Steve W; Nie, Shaoping; Phillips, Glyn O; Goff, H Douglas; Wang, Qi

    2015-03-01

    Main objective of this study was to investigate the detailed structural information about O-acetylated sugar residues in Dendronan(). A water solution (2%, w/w) of Dendronan() was treated with endo-?-mannanase to produce oligosaccharides rich in O-acetylated sugar residues. The oligosaccharides were partly recovered by ethanol precipitation (70%, w/w). The recovered sample (designated Hydrolyzed Dendrobium officinale Polysaccharide, HDOP) had a yield of 24.7% based on the dry weight of Dendronan() and was highly O-acetylated. A D2O solution of HDOP (6%, w/w) generated strong signals in (1)H, (13)C, 2D (1)H-(1)H COSY, 2D (1)H-(1)H TOCSY, 2D (1)H-(1)H NOESY, 2D (1)H-(13)C HMQC, and 2D (1)H-(13)C HMBC NMR spectra. Results of NMR analyses showed that the majority of O-acetylated mannoses were mono-substituted with acetyl groups at O-2 or O-3 position. There were small amounts of mannose residues with di-O-acetyl substitution at both O-2 and O-3 positions. Minor levels of mannoses with 6-O-acetyl, 2,6-di-O-acetyl, and 3,6-di-O-acetyl substitutions were also identified. Much information about sugar residue sequence was extracted from 2D (1)H-(13)C HMBC and 2D (1)H-(1)H NOESY spectra. (1)J(C-H) coupling constants of major sugar residues were obtained. Evidences for the existence of branches or O-acetylated glucoses in HDOP were not found. The major structure of Dendronan() is shown as follows: [Formula: see text] M: ?-D-mannopyranose; G: ?-D-glucopyranose; a: O-acetyl group. PMID:25498655

  6. Lysogenic conversion of Salmonella typhimurium bacteriophages A3 and A4 consists of O-acetylation of rhamnose of the repeating unit of the O-antigenic polysaccharide chain.

    PubMed

    Wollin, R; Stocker, B A; Lindberg, A A

    1987-03-01

    Lysogenization of Salmonella typhimurium with either of the bacteriophages A3 and A4 results in O-acetylation of the L-rhamnose residues of the O-polysaccharide chain of the lipopolysaccharide of the bacterial cell envelope. The O-acetyl group is found on both O-2 and O-3 of the L-rhamnosyl residues. This lysogenic conversion prevents the adsorption of the A3 and A4 phages and also greatly reduces the rate of adsorption of phage P22 to the O-polysaccharide chain as measured by binding studies with whole bacteria. Isolated lipopolysaccharide from A3- and A4-lysogenized bacteria was also inefficient in inactivating these phages: the concentration required for 50% inactivation was 10,000-fold higher than that for lipopolysaccharide from S. typhimurium not lysogenized by any A phage. Binding of phages A3 and A4 is accompanied by hydrolysis of the alpha-1,3 linkage between rhamnose and galactose in the tetrasaccharide repeating unit of the O-polysaccharide. Phage hydrolysis generates saccharides of various lengths, the majority being dodecasaccharides, i.e., equivalent to three repeating units. It is surmised that O-acetylation of the rhamnosyl residue interferes with phage A3, A4, and P22 infection by preventing binding to and hydrolysis of the O-polysaccharide chain, the initial step in the phage infection cycle. The new O-acetyl-rhamnose entities did not elicit specific antibodies in rabbits in accordance with earlier experiences. The O-acetylation of O-2 and O-3 of rhamnose is a new, hitherto unknown, modification of the O-polysaccharide chain of S. typhimurium. PMID:3546260

  7. CoA synthase is in complex with p85alphaPI3K and affects PI3K signaling pathway.

    PubMed

    Breus, Oksana; Panasyuk, Ganna; Gout, Ivan T; Filonenko, Valeriy; Nemazanyy, Ivan

    2009-08-01

    The complex interplay between cellular signaling and metabolism in eukaryotic cells just start to emerge. Coenzyme A (CoA) and its derivatives play a key role in cell metabolism and also participate in regulatory processes. CoA synthase (CoASy) is a mitochondria-associated enzyme which mediates two final stages of de novo CoA biosynthesis. Here, we report that CoASy is involved in signaling events in the cell and forms a functional complex with p85alphaPI3K in vivo. Importantly, observed interaction of endogenous CoASy and p85alphaPI3K is regulated in a growth factor dependent manner. Surprisingly, both catalytic p110alpha and regulatory p85alpha subunits of PI3K were detected in mitochondrial fraction where mitochondria-localized p85alphaPI3K was found in complex with CoASy. Unexpectedly, significant changes of PI3K signaling pathway activity were observed in experiments with siRNA-mediated CoASy knockdown pointing on the role of CoA biosynthetic pathway in signal transduction. PMID:19482007

  8. Sphingolipids and cellular cholesterol homeostasis. Effect of ceramide on cholesterol trafficking and HMG CoA reductase activity

    PubMed Central

    Subbaiah, Papasani V.; Sowa, Jennifer M.; Singh, Dev K.

    2008-01-01

    We previously showed that degradation of cellular sphingomyelin (SM) by SMase C results in a greater stimulation of cholesterol translocation to endoplasmic reticulum, compared to its degradation by SMase D. Here we investigated the hypothesis that the effect of SMase C is partly due to the generation of ceramide, rather than due to depletion of SM alone. Inhibition of hydroxymethylglutaryl CoA reductase (HMGCR) activity was used as a measure of cholesterol translocation. Treatment of fibroblasts with SMase C resulted in a 90% inhibition of HMGCR, whereas SMase D treatment inhibited it by 29%. Treatment with exogenous ceramides, or increasing the endogenous ceramide levels also inhibited HMGCR by 6080%. Phosphorylation of HMGCR was stimulated by SMase C or exogenous ceramide. The effects of ceramide and SMase D were additive, indicating the independent effects of SM depletion and ceramide generation. These results show that ceramide regulates sterol trafficking independent of cellular SM levels. PMID:18395507

  9. EDC4 interacts with and regulates the dephospho-CoA kinase activity of CoA synthase.

    PubMed

    Gudkova, Daria; Panasyuk, Ganna; Nemazanyy, Ivan; Zhyvoloup, Alexander; Monteil, Pascale; Filonenko, Valeriy; Gout, Ivan

    2012-10-19

    Coenzyme A synthase (CoAsy) is a bifunctional enzyme which facilitates the last two steps of Coenzyme A biogenesis in higher eukaryotes. Here we describe that CoAsy forms a complex with enhancer of mRNA-decapping protein 4 (EDC4), a central scaffold component of processing bodies. CoAsy/EDC4 complex formation is regulated by growth factors and is affected by cellular stresses. EDC4 strongly inhibits the dephospho-CoA kinase activity of CoAsy in vitro. Transient overexpression of EDC4 decreases cell proliferation, and further co-expression of CoAsy diminishes this effect. Here we report that EDC4 might contribute to regulation of CoA biosynthesis in addition to its scaffold function in processing bodies. PMID:22982864

  10. Enzymatic hydrolysis of spent coffee ground.

    PubMed

    Jooste, T; Garca-Aparicio, M P; Brienzo, M; van Zyl, W H; Grgens, J F

    2013-04-01

    Spent coffee ground (SCG) is the main residue generated during the production of instant coffee by thermal water extraction from roasted coffee beans. This waste is composed mainly of polysaccharides such as cellulose and galactomannans that are not solubilised during the extraction process, thus remaining as unextractable, insoluble solids. In this context, the application of an enzyme cocktail (mannanase, endoglucanase, exoglucanase, xylanase and pectinase) with more than one component that acts synergistically with each other is regarded as a promising strategy to solubilise/hydrolyse remaining solids, either to increase the soluble solids yield of instant coffee or for use as raw material in the production of bioethanol and food additives (mannitol). Wild fungi were isolated from both SCG and coffee beans and screened for enzyme production. The enzymes produced from the selected wild fungi and recombinant fungi were then evaluated for enzymatic hydrolysis of SCG, in comparison to commercial enzyme preparations. Out of the enzymes evaluated on SCG, the application of mannanase enzymes gave better yields than when only cellulase or xylanase was utilised for hydrolysis. The recombinant mannanase (Man1) provided the highest increments in soluble solids yield (17 %), even when compared with commercial preparations at the same protein concentration (0.5 mg/g SCG). The combination of Man1 with other enzyme activities revealed an additive effect on the hydrolysis yield, but not synergistic interaction, suggesting that the highest soluble solid yields was mainly due to the hydrolysis action of mannanase. PMID:23436225

  11. Phosphatase Hydrolysis of Soil Organic Phosphorus Fractions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant available inorganic phosphorus (Pi) is usually limited in highly weathered Ultisols. The high Fe, Al, and Mn contents in these soils enhance Pi retention and fixation. The metals are also known to form complexes with organic phosphorus (Po) compounds. Hydrolysis of Po compounds is needed for P...

  12. COMPUTERIZED EXTRAPOLATION OF HYDROLYSIS RATE DATA

    EPA Science Inventory

    The program RATE was developed to aid in the extrapolation and interpretation of hydrolysis rate data to a format that is useful for environmental risk assessment. ydrolysis data typically are reported in the literature as pseudo-first-order rate constants at the temperature and ...

  13. Phosphatase hydrolysis of organic phosphorus compounds

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Phosphatases are diverse groups of enzymes that deserve special attention because of the significant roles they play in mineralizing organic phosphorus (P) into inorganic available form. For getting more insight on the enzymatically hydrolysis of organic P, in this work, we compared the catalytic pa...

  14. Optimization of dilute acid hydrolysis of Enteromorpha

    NASA Astrophysics Data System (ADS)

    Feng, Dawei; Liu, Haiyan; Li, Fuchao; Jiang, Peng; Qin, Song

    2011-11-01

    Acid hydrolysis is a simple and direct way to hydrolyze polysaccharides in biomass into fermentable sugars. To produce fermentable sugars effectively and economically for fuel ethanol, we have investigated the hydrolysis of Enteromorpha using acids that are typically used to hydrolyze biomass: H2SO4, HCl, H3PO4 and C4H4O4 (maleic acid). 5%(w/w) Enteromorpha biomass was treated for different times (30, 60, and 90 min) and with different acid concentrations (0.6, 1.0, 1.4, 1.8, and 2.2%, w/w) at 121C. H2SO4 was the most effective acid in this experiment. We then analyzed the hydrolysis process in H2SO4 in detail using high performance liquid chromatography. At a sulfuric acid concentration of 1.8% and treatment time of 60 min, the yield of ethanol fermentable sugars (glucose and xylose) was high, (230.5 mg/g dry biomass, comprising 175.2 mg/g glucose and 55.3 mg/g xylose), with 48.6% of total reducing sugars being ethanol fermentable. Therefore, Enteromorpha could be a good candidate for production of fuel ethanol. In future work, the effects of temperature and biomass concentration on hydrolysis, and also the fermentation of the hydrolysates to ethanol fuel should be focused on.

  15. Thioglycoside hydrolysis catalyzed by {beta}-glucosidase

    SciTech Connect

    Shen Hong; Byers, Larry D.

    2007-10-26

    Sweet almond {beta}-glucosidase (EC 3.2.1.21) has been shown to have significant thioglycohydrolase activity. While the K{sub m} values for the S- and O-glycosides are similar, the k{sub cat} values are about 1000-times lower for the S-glycosides. Remarkably, the pH-profile for k{sub cat}/K{sub m} for hydrolysis of p-nitrophenyl thioglucoside (pNPSG) shows the identical dependence on a deprotonated carboxylate (pK{sub a} 4.5) and a protonated group (pK{sub a} 6.7) as does the pH-profile for hydrolysis of the corresponding O-glycoside. Not surprisingly, in spite of the requirement for the presence of this protonated group in catalytically active {beta}-glucosidase, thioglucoside hydrolysis does not involve general acid catalysis. There is no solvent kinetic isotope effect on the enzyme-catalyzed hydrolysis of pNPSG.

  16. The diversity of lysine-acetylated proteins in Escherichia coli.

    PubMed

    Yu, Byung Jo; Kim, Jung Ae; Moon, Jeong Hee; Ryu, Seong Eon; Pan, Jae-Gu

    2008-09-01

    Acetylation of lysine residues in proteins is a reversible and highly regulated posttranslational modification. However, it has not been systematically studied in prokaryotes. By affinity immunoseparation using an anti-acetyllysine antibody together with nano-HPLC/MS/MS, we identified 125 lysineacetylated sites in 85 proteins among proteins derived from Escherichia coli. The lysine-acetylated proteins identified are involved in diverse cellular functions including protein synthesis, carbohydrate metabolism, the TCA cycle, nucleotide and amino acid metabolism, chaperones, and transcription. Interestingly, we found a higher level of acetylation during the stationary phase than in the exponential phase; proteins acetylated during the stationary phase were immediately deacetylated when the cells were transferred to fresh LB culture medium. These results demonstrate that lysine acetylation is abundant in E. coli and might be involved in modifying or regulating the activities of various enzymes involved in critical metabolic processes and the synthesis of building blocks in response to environmental changes. PMID:18852508

  17. Antemortem stress regulates protein acetylation and glycolysis in postmortem muscle.

    PubMed

    Li, Zhongwen; Li, Xin; Wang, Zhenyu; Shen, Qingwu W; Zhang, Dequan

    2016-07-01

    Although exhaustive research has established that preslaughter stress is a major factor contributing to pale, soft, exudative (PSE) meat, questions remain regarding the biochemistry of postmortem glycolysis. In this study, the influence of preslaughter stress on protein acetylation in relationship to glycolysis was studied. The data show that antemortem swimming significantly enhanced glycolysis and the total acetylated proteins in postmortem longissimus dorsi (LD) muscle of mice. Inhibition of protein acetylation by histone acetyltransferase (HAT) inhibitors eliminated stress induced increase in glycolysis. Inversely, antemortem injection of histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and nicotinamide (NAM), further increased protein acetylation early postmortem and the glycolysis. These data provide new insight into the biochemistry of postmortem glycolysis by showing that protein acetylation regulates glycolysis, which may participate in the regulation of preslaughter stress on glycolysis in postmortem muscle. PMID:26920270

  18. Incorporation of [2(14)C]malonyl CoA into fatty acids by a cell-free extract of Catharanthus roseus suspension culture cells.

    PubMed

    Maccarthy, J J; Stumpf, P K

    1980-12-01

    A cell-free extract containing the enzymes for de-novo synthesis, elongation and desaturation of fatty acids was prepared from cultured cells of Catharanthus roseus G. Don. (14)C-Fatty acids synthesized by the extract from [2-(14)C]malonyl CoA substrate were palmitic (16:0), stearic (18:0) and oleic (18:1). Dialyzed extract was active and stable at room temperature and at 4° C, but was inactivated on boiling. There was an absolute requirement for NADPH for incorporation of [2-(14)C]malonyl CoA into total fatty acids. Escherichia coli acyl carrier protein stimulated total fatty-acid synthesis without affecting the relative ratio of individual fatty acids. Total fatty-acid synthesis at a rate of 45 nmol·mg(-1) protein·h(-1) occurred at a substrate level of 73 μM malonyl CoA, cofactor levels of 500 μM NADPH, 30 μg·ml(-1) E. coli ACP, and 1.0 mg·ml(-1) extract protein. Total fatty acid synthesis was also sensitive to cerulenin and CoA levels. Variations in the relative abundance of individual (14)C-fatty acids were regulated by concentrations of [(14)C]malonyl CoA. NADPH and ferredoxin, as well as by pH, temperature and length of incubation. Fatty-acid synthetase enzymes responsible for [(14)C]palmitic acid were rapidly saturated at a low substrate level (0.3 μM malonyl CoA). Increasing the level of [2-(14)C]malonyl CoA permitted further synthesis of [(14)C]stearate and [(14)C]oleate. Desaturation of [(14)C]stearate to [(14)C]oleate was stimulated by increasing the levels of NADPH and ferredoxin. The desaturase and elongase enzymes were sensitive to acidic pH. The desaturase was also unstable at 41° C, although fatty acid synthetase and elongase were unaffected by this temperature. PMID:24306892

  19. Reversible acetylation regulates acetate and propionate metabolism in Mycobacterium smegmatis.

    PubMed

    Hayden, Jennifer D; Brown, Lanisha R; Gunawardena, Harsha P; Perkowski, Ellen F; Chen, Xian; Braunstein, Miriam

    2013-09-01

    Carbon metabolic pathways are important to the pathogenesis of Mycobacterium tuberculosis, the causative agent of tuberculosis. However, extremely little is known about metabolic regulation in mycobacteria. There is growing evidence for lysine acetylation being a mechanism of regulating bacterial metabolism. Lysine acetylation is a post-translational modification in which an acetyl group is covalently attached to the side chain of a lysine residue. This modification is mediated by acetyltransferases, which add acetyl groups, and deacetylases, which remove the acetyl groups. Here we set out to test whether lysine acetylation and deacetylation impact acetate metabolism in the model mycobacteria Mycobacterium smegmatis, which possesses 25 candidate acetyltransferases and 3 putative lysine deacetylases. Using mutants lacking predicted acetyltransferases and deacetylases we showed that acetate metabolism in M. smegmatis is regulated by reversible acetylation of acetyl-CoA synthetase (Ms-Acs) through the action of a single pair of enzymes: the acetyltransferase Ms-PatA and the sirtuin deacetylase Ms-SrtN. We also confirmed that the role of Ms-PatA in regulating Ms-Acs regulation depends on cAMP binding. We additionally demonstrated a role for Ms-Acs, Ms-PatA and Ms-SrtN in regulating the metabolism of propionate in M. smegmatis. Finally, along with Ms-Acs, we identified a candidate propionyl-CoA synthetase, Ms5404, as acetylated in whole-cell lysates. This work lays the foundation for studying the regulatory circuit of acetylation and deacetylation in the cellular context of mycobacteria. PMID:23813678

  20. Acetylated histone H3 increases nucleosome dissociation

    NASA Astrophysics Data System (ADS)

    Simon, Marek; Manohar, Mridula; Ottesen, Jennifer; Poirier, Michael

    2009-03-01

    Chromatin's basic unit structure is the nucleosome, i.e. genomic DNA wrapped around a particular class of proteins -- histones -- which due to their physical hindrance, block vital biological processes, such as DNA repair, DNA replication, and RNA transcription. Histone post-translational modifications, which are known to exist in vivo, are hypothesized to regulate these biological processes by directly altering DNA-histone interactions and thus nucleosome structure and stability. Using magnetic tweezers technique we studied the acetylation of histone H3 in the dyad region, i.e. at K115 and K122, on reconstituted arrays of nucleosomes under constant external force. Based on the measured increase in the probability of dissociation of modified nucleosomes, we infer that this double modification could facilitate histone chaperone mediated nucleosome disassembly in vivo.

  1. Non-catalytic steam hydrolysis of fats

    SciTech Connect

    Deibert, M.C.

    1992-08-28

    Hydrolysis of fats and oils produces fatty acid and glycerol. The catalyzed, liquid phase Colgate-Emry process, state-of-the-art, produces impure products that require extensive energy investment for their purification to commercial grade. Non-catalytic steam hydrolysis may produce products more easily purified. A bench-scale hydrolyzer was designed and constructed to contact descending liquid fat or oil with rising superheated steam. Each of the five stages in the reactor was designed similar to a distillation column stage to promote intimate liquid-gas contact. Degree of hydrolysis achieved in continuous tests using tallow feed were 15% at 280C and 35% at 300C at a tallow-to-steam mass feed ratio of 4.2. At a feed ratio of 9.2, the degree of hydrolysis was 21% at 300C. Decomposition was strongly evident at 325C but not at lower temperatures. Soybean oil rapidly polymerized under reaction conditions. Batch tests at 320C produced degrees of hydrolyses of between 44% and 63% using tallow and palm oil feeds. Over 95% fatty acids were present in a clean, readily separated organic portion of the overhead product from most tests. The test reactor had serious hydraulic resistance to liquid down-flow which limited operation to very long liquid residence times. These times are in excess of those that tallow and palm oil are stable at the reaction temperature. Little glycerol and extensive light organics were produced indicating that unexplained competing reactions to hydrolysis occurred in the experimental system. Further tests using an improved reactor will be required.

  2. Comprehensive profiling of lysine acetylation suggests the widespread function is regulated by protein acetylation in the silkworm, Bombyx mori.

    PubMed

    Nie, Zuoming; Zhu, Honglin; Zhou, Yong; Wu, Chengcheng; Liu, Yue; Sheng, Qing; Lv, Zhengbing; Zhang, Wenping; Yu, Wei; Jiang, Caiying; Xie, Longfei; Zhang, Yaozhou; Yao, Juming

    2015-09-01

    Lysine acetylation in proteins is a dynamic and reversible PTM and plays an important role in diverse cellular processes. In this study, using lysine-acetylation (Kac) peptide enrichment coupled with nano HPLC/MS/MS, we initially identified the acetylome in the silkworms. Overall, a total of 342 acetylated proteins with 667 Kac sites were identified in silkworm. Sequence motifs analysis around Kac sites revealed an enrichment of Y, F, and H in the +1 position, and F was also enriched in the +2 and -2 positions, indicating the presences of preferred amino acids around Kac sites in the silkworm. Functional analysis showed the acetylated proteins were primarily involved in some specific biological processes. Furthermore, lots of nutrient-storage proteins, such as apolipophorin, vitellogenin, storage proteins, and 30 K proteins, were highly acetylated, indicating lysine acetylation may represent a common regulatory mechanism of nutrient utilization in the silkworm. Interestingly, Ser2 proteins, the coating proteins of larval silk, were found to contain many Kac sites, suggesting lysine acetylation may be involved in the regulation of larval silk synthesis. This study is the first to identify the acetylome in a lepidoptera insect, and expands greatly the catalog of lysine acetylation substrates and sites in insects. PMID:26046922

  3. Formation of the thioester, N-acetyl, S-lactoylcysteine, by reaction of N-acetylcysteine with pyruvaldehyde in aqueous solution. [in prebiotic evolution

    NASA Technical Reports Server (NTRS)

    Weber, A. L.

    1982-01-01

    N-acetylcysteine reacts efficiently with pyruvaldehyde (methylglyoxal) in aqueous solution (pH 7.0) in the presence of a weak base, like imidazole or phosphate, to give the thioester, N-acetyl, S-lactoylcysteine. Reactions of 100 mM N-acetylcysteine with 14 mM, 24 mM and 41 mM pyruvaldehyde yield, respectively, 86%, 76% and 59% N-acetyl, S-lactoylcysteine based on pyruvaldehyde. The decrease in the percent yield at higher pyruvaldehyde concentrations suggests that during its formation the thioester is not only consumed by hydrolysis, but also by reaction with some substance in the pyruvaldehyde preparation. Indeed, purified N-acetyl, S-lactoylcysteine disappears much more rapidly in the presence of pyruvaldehyde than in its absence. Presumably, N-acetyl, S-lactoylcysteine synthesis occurs by rearrangement of the hemithioacetal of N-acetylcysteine and pyruvaldehyde. The significance of this pathway of thioester formation to molecular evolution is discussed.

  4. Semi-synthetic preparation of 1-O-(1'-/sup 14/C)hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) using plant cell cultures

    SciTech Connect

    Weber, N.; Mangold, H.K.

    1985-04-01

    Incubation of photomixotrophic cell suspension cultures of rape (Brassica napus) and heterotrophic cell suspension cultures of soya (Glycine max) with 1-O-(1'-/sup 14/C)hexadecyl-sn-glycerol or rac-1-O-(1'-/sup 14/C)hexadecylglycerol leads in high yield (up to 78%) to labeled 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines. Alkaline hydrolysis of the choline glycerophospholipids yields pure 1-O-(1'-/sup 14/C)hexadecyl-sn-glycero-3-phosphocholine. 1-O-(1'-14C)Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) is obtained by acetylating the lyso compound. The semi-synthetic preparation described leads to labeled platelet activating factor in an overall yield of 50-60% without loss of specific activity.

  5. Different modes of carbon monoxide binding to acetyl-CoA synthase and the role of a conserved phenylalanine in the coordination environment of nickel.

    PubMed

    Gencic, Simonida; Kelly, Kayla; Ghebreamlak, Selamawit; Duin, Evert C; Grahame, David A

    2013-03-12

    Acetyl-CoA synthase (ACS) catalyzes the reversible condensation of CO and CH3 units at a unique Ni-Fe cluster, the A cluster, to form an acetyl-Ni intermediate that subsequently reacts with CoA to produce acetyl-CoA. ACS is a component of the multienzyme complex acetyl-CoA decarbonylase/synthase (ACDS) in Archaea and CO dehydrogenase/ACS (CODH/ACS) in bacteria; in both systems, intraprotein CO channeling takes place between the CODH and ACS active sites. Previous studies indicated that protein conformational changes control the chemical reactivity of the A cluster and suggested the involvement of a conserved Phe residue that moves concomitantly into and out of the coordination environment of Ni. Herein, steady-state rate measurements in which both CO and CH3-corrinoid are varied, and rapid methylation reactions of the ACDS β subunit, measured by stopped-flow methods, provide a kinetic model for acetyl-CoA synthesis that includes a description of the inhibitory effects of CO explained by competition of CO and CH3 for the same form of the enzyme. Electron paramagnetic resonance titrations revealed that the formation of a paramagnetic Ni(+)-CO species does not match the kinetics of CO interaction as a substrate but instead correlates well with an inhibited state of the enzyme, which requires revision of previous models that postulate that this species is an intermediate. Characterization of the β subunit F195A variant showed markedly increased substrate reactivity with CO, which provides biochemical functional evidence of steric shielding of the CO substrate interaction site by the phenyl group side chain. The phenyl group also likely enhances the nucleophilicity of the Ni center to facilitate CH3 group transfer. A model was developed for how the catalytic properties of the A cluster are optimized by linking conformational changes to a repositionable aromatic shield able to modulate the nucleophilicity of Ni, sterically select the most productive order of substrate addition, and overcome intrinsic inhibition by CO. PMID:23394607

  6. Acetylation phenotypes and biological variation in a French Caucasian population.

    PubMed

    Pontes, Z B; Vincent-Viry, M; Gueguen, R; Galteau, M M; Siest, G

    1993-02-01

    Factors affecting the caffeine acetylation phenotype were investigated in a French Caucasian population of 150 unrelated supposedly healthy subjects, aged 18 to 63 years. This population, including 75 men and 75 women, was used to determine whether the acetylation polymorphism is related to environmental influences such as smoking habits, intake of alcohol, use of oral contraceptives, use of certain drugs. The acetylation phenotype was assessed from the molar ratio of two caffeine metabolites: 5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine. For values less than 0.85, the subjects were classified as poor acetylators (frequency, mean +/- SD: 61.3 +/- 7.9%) in this study. Dose recoveries of 5-acetylamino-6-formylamino-3-methyluracil (mean +/- SD) were 1.26 +/- 0.85% and 3.58 +/- 1.64% in slow and rapid acetylators, respectively. The recovery (mean +/- SD) of 1-methylxanthine in the 3 hour-urine was 2.86 +/- 1.51% in slow acetylators and 2.36 +/- 1.27% in rapid acetylators. The mean value (and SD) of the molar ratio was 0.437 (0.177) and 1.669 (0.651) for slow and rapid acetylators. Three other metabolite ratios can also provide an acetylation index: 5-acetylamino-6-formylamino-3-methyluracil/5-acetylamino-6-formylamino-3 - methyluracil + 1-methylxanthine + 1-methyluric acid; 5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine + 1-methyluric acid + 1,7-dimethyluric acid; and 5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine + 1-methyluric acid + 1,7-dimethyluric acid + 1,7-dimethylxanthine with a bimodal distribution for the former and a trimodal distribution for the two latter ratios, both showing about 95% concordance with the 5-acetylamino-6-formylamino-3-methyluracil/1-methylxanthine ratio. Age did not influence the excretion of caffeine and its five major metabolites. A marked influence of sex was observed only on the unchanged caffeine excretion, and the effect was greater in slow acetylators than in rapid acetylators. The 5-acetylamino-6-formylamino-3-methyluracil excretion was about three times higher in rapid acetylators than in slow acetylators in both sexes. PMID:8467011

  7. Role of Histone Acetylation in Cell Cycle Regulation.

    PubMed

    Koprinarova, Miglena; Schnekenburger, Michael; Diederich, Marc

    2016-01-01

    Core histone acetylation is a key prerequisite for chromatin decondensation and plays a pivotal role in regulation of chromatin structure, function and dynamics. The addition of acetyl groups disturbs histone/DNA interactions in the nucleosome and alters histone/histone interactions in the same or adjacent nucleosomes. Acetyl groups can also provide binding sites for recruitment of bromodomain (BRD)-containing non-histone readers and regulatory complexes to chromatin allowing them to perform distinct downstream functions. The presence of a particular acetylation pattern influences appearance of other histone modifications in the immediate vicinity forming the "histone code". Although the roles of the acetylation of particular lysine residues for the ongoing chromatin functions is largely studied, the epigenetic inheritance of histone acetylation is a debated issue. The dynamics of local or global histone acetylation is associated with fundamental cellular processes such as gene transcription, DNA replication, DNA repair or chromatin condensation. Therefore, it is an essential part of the epigenetic cell response to processes related to internal and external signals. PMID:26303420

  8. Nuclear matrix, dynamic histone acetylation and transcriptionally active chromatin.

    PubMed

    Davie, J R

    1997-08-01

    The nuclear matrix, the RNA-protein skeleton of the nucleus, has a role in the organization and function of nuclear DNA. Nuclear processes associated with the nuclear matrix include transcription, replication and dynamic histone acetylation. Nuclear matrix proteins, which are tissue and cell type specific, are altered with transformation and state of differentiation. Transcription factors are associated with the nuclear matrix, with the spectra of nuclear matrix bound factors being cell type specific. There is compelling evidence that the transcription machinery is anchored to the nuclear matrix, and the chromatin fiber is spooled through this complex. Transcriptionally active chromatin domains are associated with dynamically acetylated histones. The energy exhaustive process of dynamic histone acetylation has several functions. Acetylation of the N-terminal tails of the core histones alters nucleosome and higher order chromatin structure, aiding transcriptional elongation and facilitating the binding of transcription factors to nucleosomes associated with regulatory DNA sequences. Histone acetylation can manipulate the interactions of regulatory proteins that bind to the N-terminal tails of the core histones. Lastly, dynamic acetylation may contribute to the transient attachment of transcriptionally active chromatin to the nuclear matrix. Reversible histone acetylation is catalyzed by histone acetyltransferase and deacetylase, enzymes associated with the nuclear matrix. The recent isolation and characterization of histone acetyltransferase and deacetylase reveals that these enzymes are related to transcriptional regulators, providing us with new insights about how these enzymes are targeted to nuclear matrix sites engaged in transcription. PMID:9291093

  9. Chitosan Molecular Structure as a Function of N-Acetylation

    SciTech Connect

    Franca, Eduardo F.; Freitas, Luiz C.; Lins, Roberto D.

    2011-07-01

    Molecular dynamics simulations have been carried out to characterize the structure and solubility of chitosan nanoparticle-like structures as a function of the deacetylation level (0, 40, 60, and 100%) and the spatial distribution of the N-acetyl groups in the particles. The polysaccharide chains of highly N-deacetylated particles where the N-acetyl groups are uniformly distributed present a high flexibility and preference for the relaxed two-fold helix and five-fold helix motifs. When these groups are confined to a given region of the particle, the chains adopt preferentially a two-fold helix with f and w values close to crystalline chitin. Nanoparticles with up to 40% acetylation are moderately soluble, forming stable aggregates when the N-acetyl groups are unevenly distributed. Systems with 60% or higher N-acetylation levels are insoluble and present similar degrees of swelling regardless the distribution of their N-acetyl groups. Overall particle solvation is highly affected by electrostatic forces resulting from the degree of acetylation. The water mobility and orientation around the polysaccharide chains affects the stability of the intramolecular O3- HO3(n) ... O5(n+ 1) hydrogen bond, which in turn controls particle aggregation.

  10. Regulation of Autophagy and Mitophagy by Nutrient Availability and Acetylation

    PubMed Central

    Webster, Bradley R.; Scott, Iain; Traba, Javier; Han, Kim; Sack, Michael N.

    2014-01-01

    Normal cellular function is dependent on a number of highly regulated homeostatic mechanisms, which act in concert to maintain conditions suitable for life. During periods of nutritional deficit, cells initiate a number of recycling programs which break down complex intracellular structures, thus allowing them to utilize the energy stored within. These recycling systems, broadly named “autophagy”, enable the cell to maintain the flow of nutritional substrates until they can be replenished from external sources. Recent research has shown that a number of regulatory components of the autophagy program are controlled by lysine acetylation. Lysine acetylation is a reversible post-translational modification that can alter the activity of enzymes in a number of cellular compartments. Strikingly, the main substrate for this modification is a product of cellular energy metabolism: acetyl-CoA. This suggests a direct and intricate link between fuel metabolites and the systems which regulate nutritional homeostasis. In this review, we examine how acetylation regulates the systems that control cellular autophagy, and how global protein acetylation status may act as a trigger for recycling of cellular components in a nutrient-dependent fashion. In particular, we focus on how acetylation may control the degradation and turnover of mitochondria, the major source of fuel-derived acetyl-CoA. PMID:24525425

  11. Obesity, cancer, and acetyl-CoA metabolism.

    PubMed

    Lee, Joyce V; Shah, Supriya A; Wellen, Kathryn E

    2013-06-01

    As rates of obesity soar in the Unites States and around the world, cancer attributed to obesity has emerged as major threat to public health. The link between obesity and cancer can be attributed in part to the state of chronic inflammation that develops in obesity. Acetyl-CoA production and protein acetylation patterns are highly sensitive to metabolic state and are significantly altered in obesity. In this article, we explore the potential role of nutrient-sensitive lysine acetylation in regulating inflammatory processes in obesity-linked cancer. PMID:23878588

  12. Erasers of Histone Acetylation: The Histone Deacetylase Enzymes

    PubMed Central

    Seto, Edward; Yoshida, Minoru

    2014-01-01

    Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl functional groups from the lysine residues of both histone and nonhistone proteins. In humans, there are 18 HDAC enzymes that use either zinc- or NAD+-dependent mechanisms to deacetylate acetyl lysine substrates. Although removal of histone acetyl epigenetic modification by HDACs regulates chromatin structure and transcription, deacetylation of nonhistones controls diverse cellular processes. HDAC inhibitors are already known potential anticancer agents and show promise for the treatment of many diseases. PMID:24691964

  13. Altered acetylation in polyglutamine disease: an opportunity for therapeutic intervention?

    PubMed

    Taylor, J Paul; Fischbeck, Kenneth H

    2002-05-01

    Recent investigations into polyglutamine diseases suggest that aberrant transcriptional regulation might be central to the molecular pathogenesis, perhaps because of inappropriate interaction between mutant proteins and important nuclear factors. Several groups have reported an interaction of mutant polyglutamine with histone acetylases, implicating defective acetylation as a cause of abnormal transcription. An important recent observation is that reversal of the acetylation defect with histone deacetylase inhibitors ameliorates polyglutamine toxicity in yeast, mammalian cell culture, and animal models. These encouraging findings suggest that a novel strategy--pharmacological restoration of histone acetylation-- could prove effective in treating this group of devastating illnesses. PMID:12067622

  14. Pretreatment of sallow prior to enzymatic hydrolysis

    SciTech Connect

    Galbe, M.; Zacchi, G.; Scott, C.D.

    1986-01-01

    Pretreatment of fast-growing sallow by steam explosion prior to enzymic hydrolysis was investigated to find optimum conditions regarding pretreatment temperature and time. Some preliminary experiments with impregnation of the material with H/sub 2/SO/sub 4/ or Na/sub 2/SO/sub 3/ were performed to reduce the byproduct formation and to increase the xylose yield. A temperature of 220 degrees for 15 minutes gave the highest yield, approximately 80% of the glucose available based on raw material. The xylose recovered was equal to or less than 20% when no chemicals were added. Impregnation with Na/sub 2/SO/sub 3/ gave an improvement compared with the unimpregnated material. About 30% of the xylose content could thus be recovered after the enzymic hydrolysis. The results are promising. (Refs. 5).

  15. Continuous steam hydrolysis of tulip poplar

    SciTech Connect

    Fieber, C.; Colcord, A.R.; Faass, S.; Muzzy, J.D.; Roberts, R.S.

    1982-08-01

    To produce ethanol from hardwood it is desirable to fractionate the hardwood in order to produce a relatively pure cellulosic pulp for dilute acid hydrolysis. An experimental investigation of continuous steam hydrolysis of tulip poplar wood chips indicates that over 90% of the lignin present can be extracted by 0.1N sodium hydroxide, resulting in a cellulose pulp containing over 90% hexosan. The study was performed using a Stake Technology, Ltd., continuous digester rated at one oven dry ton per hour of wood chips. The yields of hexosans, hexoses, xylan, xylose, lignin, furfural, acetic acid and methanol were determined as a function of residence time and steam pressure in the digester. The information provides a basis for establishing a material and energy balance for a hardwood to ethanol plant.

  16. ?-cyclodextrin assistant flavonoid glycosides enzymatic hydrolysis

    PubMed Central

    Jin, Xin; Zhang, Zhen-hai; Sun, E.; Jia, Xiao-Bin

    2013-01-01

    Background: The content of icaritin and genistein in herba is very low, preparation with relatively large quantities is an important issue for extensive pharmacological studies. Objective: This study focuses on preparing and enzymic hydrolysis of flavonoid glycosides /?-cyclodextrin inclusion complex to increase the hydrolysis rate. Materials and Methods: The physical property of newly prepared inclusion complex was tested by differential scanning calorimetry (DSC). The conditions of enzymatic hydrolysis were optimized for the bioconversion of flavonoid glycosides /?-cyclodextrin inclusion complex by mono-factor experimental design. The experiments are using the icariin and genistein as the model drugs. Results: The solubility of icariin and genistein were increased almost 17 times from 29.2 ?g/ml to 513.5 ?g/ml at 60C and 28 times from 7.78 ?g/ml to 221.46 ?g/ml at 50C, respectively, demonstrating that the inclusion complex could significantly increase the solubility of flavonoid glycosides. Under the optimal conditions, the reaction time of icariin and genistin decreased by 68% and 145%, when compared with that without ?-CD inclusion. By using this enzymatic condition, 473 mg icaritin (with the purity of 99.34%) and 567 mg genistein(with the purity of 99.46%), which was finally determined by melt point, ESI-MS, UV, IR, 1H NMR and 13C NMR, was obtained eventually by transforming the inclusion complex(contains 1.0 g substrates). Conclusion: This study can clearly indicate a new attempt to improve the speed of enzyme-hydrolysis of poorly water-soluble flavonoid glycosides and find a more superior condition which is used to prepare icaritin and genistein. PMID:24143039

  17. 9-O-Acetylation of sialic acids is catalysed by CASD1 via a covalent acetyl-enzyme intermediate

    PubMed Central

    Baumann, Anna-Maria T.; Bakkers, Mark J. G.; Buettner, Falk F. R.; Hartmann, Maike; Grove, Melanie; Langereis, Martijn A.; de Groot, Raoul J.; Mühlenhoff, Martina

    2015-01-01

    Sialic acids, terminal sugars of glycoproteins and glycolipids, play important roles in development, cellular recognition processes and host–pathogen interactions. A common modification of sialic acids is 9-O-acetylation, which has been implicated in sialoglycan recognition, ganglioside biology, and the survival and drug resistance of acute lymphoblastic leukaemia cells. Despite many functional implications, the molecular basis of 9-O-acetylation has remained elusive thus far. Following cellular approaches, including selective gene knockout by CRISPR/Cas genome editing, we here show that CASD1—a previously identified human candidate gene—is essential for sialic acid 9-O-acetylation. In vitro assays with the purified N-terminal luminal domain of CASD1 demonstrate transfer of acetyl groups from acetyl-coenzyme A to CMP-activated sialic acid and formation of a covalent acetyl-enzyme intermediate. Our study provides direct evidence that CASD1 is a sialate O-acetyltransferase and serves as key enzyme in the biosynthesis of 9-O-acetylated sialoglycans. PMID:26169044

  18. Xylan hydrolysis in zinc chloride solution

    SciTech Connect

    Cao, N.J.; Xu, Q.; Chen, L.F

    1995-12-31

    Xylan is the major component of hemicellulose, which consists of up to one-third of the lignocellulosic biomass. When the zinc chloride solution was used as a pretreatment agent to facilitate cellulose hydrolysis, hemicellulose was hydrolyzed during the pretreatment stage. In this study, xylan was used as a model to study the hydrolysis of hemicellulose in zinc chloride solution. The degradation of xylose that is released from xylan was reduced by the formation of zinc-xylose complex. The xylose yield was > 90% (w/w) at 70{degrees}C. The yield and rate of hydrolysis were a function of temperature and the concentration of zinc chloride. The ratio of zinc chloride can be decreased from 9 to 1.3 (w/w). At this ratio, 76% of xylose yield was obtained. When wheat straw was pretreated with a concentrated zinc chloride solution, the hemicellulose hydrolysate contained only xylose and trace amounts of arabinose and oligosaccharides. With this approach, the hemicellulose hydrolysate can be separated from cellulose residue, which would be hydrolyzed subsequently to glucose by acid or enzymes to produce glucose. This production scheme provided a method to produce glucose and xylose in different streams, which can be fermented in separated fermenters.

  19. Mechanistic insights for ?-cyclodextrin catalyzed phosphodiester hydrolysis.

    PubMed

    Rahimian, Mahboobeh; Yeole, Sachin D; Gejji, Shridhar P

    2014-04-01

    Hydrolysis of phosphodiester bond in different substrates containing alkyl or aryl substituents, in the presence of ?-cyclodextrin (?-CD) as a catalyst, has been investigated employing the density functional theory. It has been shown that the mechanism of ?-CD catalyzed phosphodiester hydrolysis in modeled substrates viz. [p-nitrophenyl][(2,2) methylpropan] phosphodiester (G1); [p-nitrophenyl] [(2,2)methyl butan] phosphodiester (G2); (p-nitrophenyl) (2-methyl pentan) phosphodiester (G3); (p-nitrophenyl) (phenyl) phosphodiester (G4); (p-nitrophenyl) (m-tert-butyl phenyl) phosphodiester (G5) and (p-nitrophenyl) (p-nitrophenyl) phosphodiester (G6) involves net phosphoryl transfer from p-nitrophenyl to the catalyst. The hydrolysis occurs in a single-step D(N)A(N) mechanism wherein the ?-CD acts as a competitive general base. The nucleophile addition is facilitated via face-to-face hydrogen-bonded interactions from the secondary hydroxyl groups attached to the top rim of ?-CD. The insights for cleavage of phosphodiester along the dissociative pathway have been derived using the molecular electrostatic potential studies as a tool. The activation barrier of substrates containing alkyl group (G2 and G3) are found to be lower than those containing aryl groups (G4, G5 and G6). PMID:24652502

  20. Sugarcane bagasse hydrolysis using yeast cellulolytic enzymes.

    PubMed

    Souza, Angelica Cristina de; Carvalho, Fernanda Paula; Silva e Batista, Cristina Ferreira; Schwan, Rosane Freitas; Dias, Disney Ribeiro

    2013-10-28

    Ethanol fuel production from lignocellulosic biomass is emerging as one of the most important technologies for sustainable development. To use this biomass, it is necessary to circumvent the physical and chemical barriers presented by the cohesive combination of the main biomass components, which hinders the hydrolysis of cellulose and hemicellulose into fermentable sugars. This study evaluated the hydrolytic capacity of enzymes produced by yeasts, isolated from the soils of the Brazilian Cerrado biome (savannah) and the Amazon region, on sugarcane bagasse pre-treated with H2SO4. Among the 103 and 214 yeast isolates from the Minas Gerais Cerrado and the Amazon regions, 18 (17.47%) and 11 (5.14%) isolates, respectively, were cellulase-producing. Cryptococcus laurentii was prevalent and produced significant ?- glucosidase levels, which were higher than the endo- and exoglucanase activities. In natura sugarcane bagasse was pre-treated with 2% H2SO4 for 30 min at 150oC. Subsequently, the obtained fibrous residue was subjected to hydrolysis using the Cryptococcus laurentii yeast enzyme extract for 72 h. This enzyme extract promoted the conversion of approximately 32% of the cellulose, of which 2.4% was glucose, after the enzymatic hydrolysis reaction, suggesting that C. laurentii is a good ?-glucosidase producer. The results presented in this study highlight the importance of isolating microbial strains that produce enzymes of biotechnological interest, given their extensive application in biofuel production. PMID:23851270

  1. Fungal secretomes enhance sugar beet pulp hydrolysis.

    PubMed

    Kracher, Daniel; Oros, Damir; Yao, Wanying; Preims, Marita; Rezic, Iva; Haltrich, Dietmar; Rezic, Tonci; Ludwig, Roland

    2014-04-01

    The recalcitrance of lignocellulose makes enzymatic hydrolysis of plant biomass for the production of second generation biofuels a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table sugar industry. Three fungal strains were grown on different substrates and the production of various extracellular hydrolytic and oxidative enzymes involved in pectin, hemicellulose, and cellulose breakdown were monitored. In a second step, the ability of the culture supernatants to hydrolyze thermally pretreated SBP was tested in batch experiments. The supernatant of Sclerotium rolfsii, a soil-borne facultative plant pathogen, was found to have the highest hydrolytic activity on SBP and was selected for further hydrolyzation experiments. A low enzyme load of 0.2 mg g(-1) protein from the culture supernatant was sufficient to hydrolyze a large fraction of the pectin and hemicelluloses present in SBP. The addition of Trichoderma reesei cellulase (1-17.5 mg g(-1) SBP) resulted in almost complete hydrolyzation of cellulose. It was found that the combination of pectinolytic, hemicellulolytic, and cellulolytic activities works synergistically on the complex SBP composite, and a combination of these hydrolytic enzymes is required to achieve a high degree of enzymatic SBP hydrolysis with a low enzyme load. PMID:24677771

  2. Fungal secretomes enhance sugar beet pulp hydrolysis

    PubMed Central

    Kracher, Daniel; Oros, Damir; Yao, Wanying; Preims, Marita; Rezic, Iva; Haltrich, Dietmar; Rezic, Tonci; Ludwig, Roland

    2014-01-01

    The recalcitrance of lignocellulose makes enzymatic hydrolysis of plant biomass for the production of second generation biofuels a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table sugar industry. Three fungal strains were grown on different substrates and the production of various extracellular hydrolytic and oxidative enzymes involved in pectin, hemicellulose, and cellulose breakdown were monitored. In a second step, the ability of the culture supernatants to hydrolyze thermally pretreated SBP was tested in batch experiments. The supernatant of Sclerotium rolfsii, a soil-borne facultative plant pathogen, was found to have the highest hydrolytic activity on SBP and was selected for further hydrolyzation experiments. A low enzyme load of 0.2 mg g–1 protein from the culture supernatant was sufficient to hydrolyze a large fraction of the pectin and hemicelluloses present in SBP. The addition of Trichoderma reesei cellulase (1–17.5 mg g–1 SBP) resulted in almost complete hydrolyzation of cellulose. It was found that the combination of pectinolytic, hemicellulolytic, and cellulolytic activities works synergistically on the complex SBP composite, and a combination of these hydrolytic enzymes is required to achieve a high degree of enzymatic SBP hydrolysis with a low enzyme load. PMID:24677771

  3. Codominant expression of N-acetylation and O-acetylation activities catalyzed by N-acetyltransferase 2 in human hepatocytes.

    PubMed

    Doll, Mark A; Zang, Yu; Moeller, Timothy; Hein, David W

    2010-08-01

    Human populations exhibit genetic polymorphism in N-acetylation capacity, catalyzed by N-acetyltransferase 2 (NAT2). We investigated the relationship between NAT2 acetylator genotype and phenotype in cryopreserved human hepatocytes. NAT2 genotypes determined in 256 human samples were assigned as rapid (two rapid alleles), intermediate (one rapid and one slow allele), or slow (two slow alleles) acetylator phenotypes based on functional characterization of the NAT2 alleles reported previously in recombinant expression systems. A robust and significant relationship was observed between deduced NAT2 phenotype (rapid, intermediate, or slow) and N-acetyltransferase activity toward sulfamethazine (p < 0.0001) and 4-aminobiphenyl (p < 0.0001) and for O-acetyltransferase-catalyzed metabolic activation of N-hydroxy-4-aminobiphenyl (p < 0.0001), N-hydroxy-2-amino-3,8-dimethylimidazo[4,5-f] quinoxaline (p < 0.01), and N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (p < 0.0001). NAT2-specific protein levels also significantly associated with the rapid, intermediate, and slow NAT2 acetylator phenotypes (p < 0.0001). As a negative control, p-aminobenzoic acid (an N-acetyltransferase 1-selective substrate) N-acetyltransferase activities from the same samples did not correlate with the three NAT2 acetylator phenotypes (p > 0.05). These results clearly document codominant expression of human NAT2 alleles resulting in rapid, intermediate, and slow acetylator phenotypes. The three phenotypes reflect levels of NAT2 protein catalyzing both N- and O-acetylation. Our results suggest a significant role of NAT2 acetylation polymorphism in arylamine-induced cancers and are consistent with differential cancer risk and/or drug efficacy/toxicity in intermediate compared with rapid or slow NAT2 acetylator phenotypes. PMID:20430842

  4. Neuroprotection in rabbit retina with N-acetyl-aspartylglutamate and 2-phosphonyl-methyl pentanedioic acid

    NASA Astrophysics Data System (ADS)

    Hacker, Henry D.; Yourick, Debra L.; Koenig, Michael K.; Slusher, Barbara S.; Meyerhoff, James L.

    1999-06-01

    Retinal tissue is subject to ischemia from diabetic retinopathy and other conditions that affect the retinal vasculature such as lupus erythematosus and temporal arteritis. There is evidence in animal models of reversible ischemia that a therapeutic window exists during early recovery when agents that reduce glutamate activity at its receptor sites can rescue neurons from injury. To model ischemia, we used sodium cyanide (NaCN), to inhibit oxidative metabolism, and 2-deoxyglucose (2-DG) to inhibit glycolysis. Dissociated rabbit retina cells were studied to evaluate the potential neuroprotective effects of N-acetyl-aspartyl-glutamate (MAAG), which competes with glutamate as a low-potency agonist at the NMDA receptor complex. N-acetylated ?-linked acidic dipeptidase (NAALADase; the NAAG-hydrolyzing enzyme) is responsible for the hydrolysis of NAAG into glutamate, a neurotransmitter and potent excitotoxin, and N-acetylaspartate. 2-Phosphonyl-methyl pentanedioic acid (PMPA) and ?-linked NAAG (?-NAAG), inhibitors of NAALADase, were also tested, since inhibition of NAALADase could reduce synaptic glutamate and increase the concentration of NAAG. We found that metabolic inhibition with NaCN/2-DG for 1 hour caused 50% toxicity as assessed with the MTT assay. Co-treatment with NAAG resulted in dose-dependent protection of up to 55% (p<0.005). When the non-hydrolyzable, NAALADase inhibitor ?-NAAG was employed dose-dependent protection of up to 37% was observed (p<0.001). PMPA also showed 48% protection (p<.05-.001) against these insults. These data suggest that NAAG may antagonize the effect of glutamate at the NMDA receptor complex in retina. Inhibition of NAALADase by PMPA and ?-NAAG may increase the activity of endogenous NAAG.

  5. Histone Acetylation Modifiers in the Pathogenesis of Alzheimer's Disease.

    PubMed

    Lu, Xi; Wang, Li; Yu, Caijia; Yu, Daohai; Yu, Gang

    2015-01-01

    It is becoming more evident that histone acetylation, as one of the epigenetic modifications or markers, plays a key role in the etiology of Alzheimer's disease (AD). Histone acetylases and histone deacetylases (HDACs) are the well-known covalent enzymes that modify the reversible acetylation of lysine residues in histone amino-terminal domains. In AD, however, the roles of these enzymes are controversial. Some recent studies indicate that HDAC inhibitors are neuroprotective by regulating memory and synaptic dysfunctions in cellular and animal models of AD; while on the other hand, increase of histone acetylation have been implicated in AD pathology. In this review, we focus on the recent advances on the roles of histone acetylation covalent enzymes in AD and discuss how targeting these enzymes can ultimately lead to therapeutic approaches for treating AD. PMID:26136662

  6. Protein kinase C coordinates histone H3 phosphorylation and acetylation.

    PubMed

    Darieva, Zoulfia; Webber, Aaron; Warwood, Stacey; Sharrocks, Andrew D

    2015-01-01

    The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly. PMID:26468616

  7. Partially Acetylated Sugarcane Bagasse For Wicking Oil From Contaminated Wetlands

    EPA Science Inventory

    Sugarcane bagasse was partially acetylated to enhance its oil-wicking ability in saturated environments while holding moisture for hydrocarbon biodegradation. The water sorption capacity of raw bagasse was reduced fourfold after treatment, which indicated considerably increased ...

  8. Data detailing the platelet acetyl-lysine proteome

    PubMed Central

    Aslan, Joseph E.; David, Larry L.; McCarty, Owen J.T.

    2015-01-01

    Here we detail proteomics data that describe the acetyl-lysine proteome of blood platelets (Aslan et al., 2015 [1]). An affinity purification mass spectrometry (AP-MS) approach was used to identify proteins modified by N?-lysine acetylation in quiescent, washed human platelets. The data provide insights into potential regulatory mechanisms of platelet function mediated by protein lysine acetylation. Additionally, as platelets are anucleate and lack histone proteins, they offer a unique and valuable system to study the regulation of cytosolic proteins by lysine acetylation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaino et al., 2014 [2]) via with PRIDE partner repository with the dataset identifier PXD002332. PMID:26904711

  9. Protein kinase C coordinates histone H3 phosphorylation and acetylation

    PubMed Central

    Darieva, Zoulfia; Webber, Aaron; Warwood, Stacey; Sharrocks, Andrew D

    2015-01-01

    The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly. DOI: http://dx.doi.org/10.7554/eLife.09886.001 PMID:26468616

  10. Acetylation of C/EBPα inhibits its granulopoietic function.

    PubMed

    Bararia, Deepak; Kwok, Hui Si; Welner, Robert S; Numata, Akihiko; Sárosi, Menyhárt B; Yang, Henry; Wee, Sheena; Tschuri, Sebastian; Ray, Debleena; Weigert, Oliver; Levantini, Elena; Ebralidze, Alexander K; Gunaratne, Jayantha; Tenen, Daniel G

    2016-01-01

    CCAAT/enhancer-binding protein alpha (C/EBPα) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBPα at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBPα DNA-binding ability and modulates C/EBPα transcriptional activity. Acetylated C/EBPα is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)- mediated granulocytic differentiation of 32Dcl3 cells. C/EBPα mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBPα-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBPα and demonstrate the importance of C/EBPα acetylation in myeloid differentiation. PMID:27005833

  11. Acetylation negatively regulates glycogen phosphorylase by recruiting protein phosphatase 1

    PubMed Central

    Zhang, Tengfei; Wang, Shiwen; Lin, Yan; Xu, Wei; Ye, Dan; Xiong, Yue; Zhao, Shimin; Guan, Kun-Liang

    2012-01-01

    Glycogen phosphorylase (GP) catalyzes the rate-limiting step in glycogen catabolism and plays a key role in maintaining cellular and organismal glucose homeostasis. GP is the first protein whose function was discovered to be regulated by reversible protein phosphorylation, which is controlled by phosphorylase kinase (PhK) and protein phosphatase 1 (PP1). Here, we report that lysine acetylation negatively regulates GP activity by both inhibiting enzyme activity directly and promoting dephosphorylation. Acetylation of GP Lys470 enhances its interaction with the PP1 substrate targeting subunit, GL, and PP1, thereby promoting GP dephosphorylation and inactivation. We show that GP acetylation is stimulated by glucose and insulin and inhibited by glucagon. Our results provide molecular insights into the intricate regulation of the classical GP and a functional cross-talk between protein acetylation and phosphorylation. PMID:22225877

  12. A Non-Isotopic In Vitro Assay for Histone Acetylation

    PubMed Central

    Kuninger, David; Lundblad, James; Semirale, Anthony; Rotwein, Peter

    2007-01-01

    We describe a simple, robust, and relatively inexpensive non-radioactive in vitro assay for measuring histone acetyl-transferase activity. The assay takes advantage of easy to purify recombinant E. coli-derived fusion proteins containing the NH2-terminal tails of histones H3 and H4 linked to epitope-tagged maltose binding protein (MBP), and immunoblotting with antibodies specific to acetylated H3 and H4. Here we show the specificity and dynamic range of this assay for the histone acetyl-transferases, p300 and PCAF. This assay may be adapted readily for other substrates by simply generating new fusion proteins and for other acetyl-transferases by modifying reaction conditions. PMID:17698235

  13. A non-isotopic in vitro assay for histone acetylation.

    PubMed

    Kuninger, David; Lundblad, James; Semirale, Anthony; Rotwein, Peter

    2007-09-15

    We describe a simple, robust, and relatively inexpensive non-radioactive in vitro assay for measuring histone acetyl-transferase activity. The assay takes advantage of easy to purify recombinant E. coli-derived fusion proteins containing the NH(2)-terminal tails of histones H3 and H4 linked to epitope-tagged maltose-binding protein (MBP), and immunoblotting with antibodies specific to acetylated H3 and H4. Here we show the specificity and dynamic range of this assay for the histone acetyl-transferases, p300 and PCAF. This assay may be adapted readily for other substrates by simply generating new fusion proteins and for other acetyl-transferases by modifying reaction conditions. PMID:17698235

  14. Histone acetylation in signal transduction by growth regulatory signals.

    PubMed

    Magnaghi-Jaulin, L; Ait-Si-Ali, S; Harel-Bellan, A

    1999-04-01

    Cell fate is determined by extracellular signals which are transmitted to the nucleus and result in the transcriptional regulation of specific subsets of genes. Transcriptional regulation has been recently linked to enzymatic activities which are able to acetylate or deacetylate core histone tails. A number of transcriptional co-regulators are histone acetyl-transferases or histone deacetylases. Here, we discuss the involvement of these enzymes in critical steps of cell proliferation or cell differentiation control PMID:10441073

  15. Mechanistic insights into the regulation of metabolic enzymes by acetylation

    PubMed Central

    2012-01-01

    The activity of metabolic enzymes is controlled by three principle levels: the amount of enzyme, the catalytic activity, and the accessibility of substrates. Reversible lysine acetylation is emerging as a major regulatory mechanism in metabolism that is involved in all three levels of controlling metabolic enzymes and is altered frequently in human diseases. Acetylation rivals other common posttranslational modifications in cell regulation not only in the number of substrates it modifies, but also the variety of regulatory mechanisms it facilitates. PMID:22826120

  16. Nucleosome Dancing at the Tempo of Histone Tail Acetylation

    PubMed Central

    Galvani, Angélique; Thiriet, Christophe

    2015-01-01

    The impact of histone acetylation on transcription was revealed over 50 years ago by Allfrey and colleagues. However, it took decades for an understanding of the fine mechanism by which this posttranslational modification affects chromatin structure and promotes transcription. Here, we review breakthroughs linking histone tail acetylation, histone dynamics, and transcription. We also discuss the histone exchange during transcription and highlight the important function of a pool of non-chromatinized histones in chromatin dynamics. PMID:26184324

  17. Effect of hydrolysis time on the physicochemical and functional properties of corn glutelin by Protamex hydrolysis.

    PubMed

    Zheng, Xi-qun; Wang, Jun-tong; Liu, Xiao-lan; Sun, Ying; Zheng, Yong-jie; Wang, Xiao-jie; Liu, Yue

    2015-04-01

    The physicochemical and functional properties, such as surface hydrophobicity, disulphide bond content, thermal properties, molecular weight distribution, antioxidant properties, of corn glutelin hydrolysates catalysed by Protamex at different hydrolysis times were evaluated. The hydrolysis influenced the properties of corn glutelin significantly, and not only decreased its molecular weight and disulphide bond content, but also eventually transformed its insoluble native aggregates to soluble aggregates during the hydrolysis process. Corn glutelin hydrolysates were found to have a higher solubility, which was associated with their relatively higher foaming and emulsifying properties compared to the original glutelin. Corn glutelin and its hydrolysates maintained a high thermal stability. In addition, the hydrolysates exhibited excellent antioxidant properties measured through in vitro assays, namely DPPH and OH radical scavenging activity, Fe(2+)-chelating capacity and reducing power; the values were 58.86%, 82.64%, 29.92% and 0.236% at 2.0mg/mL, respectively. PMID:25442571

  18. An acetylation switch controls TDP-43 function and aggregation propensity

    PubMed Central

    Cohen, Todd J.; Hwang, Andrew W.; Restrepo, Clark R.; Yuan, Chao-Xing; Trojanowski, John Q.; Lee, Virginia M.Y.

    2015-01-01

    TDP-43 pathology is a disease hallmark that characterizes amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). Although a critical role for TDP-43 as an RNA-binding protein has emerged, the regulation of TDP-43 function is poorly understood. Here we identify lysine acetylation as a novel post-translational modification controlling TDP-43 function and aggregation. We provide evidence that TDP-43 acetylation impairs RNA-binding and promotes accumulation of insoluble, hyper-phosphorylated TDP-43 species that largely resemble pathological inclusions in ALS and FTLD-TDP. Moreover, biochemical and cell-based assays identify oxidative stress as a signaling cue that promotes acetylated TDP-43 aggregates that are readily engaged by the cellular defense machinery. Importantly, acetylated TDP-43 lesions are found in ALS patient spinal cord, indicating that aberrant TDP-43 acetylation and loss of RNA binding are linked to TDP-43 proteinopathy. Thus, modulating TDP-43 acetylation represents a plausible strategy to fine-tune TDP-43 activity, which could provide new therapeutic avenues for TDP-43 proteinopathies. PMID:25556531

  19. Acetyl Radical Generation in Cigarette Smoke: Quantification and Simulations.

    PubMed

    Hu, Na; Green, Sarah A

    2014-10-01

    Free radicals are present in cigarette smoke and can have a negative effect on human health. However, little is known about their formation mechanisms. Acetyl radicals were quantified in tobacco smoke and mechanisms for their generation were investigated by computer simulations. Acetyl radicals were trapped from the gas phase using 3-amino-2, 2, 5, 5-tetramethyl-proxyl (3AP) on solid support to form stable 3AP adducts for later analysis by high performance liquid chromatography (HPLC), mass spectrometry/tandem mass spectrometry (MS-MS/MS) and liquid chromatography-mass spectrometry (LC-MS). Simulations were performed using the Master Chemical Mechanism (MCM). A range of 10-150 nmol/cigarette of acetyl radical was measured from gas phase tobacco smoke of both commerial and research cigarettes under several different smoking conditions. More radicals were detected from the puff smoking method compared to continuous flow sampling. Approximately twice as many acetyl radicals were trapped when a glass filber particle filter (GF/F specifications) was placed before the trapping zone. Simulations showed that NO/NO2 reacts with isoprene, initiating chain reactions to produce hydroxyl radical, which abstracts hydrogen from acealdehyde to generate acetyl radical. These mechanisms can account for the full amount of acetyl radical detected experimentally from cigarette smoke. Similar mechanisms may generate radicals in second hand smoke. PMID:25253993

  20. Acetyl radical generation in cigarette smoke: Quantification and simulations

    NASA Astrophysics Data System (ADS)

    Hu, Na; Green, Sarah A.

    2014-10-01

    Free radicals are present in cigarette smoke and can have a negative effect on human health. However, little is known about their formation mechanisms. Acetyl radicals were quantified in tobacco smoke and mechanisms for their generation were investigated by computer simulations. Acetyl radicals were trapped from the gas phase using 3-amino-2, 2, 5, 5-tetramethyl-proxyl (3AP) on solid support to form stable 3AP adducts for later analysis by high-performance liquid chromatography (HPLC), mass spectrometry/tandem mass spectrometry (MS-MS/MS) and liquid chromatography-mass spectrometry (LC-MS). Simulations were performed using the Master Chemical Mechanism (MCM). A range of 10-150 nmol/cigarette of acetyl radical was measured from gas phase tobacco smoke of both commercial and research cigarettes under several different smoking conditions. More radicals were detected from the puff smoking method compared to continuous flow sampling. Approximately twice as many acetyl radicals were trapped when a glass fiber particle filter (GF/F specifications) was placed before the trapping zone. Simulations showed that NO/NO2 reacts with isoprene, initiating chain reactions to produce hydroxyl radical, which abstracts hydrogen from acetaldehyde to generate acetyl radical. These mechanisms can account for the full amount of acetyl radical detected experimentally from cigarette smoke. Similar mechanisms may generate radicals in second hand smoke.

  1. Acetyl Radical Generation in Cigarette Smoke: Quantification and Simulations

    PubMed Central

    Hu, Na; Green, Sarah A.

    2014-01-01

    Free radicals are present in cigarette smoke and can have a negative effect on human health. However, little is known about their formation mechanisms. Acetyl radicals were quantified in tobacco smoke and mechanisms for their generation were investigated by computer simulations. Acetyl radicals were trapped from the gas phase using 3-amino-2, 2, 5, 5-tetramethyl-proxyl (3AP) on solid support to form stable 3AP adducts for later analysis by high performance liquid chromatography (HPLC), mass spectrometry/tandem mass spectrometry (MS-MS/MS) and liquid chromatography–mass spectrometry (LC-MS). Simulations were performed using the Master Chemical Mechanism (MCM). A range of 10–150 nmol/cigarette of acetyl radical was measured from gas phase tobacco smoke of both commerial and research cigarettes under several different smoking conditions. More radicals were detected from the puff smoking method compared to continuous flow sampling. Approximately twice as many acetyl radicals were trapped when a glass filber particle filter (GF/F specifications) was placed before the trapping zone. Simulations showed that NO/NO2 reacts with isoprene, initiating chain reactions to produce hydroxyl radical, which abstracts hydrogen from acealdehyde to generate acetyl radical. These mechanisms can account for the full amount of acetyl radical detected experimentally from cigarette smoke. Similar mechanisms may generate radicals in second hand smoke. PMID:25253993

  2. Methods to detect NF-κB Acetylation and Methylation

    PubMed Central

    Chen, JinJing; Chen, Lin-Feng

    2015-01-01

    Summary Post-translational modifications of NF-κB, including acetylation and methylation, have emerged as an important regulatory mechanism for determining the duration and strength of NF-κB nuclear activity as well as its transcriptional output. Within the seven NF-κB family proteins, the RelA subunit of NF-κB is the most studied for its regulation by lysine acetylation and methylation. Acetylation or methylation at different lysine residues modulates distinct functions of NF-κB, including DNA binding and transcription activity, protein stability, and its interaction with NF-κB modulators. Here, we describe the experimental methods to monitor the in vitro and in vivo acetylated or methylated forms of NF-κB. These methods include radiolabeling the acetyl- or methyl- groups and immunoblotting with pan or site-specific acetyl- or methyl-lysine antibodies. Radiolabeling is useful in the initial validation of the modifications. Immunoblotting with antibodies provides a rapid and powerful approach to detect and analyze the functions of these modifications in vitro and in vivo. PMID:25736763

  3. Effects of peptide acetylation and dimethylation on electrospray ionization efficiency.

    PubMed

    Cho, Kyung-Cho; Kang, Jeong Won; Choi, Yuri; Kim, Tae Woo; Kim, Kwang Pyo

    2016-02-01

    Peptide acetylation and dimethylation have been widely used to derivatize primary amino groups (peptide N-termini and the ε-amino group of lysines) for chemical isotope labeling of quantitative proteomics or for affinity tag labeling for selection and enrichment of labeled peptides. However, peptide acetylation results in signal suppression during electrospray ionization (ESI) due to charge neutralization. In contrast, dimethylated peptides show increased ionization efficiency after derivatization, since dimethylation increases hydrophobicity and maintains a positive charge on the peptide under common LC conditions. In this study, we quantitatively compared the ESI efficiencies of acetylated and dimethylated model peptides and tryptic peptides of BSA. Dimethylated peptides showed higher ionization efficiency than acetylated peptides for both model peptides and tryptic BSA peptides. At the proteome level, peptide dimethylation led to better protein identification than peptide acetylation when tryptic peptides of mouse brain lysate were analyzed with LC-ESI-MS/MS. These results demonstrate that dimethylation of tryptic peptides enhanced ESI efficiency and provided up to two-fold improved protein identification sensitivity in comparison with acetylation. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26889926

  4. Nonthermal rotational distribution of CO/A 1Pi/ fragments produced by dissociative excitation of CO2 by electron impact. [in Mars atmosphere

    NASA Technical Reports Server (NTRS)

    Mumma, M. J.; Stone, E. J.; Zipf, E. C.

    1975-01-01

    Measurements were made of the rotational profiles of specific bands of the CO fourth-positive group (4PG). The CO 4PG bands were excited by electron impact dissociative excitation of CO2. The results are applicable to analysis of the Mariner observations of the CO 4PG in the dayglow of Mars. The results indicate that dissociative excitation of CO2 by electron impact leads to CO(A 1Pi) fragments with a rotational distribution that is highly nonthermal. The parent CO2 temperature was about 300 K in the experiment, while the fragment CO(A 1Pi) showed emission band profiles consistent with a rotational temperature greater than about 1500 K. Laboratory measurement of the reduced transmission of the hot bands by thermal CO appears to be the most direct way of determining the column density responsible for the CO(v',0) absorption of Mars.

  5. Widespread N-Acetyl-d-Glucosamine Uptake among Pelagic Marine Bacteria and Its Ecological Implications

    PubMed Central

    Riemann, Lasse; Azam, Farooq

    2002-01-01

    Dissolved free and combined N-acetyl-d-glucosamine (NAG) is among the largest pools of amino sugars in the ocean. NAG is a main structural component in chitin and a substantial constituent of bacterial peptidoglycan and lipopolysaccharides. We studied the distribution and kinetics of NAG uptake by the phosphoenolpyruvate:NAG phosphotransferase systems (PTS) in marine bacterial isolates and natural bacterial assemblages in near-shore waters. Of 78 bacterial isolates examined, 60 took up 3H-NAG, while 18 showed no uptake. No systematic pattern in NAG uptake capability relative to phylogenetic affiliation was found, except that all isolates within Vibrionaceae took up NAG. Among 12 isolates, some showed large differences in the relationship between polymer hydrolysis (measured as chitobiase activity) and uptake of the NAG, the hydrolysis product. Pool turnover time and estimated maximum ambient concentration of dissolved NAG in samples off Scripps Pier (La Jolla, Calif.) were 5.9 3.0 days (n = 10) and 5.2 0.9 nM (n = 3), respectively. Carbohydrate competition experiments indicated that glucose, glucosamine, mannose, and fructose were taken up by the same system as NAG. Sensitivity to the antibiotic and NAG structural analog streptozotocin (STZ) was developed into a culture-independent approach, which demonstrated that approximately one-third of bacteria in natural marine assemblages that were synthesizing DNA took up NAG. Isolates possessing a NAG PTS system were found to be predominantly facultative anaerobes. These results suggest the hypothesis that a substantial fraction of bacteria in natural pelagic assemblages are facultative anaerobes. The adaptive value of fermentative metabolism in the pelagic environment is potentially significant, e.g., to bacteria colonizing microenvironments such as marine snow that may experience periodic O2-limitation. PMID:12406749

  6. Studies of Human 2,4-Dienoyl CoA Reductase Shed New Light on Peroxisomal β-Oxidation of Unsaturated Fatty Acids

    SciTech Connect

    Hua, Tian; Wu, Dong; Ding, Wei; Wang, Jiangyun; Shaw, Neil; Liu, Zhi-Jie

    2012-10-15

    Peroxisomes play an essential role in maintaining fatty acid homeostasis. Although mitochondria are also known to participate in the catabolism of fatty acids via β-oxidation, differences exist between the peroxisomal and mitochondrial β-oxidation. Only peroxisomes, but not mitochondrion, can shorten very long chain fatty acids. Here, we describe the crystal structure of a ternary complex of peroxisomal 2,4-dienoyl CoA reductases (pDCR) with hexadienoyl CoA and NADP, as a prototype for comparison with the mitochondrial 2,4-dienoyl CoA reductase (mDCR) to shed light on the differences between the enzymes from the two organelles at the molecular level. Unexpectedly, the structure of pDCR refined to 1.84 Å resolution reveals the absence of the tyrosine-serine pair seen in the active site of mDCR, which together with a lysine and an asparagine have been deemed a hallmark of the SDR family of enzymes. Instead, aspartate hydrogen-bonded to the Cα hydroxyl via a water molecule seems to perturb the water molecule for protonation of the substrate. Our studies provide the first structural evidence for participation of water in the DCR-catalyzed reactions. Biochemical studies and structural analysis suggest that pDCRs can catalyze the shortening of six-carbon-long substrates in vitro. However, the Km values of pDCR for short chain acyl CoAs are at least 6-fold higher than those for substrates with 10 or more aliphatic carbons. Unlike mDCR, hinge movements permit pDCR to process very long chain polyunsaturated fatty acids.

  7. Kinetically and Crystallographically Guided Mutations of a Benzoate CoA Ligase (BadA) Elucidate Mechanism and Expand Substrate Permissivity.

    PubMed

    Thornburg, Chelsea K; Wortas-Strom, Susan; Nosrati, Meisam; Geiger, James H; Walker, Kevin D

    2015-10-13

    A benzoate CoA ligase (BadA), isolated from the bacterium Rhodopseudomonas palustris, catalyzes the conversion of benzoate to benzoyl CoA on the catabolic pathway of aromatic carboxylic acids. Herein, apparent Michaelis constants K(app)cat and K(app)M were determined for an expanded array of 31 substrates chosen to systematically probe the active site architecture of the enzyme and provide a baseline for expansion of wild-type substrate specificity. Acyl CoA products were observed for 25 of the 31 substrates; in general, BadA converted ortho-substituted substrates better than the corresponding meta and para regioisomers, and the turnover number was more affected by steric rather than electronic effects. The kinetic data are interpreted in relation to six crystal structures of BadA in complex with several substrates and a benzoyl-AMP reaction intermediate. In contrast to other known natural substrate-bound benzoate ligase structures, all substrate-bound BadA structures adopted the thiolation conformation instead of the adenylation conformation. We also observed all the aryl carboxylates to be uniquely oriented within the active site, relative to other structures. Together, the kinetics and structural data suggested a mechanism that involves substrate binding in the thiolation conformation, followed by substrate rotation to an active orientation upon the transition to the adenylation conformation. On the basis of this hypothesis and the structural data, sterically demanding active site residues were mutated, and the substrate specificity was expanded substantially versus that of BadA. Novel activities were seen for substrates with larger substituents, including phenyl acetate. Additionally, the mutant Lys427Ala identified this nonconserved residue as essential for the thiolation step of BadA, but not adenylation. These variously acylated CoAs can serve as novel substrates of acyl CoA-dependent acyltransferases in coupled enzyme assays to produce analogues of bioactive natural products. PMID:26378464

  8. Synergy between cellulases and pectinases in the hydrolysis of hemp.

    PubMed

    Zhang, Junhua; Pakarinen, Annukka; Viikari, Liisa

    2013-02-01

    The impact of pectinases in the hydrolysis of fresh, steam-exploded and ensiled hemp was investigated and the synergy between cellulases, pectinases and xylanase in the hydrolysis was evaluated. About half; 59.3% and 46.1% of pectin in the steam-exploded and ensiled hemp, respectively, could be removed by a low dosage of pectinases used. Pectinases were more efficient than xylanase in the hydrolysis of fresh and ensiled hemp whereas xylanase showed higher hydrolytic efficiency than the pectinase preparation used in the hydrolysis of steam-exploded hemp. Clear synergistic action between cellulases and xylanase could be observed in the hydrolysis of steam-exploded hemp. Supplementation of pectinase resulted in clear synergism with cellulases in the hydrolysis of all hemp substrates. Highest hydrolysis yield of steam-exploded hemp was obtained in the hydrolysis with cellulases and xylanase. In the hydrolysis of ensiled hemp, the synergistic action between cellulases and pectinases was more obvious for efficient hydrolysis. PMID:23262004

  9. A Method for Genetically Installing Site-Specific Acetylation in Recombinant Histones Defines the Effects of H3 K56 Acetylation

    PubMed Central

    Neumann, Heinz; Hancock, Susan M.; Buning, Ruth; Routh, Andrew; Chapman, Lynda; Somers, Joanna; Owen-Hughes, Tom; van Noort, John; Rhodes, Daniela; Chin, Jason W.

    2009-01-01

    Summary Lysine acetylation of histones defines the epigenetic status of human embryonic stem cells and orchestrates DNA replication, chromosome condensation, transcription, telomeric silencing, and DNA repair. A detailed mechanistic explanation of these phenomena is impeded by the limited availability of homogeneously acetylated histones. We report a general method for the production of homogeneously and site-specifically acetylated recombinant histones by genetically encoding acetyl-lysine. We reconstitute histone octamers, nucleosomes, and nucleosomal arrays bearing defined acetylated lysine residues. With these designer nucleosomes, we demonstrate that, in contrast to the prevailing dogma, acetylation of H3 K56 does not directly affect the compaction of chromatin and has modest effects on remodeling by SWI/SNF and RSC. Single-molecule FRET experiments reveal that H3 K56 acetylation increases DNA breathing 7-fold. Our results provide a molecular and mechanistic underpinning for cellular phenomena that have been linked with K56 acetylation. PMID:19818718

  10. Genetic Construction of Truncated and Chimeric Metalloproteins Derived from the Alpha Subunit of Acetyl-CoA Synthase from Clostridium thermoaceticum

    SciTech Connect

    Huay-Keng Loke; Xiangshi Tan; Paul A. Lindahl

    2002-06-28

    In this study, a genetics-based method is used to truncate acetyl-coenzyme A synthase from Clostridium thermoaceticum (ACS), an alpha2beta2 tetrameric 310 kda bifunctional enzyme. ACS catalyzes the reversible reduction of CO2 to CO and the synthesis of acetyl-CoA from CO (or CO2 in the presence of low-potential reductants), CoA, and a methyl group bound to a corrinoid-iron sulfur protein (CoFeSP). ACS contains 7 metal-sulfur clusters of 4 different types called A, B, C, and D. The B, C, and D clusters are located in the 72 kda beta subunit while the A-cluster, a Ni-X-Fe4S4 cluster that serves as the active site for acetyl-CoA synthase activity, is located in the 82 kda alpha subunit. The extent to which the essential properties of the cluster, including catalytic, redox, spectroscopic, and substrate-binding properties, were retained as ACS was progressively truncated was determined. Acetyl-CoA synthase catalytic activity remained when the entire alpha subunit was removed, as long as CO, rather than CO2 and a low-potential reductant, was used as a substrate. Truncating an {approx} 30 kda region from the N-terminus of the alpha subunit yielded a 49 kda protein that lacked catalytic activity but exhibited A-cluster-like spectroscopic, redox, and CO binding properties. Further truncation afforded a 23 kda protein that lacked recognizable A-cluster properties except for UV-vis spectra typical of [Fe4S4]2+ clusters. Two chimeric proteins were constructed by fusing the gene encoding a ferredoxin from Chromatium vinosum to genes encoding the 49 kda and 82 kda fragments of the alpha subunit. The chimeric proteins exhibited EPR signals that were not the simple sum of the signals from the separate proteins, suggesting magnetic interactions between clusters. This study highlights the potential for using genetics to simplify the study of complex multi-centered metalloenzymes and to generate new complex metalloenzymes with interesting properties.

  11. Purification and properties of an O-acetyl-transferase from Escherichia coli that can O-acetylate polysialic acid sequences

    SciTech Connect

    Higa, H.; Varki, A.

    1986-05-01

    Certain strains of bacteria synthesize an outer polysialic acid (K1) capsule. Some strains of K1/sup +/ E.coli are also capable of adding O-acetyl-esters to the exocyclic hydroxyl groups of the sialic acid residues. Both the capsule and the O-acetyl modification have been correlated with differences in antigenicity and pathogenicity. The authors have developed an assay for an O-acetyl-transferase in E.coli that transfers O-(/sup 3/H)acetyl groups from (/sup 3/H)acetyl-Coenzyme A to colominic acid (fragments of the polysialic acid capsule). Using this assay, the enzyme was solubilized, and purified approx. 600-fold using a single affinity chromatography step with Procion Red-A Agarose. The enzyme also binds to Coenzyme A Sepharose, and can be eluted with high salt or Coenzyme A. The partially purified enzyme has a pH optimum of 7.0 - 7.5, is unaffected by divalent cations, is inhibited by high salt concentrations, is inhibited by Coenzyme A (50% inhibition at 100 ..mu..M), and shows an apparent Km for colominic acid of 3.7 mM (sialic acid concentration). This enzyme could be involved in the O-acetyl +/- form variation seen in some strains of K1/sup +/ E.coli.

  12. Hydrolysis of ferric chloride in solution

    SciTech Connect

    Lussiez, G.; Beckstead, L.

    1996-11-01

    The Detox{trademark} process uses concentrated ferric chloride and small amounts of catalysts to oxidize organic compounds. It is under consideration for oxidizing transuranic organic wastes. Although the solution is reused extensively, at some point it will reach the acceptable limit of radioactivity or maximum solubility of the radioisotopes. This solution could be cemented, but the volume would be increased substantially because of the poor compatibility of chlorides and cement. A process has been developed that recovers the chloride ions as HCl and either minimizes the volume of radioactive waste or permits recycling of the radioactive chlorides. The process involves a two-step hydrolysis at atmospheric pressure, or preferably under a slight vacuum, and relatively low temperature, about 200{degrees}C. During the first step of the process, hydrolysis occurs according to the reaction below: FeCl{sub 3 liquid} + H{sub 2}O {r_arrow} FeOCl{sub solid} + 2 HCl{sub gas} During the second step, the hot, solid, iron oxychloride is sprayed with water or placed in contact with steam, and hydrolysis proceeds to the iron oxide according to the following reaction: 2 FeOCl{sub solid} + H{sub 2}O {r_arrow} Fe{sub 2}O{sub 3 solid} + 2 HCl{sub gas}. The iron oxide, which contains radioisotopes, can then be disposed of by cementation or encapsulation. Alternately, these chlorides can be washed off of the solids and can then either be recycled or disposed of in some other way.

  13. QSAR for cholinesterase inhibition by organophosphorus esters and CNDO/2 calculations for organophosphorus ester hydrolysis. [quantitative structure-activity relationship, complete neglect of differential overlap

    NASA Technical Reports Server (NTRS)

    Johnson, H.; Kenley, R. A.; Rynard, C.; Golub, M. A.

    1985-01-01

    Quantitative structure-activity relationships were derived for acetyl- and butyrylcholinesterase inhibition by various organophosphorus esters. Bimolecular inhibition rate constants correlate well with hydrophobic substituent constants, and with the presence or absence of cationic groups on the inhibitor, but not with steric substituent constants. CNDO/2 calculations were performed on a separate set of organophosphorus esters, RR-primeP(O)X, where R and R-prime are alkyl and/or alkoxy groups and X is fluorine, chlorine or a phenoxy group. For each subset with the same X, the CNDO-derived net atomic charge at the central phosphorus atom in the ester correlates well with the alkaline hydrolysis rate constant. For the whole set of esters with different X, two equations were derived that relate either charge and leaving group steric bulk, or orbital energy and bond order to the hydrolysis rate constant.

  14. Pretreatment and enzymatic hydrolysis of lignocellulosic biomass

    NASA Astrophysics Data System (ADS)

    Corredor, Deisy Y.

    The performance of soybean hulls and forage sorghum as feedstocks for ethanol production was studied. The main goal of this research was to increase fermentable sugars' yield through high-efficiency pretreatment technology. Soybean hulls are a potential feedstock for production of bio-ethanol due to their high carbohydrate content (≈50%) of nearly 37% cellulose. Soybean hulls could be the ideal feedstock for fuel ethanol production, because they are abundant and require no special harvesting and additional transportation costs as they are already in the plant. Dilute acid and modified steam-explosion were used as pretreatment technologies to increase fermentable sugars yields. Effects of reaction time, temperature, acid concentration and type of acid on hydrolysis of hemicellulose in soybean hulls and total sugar yields were studied. Optimum pretreatment parameters and enzymatic hydrolysis conditions for converting soybean hulls into fermentable sugars were identified. The combination of acid (H2SO4, 2% w/v) and steam (140°C, 30 min) efficiently solubilized the hemicellulose, giving a pentose yield of 96%. Sorghum is a tropical grass grown primarily in semiarid and dry parts of the world, especially in areas too dry for corn. The production of sorghum results in about 30 million tons of byproducts mainly composed of cellulose, hemicellulose, and lignin. Forage sorghum such as brown midrib (BMR) sorghum for ethanol production has generated much interest since this trait is characterized genetically by lower lignin concentrations in the plant compared with conventional types. Three varieties of forage sorghum and one variety of regular sorghum were characterized and evaluated as feedstock for fermentable sugar production. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM) and X-Ray diffraction were used to determine changes in structure and chemical composition of forage sorghum before and after pretreatment and enzymatic hydrolysis process. Up to 72% of hexose yield and 94% of pentose yield were obtained using "modified" steam explosion with 2% sulfuric acid at 140°C for 30 min and enzymatic hydrolysis with cellulase (15 FPU/g cellulose) and beta-glucosidase (50 CBU/g cellulose).

  15. Cytoskeleton Dynamics: A Continuum Cooperative Hydrolysis Model

    NASA Astrophysics Data System (ADS)

    Xu, Jian-Wei; Cheng, Bo; Feng, Yu-Yu; Wang, Zi-Qing; Wang, Guo-Dong

    2015-05-01

    Cytoskeleton is a network of filamentous proteins, such as actin filaments and microtubules. We propose a continuum cooperative hydrolysis model which possesses exactly analytical solution to describe the dynamics of filament. The results show that the cooperativity leads to non negative-exponential distribution of T (ATP or GTP) subunits. As an application, we investigate the treadmilling phenomenon using our model. It is shown that the cooperativity remarkably affects the length of filament. Supported by Chinese Universities Scientific Fund under Grant No. 2014YB029 and National Natural Science Foundation of China under Grant No. 11205123

  16. Improved method for detection of starch hydrolysis

    SciTech Connect

    Ohawale, M.R.; Wilson, J.J.; Khachatourians, G.G.; Ingledew, W.M.

    1982-09-01

    A new starch hydrolysis detection method which does not rely on iodine staining or the use of color-complexed starch is described. A linear relationship was obtained with agar-starch plates when net clearing zones around colonies of yeasts were plotted against enzyme levels (semilogarithm scale) produced by the same yeast strains in liquid medium. A similar relationship between starch clearing zones and alpha-amylase levels from three different sources was observed. These observations suggest that the method is useful in mutant isolations, strain improvement programs, and the prediction of alpha-amylase activities in culture filtrates or column effluents. (Refs. 18).

  17. Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases

    PubMed Central

    Hamer, Stefanie Nicole; Cord-Landwehr, Stefan; Biarns, Xevi; Planas, Antoni; Waegeman, Hendrik; Moerschbacher, Bruno Maria; Kolkenbrock, Stephan

    2015-01-01

    Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture. PMID:25732514

  18. Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases.

    PubMed

    Hamer, Stefanie Nicole; Cord-Landwehr, Stefan; Biarnés, Xevi; Planas, Antoni; Waegeman, Hendrik; Moerschbacher, Bruno Maria; Kolkenbrock, Stephan

    2015-01-01

    Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture. PMID:25732514

  19. Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases

    NASA Astrophysics Data System (ADS)

    Hamer, Stefanie Nicole; Cord-Landwehr, Stefan; Biarnés, Xevi; Planas, Antoni; Waegeman, Hendrik; Moerschbacher, Bruno Maria; Kolkenbrock, Stephan

    2015-03-01

    Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture.

  20. Unusual Extra Space at the Active Site and High Activity for Acetylated Hydroxyproline of Prolyl Aminopeptidase from Serratia marcescens

    PubMed Central

    Nakajima, Yoshitaka; Ito, Kiyoshi; Sakata, Makoto; Xu, Yue; Nakashima, Kanako; Matsubara, Futoshi; Hatakeyama, Susumi; Yoshimoto, Tadashi

    2006-01-01

    The prolyl aminopeptidase complexes of Ala-TBODA [2-alanyl-5-tert-butyl-(1, 3, 4)-oxadiazole] and Sar-TBODA [2-sarcosyl-5-tert-butyl-(1, 3, 4)-oxadiazole] were analyzed by X-ray crystallography at 2.4 resolution. Frames of alanine and sarcosine residues were well superimposed on each other in the pyrrolidine ring of proline residue, suggesting that Ala and Sar are recognized as parts of this ring of proline residue by the presence of a hydrophobic proline pocket at the active site. Interestingly, there was an unusual extra space at the bottom of the hydrophobic pocket where proline residue is fixed in the prolyl aminopeptidase. Moreover, 4-acetyloxyproline-?NA (4-acetyloxyproline ?-naphthylamide) was a better substrate than Pro-?NA. Computer docking simulation well supports the idea that the 4-acetyloxyl group of the substrate fitted into that space. Alanine scanning mutagenesis of Phe139, Tyr149, Tyr150, Phe236, and Cys271, consisting of the hydrophobic pocket, revealed that all of these five residues are involved significantly in the formation of the hydrophobic proline pocket for the substrate. Tyr149 and Cys271 may be important for the extra space and may orient the acetyl derivative of hydroxyproline to a preferable position for hydrolysis. These findings imply that the efficient degradation of collagen fragment may be achieved through an acetylation process by the bacteria. PMID:16452443

  1. The Hydrolysis of Carbonyl Sulfide at Low Temperature: A Review

    PubMed Central

    Zhao, Shunzheng; Yi, Honghong; Tang, Xiaolong; Jiang, Shanxue; Gao, Fengyu; Zhang, Bowen; Zuo, Yanran; Wang, Zhixiang

    2013-01-01

    Catalytic hydrolysis technology of carbonyl sulfide (COS) at low temperature was reviewed, including the development of catalysts, reaction kinetics, and reaction mechanism of COS hydrolysis. It was indicated that the catalysts are mainly involved metal oxide and activated carbon. The active ingredients which can load on COS hydrolysis catalyst include alkali metal, alkaline earth metal, transition metal oxides, rare earth metal oxides, mixed metal oxides, and nanometal oxides. The catalytic hydrolysis of COS is a first-order reaction with respect to carbonyl sulfide, while the reaction order of water changes as the reaction conditions change. The controlling steps are also different because the reaction conditions such as concentration of carbonyl sulfide, reaction temperature, water-air ratio, and reaction atmosphere are different. The hydrolysis of carbonyl sulfide is base-catalyzed reaction, and the force of the base site has an important effect on the hydrolysis of carbonyl sulfide. PMID:23956697

  2. Acetylation of nuclear receptors in cellular growth and apoptosis.

    PubMed

    Fu, Maofu; Wang, Chenguang; Zhang, Xueping; Pestell, Richard G

    2004-09-15

    Post-translational modification of chromatin histones governs a key mechanism of transcriptional regulation. Histone acetylation, together with methylation, phosphorylation, ubiquitylation, sumoylation, glycosylation, and ADP ribosylation, modulate the activity of many genes by modifying both core histones and non-histone transcription factors. Epigenetic protein modification plays an important role in multiple cellular processes including DNA repair, protein stability, nuclear translocation, protein-protein interactions, and in regulation of cellular proliferation, differentiation and apoptosis. Histone acetyltransferases modify histones, coactivators, nuclear transport proteins, structural proteins, cell cycle components and transcription factors including p53 and nuclear receptors. The estrogen, PPARgamma and androgen receptor are members of the nuclear receptor (NR) superfamily. The androgen receptor (AR) and estrogen receptor alpha (ERalpha) are directly acetylated by histone acetyltransferases at a motif that is conserved between species and other NR. Point mutations at the lysine residue within the acetylation motif of the AR and ERalpha have been identified in prostate cancer as well as in breast cancer tissue. Acetylation of the NR governs ligand sensitivity and hormone antagonist responses. The AR is acetylated by p300, P/CAF and TIP60 and acetylation of the AR regulates co-regulator recruitment and growth properties of the receptors in cultured cells and in vivo. AR acetylation mimic mutants convey reduced apoptosis and enhanced growth properties correlating with altered promoter specificity for cell-cycle target genes. Cell-cycle control proteins, including cyclins, in turn alter the access of transcription factors and nuclear receptors to the promoters of target genes. PMID:15313417

  3. Mutations Affecting Peptidoglycan Acetylation in Neisseria gonorrhoeae and Neisseria meningitidis

    PubMed Central

    Dillard, Joseph P.; Hackett, Kathleen T.

    2005-01-01

    Neisseria gonorrhoeae acetylates its cell wall peptidoglycan (PG) at the C-6 position on N-acetylmuramic acid. To understand the effects of PG acetylation on PG metabolism and release of PG fragments, we have made mutations in the genes responsible for PG acetylation. An insertion mutation in a putative PG acetylase gene (designated pacA) resulted in loss of PG acetylation as detected by a high-performance liquid chromatography-based assay. Sequence analysis of a naturally occurring nonacetylating strain revealed the presence of a 26-bp deletion in pacA. Introduction of the deletion mutation into wild-type gonococci resulted in lack of acetylation, and the phenotype was complemented by the addition of a wild-type copy of pacA at a distant location on the chromosome. Mutations were also introduced into three genes downstream of pacA. The gene directly downstream of pacA was required for acetylation and was designated pacB, whereas the next two genes were not required. Sequences highly similar to pacA and pacB were also found in N. meningitidis and N. lactamica strains, and an insertion in the meningococcal pacA eliminated PG acetylation. Phenotypic analyses of an N. gonorrhoeae pacA mutant did not show any decrease in lysozyme resistance or serum resistance, and the release of PG fragments during growth was unchanged. However, purified PG from the wild-type strain was significantly more resistant to the action of human lysozyme than was PG purified from the pacA mutant. Interestingly, the pacA mutant was more sensitive to EDTA, a compound known to trigger autolysis. PMID:16113287

  4. ESTIMATION OF PHOSPHATE ESTER HYDROLYSIS RATE CONSTANTS. II. ACID AND GENERAL BASE CATALYZED HYDROLYSIS

    EPA Science Inventory

    SPARC (SPARC Performs Automated Reasoning in Chemistry) chemical reactivity models were extended to calculate acid and neutral hydrolysis rate constants of phosphate esters in water. The rate is calculated from the energy difference between the initial and transition states of a ...

  5. Simulation of continuous and batch hydrolysis of willow

    SciTech Connect

    Zacchi, G.; Dahlbom, J.; Scott, C.D.

    1986-01-01

    The influence of product and enzyme concentrations on the kinetics of the enzymic hydrolysis of alkali-pretreated willow is studied. The hydrolysis was performed in a UF-membrane reactor in which the product concentration was kept constant. An empirical 4-parameter rate equation that gives a good correlation to both continuous and batch hydrolysis data is presented. The model comprises the effects of enzyme concentration and product inhibition. (Refs. 11).

  6. [The study of hydrolysis of new lipid-like substrates and trilaurin in monolayers catalyzed with the lipase from Pseudomonas fluorescens].

    PubMed

    Za?tsev, S Iu; Aha, B; Volchenkova, T A; Belov, S V; Schneider, M P; Ivanov, A E

    2000-03-01

    A simple method for determining the enzymic hydrolysis parameters of lipid-like substrates and trilaurin assembled in monolayers at the water-air interface was suggested. At a surface pressure of 10 mN/m, the initial rates of lipolysis were found to be proportional to the decrease in area of the substrate monolayer caused by the enzymic hydrolysis in a single-compartment Langmuir balance. The kinetic parameters for the hydrolysis of trilaurin and three 1,3-dilaurylpseudoglycerides acetylated in position 2 with an amino acid (phenylalanine, leucine, or valine) catalyzed with lipase from Pseudomonas fluorescens were determined. Unlike models of enzymic hydrolysis that neglect the thickness of the substrate monolayer, our method allows the determination of kinetic parameters in standard dimensions. The values of kcat for the synthetic pseudoglycerides were found to be significantly higher than that for trilaurin, while the values of Km(app) were close. This may be due to the presence of positively charged primary amino groups in the molecules of pseudoglycerides. PMID:10816821

  7. Effect of Zn on acetyl coenzyme a synthase: evidence for a conformational change in the alpha subunit during catalysis.

    PubMed

    Tan, Xiangshi; Bramlett, Matthew R; Lindahl, Paul A

    2004-05-19

    Acetyl coenzyme A synthase (ACS) is an alpha2beta2 tetramer in which the active-site A-cluster, located in the alpha subunits, consists of an Fe4S4 cubane bridged to a {Nip Nid} binuclear site. The alpha subunits exist in two conformations. In the open conformation, Nip is surface-exposed, while the proximal metal is buried in the closed conformation. Nip is labile and can be replaced by Cu. In this study, the effects of Zn are reported. ACS in which Zn replaced Nip was inactive and did not exhibit the so-called NiFeC EPR signal nor the ability to accept a methyl group from the corrinoid-iron-sulfur protein (CoFeSP). Once Zn-bound, it could not be replaced by subsequently adding Ni. The Zn-bound A-cluster cannot be reduced and bound with CO or become methylated, probably because Zn (like Cu) is insufficiently nucleophilic for these functions. Unexpectedly, Zn replaced Nip only while ACS was engaged in catalysis. Under these conditions, replacement occurred with kapp approximately 0.6 min-1. Replacement was blocked by including EDTA in the assay mix. Zn appears to replace Nip when ACS is in an intermediate state (or states) of catalysis but this(these) state(s) must not be present when ACS is reduced in CO alone, or in the presence of CoA, CoFeSP, or reduced methyl viologen. Nip appears susceptible to Zn-attack when the alpha subunit is in the open conformation and protected from attack when it is in the closed conformation. This is the first evidence that the structurally-characterized conformations of the alpha subunit change during catalysis, indicating a mechanistic role for this conformational change. PMID:15137746

  8. Hydrolysis of lignocelluloses by penicillium funiculosum cellulase

    SciTech Connect

    Mishra, C.; Rao, M.; Seeta, R.; Srinivasan, M.C.; Deshpande, V.

    1984-04-01

    Enzymatic hydrolysis of cellulose is a promising method for the conversion of waste cellulose to glucose. During the past few years, the development of this technology has proceeded rapidly, with significant advances made in enzyme production, pretreatment, and hydrolysis. A variety of fungi are reported to produce cellulases but among these Trichoderma reesei and its mutants are powerful producers of cellulases. However, the search for new and possibly better sources of cellulase is continued due to the low levels of beta-glucosidase of T. reesei. Penicillium funiculosum produces a complete cellulase having endo-beta-1,4-glucanase (15-20 U/mL), exo-beta-1,4-glucanase (1.5-2.0 U/mL), and high beta-glucosidase (8-10 U/mL). The saccharification of alkali-treated cotton and bagasse by P. funiculosum enzyme was 70 and 63%, respectively. It was possible to obtain glucose concentration as high as 30% using 50% bagasse. It is of interest that the percent saccharification of cellulosic substrates with the Penicillium enzyme is comparable to that of T. reesei cellulase when the same amount of filter paper activity is used, although the endo-glucanase activity of the latter is two to three times higher. This communication reports the studies on saccharification of lignocelluloses by P. funiculosum cellulase and certain studies on the kinetic aspects. (Refs. 15).

  9. 4-O-Acetyl-sialic acid (Neu4,5Ac2) in acidic milk oligosaccharides of the platypus (Ornithorhynchus anatinus) and its evolutionary significance.

    PubMed

    Urashima, Tadasu; Inamori, Hiroaki; Fukuda, Kenji; Saito, Tadao; Messer, Michael; Oftedal, Olav T

    2015-06-01

    Monotremes (echidnas and platypus) retain an ancestral form of reproduction: egg-laying followed by secretion of milk onto skin and hair in a mammary patch, in the absence of nipples. Offspring are highly immature at hatching and depend on oligosaccharide-rich milk for many months. The primary saccharide in long-beaked echidna milk is an acidic trisaccharide Neu4,5Ac2(?2-3)Gal(?1-4)Glc (4-O-acetyl 3'-sialyllactose), but acidic oligosaccharides have not been characterized in platypus milk. In this study, acidic oligosaccharides purified from the carbohydrate fraction of platypus milk were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and (1)H-nuclear magnetic resonance spectroscopy. All identified structures, except Neu5Ac(?2-3)Gal(?1-4)Glc (3'-sialyllactose) contained Neu4,5Ac2 (4-O-acetyl-sialic acid). These include the trisaccharide 4-O-acetyl 3'-sialyllactose, the pentasaccharide Neu4,5Ac2(?2-3)Gal(?1-4)GlcNAc(?1-3)Gal(?1-4)Glc (4-O-acetyl-3'-sialyllacto-N-tetraose d) and the hexasaccharide Neu4,5Ac2(?2-3)Gal(?1-4)[Fuc(?1-3)]GlcNAc(?1-3)Gal(?1-4)Glc (4-O-acetyl-3'-sialyllacto-N-fucopentaose III). At least seven different octa- to deca-oligosaccharides each contained a lacto-N-neohexaose core (LNnH) and one or two Neu4,5Ac2 and one to three fucose residues. We conclude that platypus milk contains a diverse (? 20) array of neutral and acidic oligosaccharides based primarily on lactose, lacto-N-neotetraose (LNnT) and LNnH structural cores and shares with echidna milk the unique feature that all identified acidic oligosaccharides (other than 3'-sialyllactose) contain the 4-O-acetyl-sialic acid moiety. We propose that 4-O-acetylation of sialic acid moieties protects acidic milk oligosaccharides secreted onto integumental surfaces from bacterial hydrolysis via steric interference with bacterial sialidases. This may be of evolutionary significance since taxa ancestral to monotremes and other mammals are thought to have secreted milk, or a milk-like fluid containing oligosaccharides, onto skin surfaces. PMID:25601457

  10. Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism

    SciTech Connect

    Martinson, E.A.; Goldstein, D.; Brown, J.H. )

    1989-09-05

    We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of (3H)choline and (3H)phosphorylcholine ((3H)Pchol) from cells containing (3H)choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of (3H)phosphatidic acid ((3H)PA) in cells containing (3H)myristate-labeled PC. (3H)Diacylglycerol ((3H)DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with (3H)myristate and (14C)arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol.

  11. Reduced inhibition of enzymatic hydrolysis of steam-pretreated softwood.

    PubMed

    Tengborg, C; Galbe, M; Zacchi, G

    2001-06-01

    Softwood constitutes the main source of lignocellulosic material in Sweden which can be used for ethanol production from renewable resources. To make the biomass-to-ethanol process more economically feasible, it is preferable to include the sugar-rich prehydrolysate, i.e. the liquid obtained after the pretreatment step, in the enzymatic hydrolysis of the solid fraction. This study shows that the prehydrolysate inhibits cellulose conversion in the enzymatic hydrolysis step. When the prehydrolysate was included in the enzymatic hydrolysis, the cellulose conversion was reduced by up to 36%. However, this inhibition can be overcome by fermentation of the prehydrolysate prior to enzymatic hydrolysis. PMID:11397466

  12. Acetylation of HDAC1 and degradation of SIRT1 form a positive feedback loop to regulate p53 acetylation during heat-shock stress

    PubMed Central

    Yang, H; Yan, B; Liao, D; Huang, S; Qiu, Y

    2015-01-01

    The tumor suppressor p53 is an essential transcription factor that sensitively regulates cellular responses to various stresses. Acetylation, a critically important posttranslational modification of p53, is induced in response to cellular stresses. P53 acetylation level strongly correlates with protein stability and activity. The steady-state level of p53 acetylation is balanced by dynamic acetylation and deacetylation. Despite the function of p53 acetylation being well studied, how the steady state of p53 acetylation level is regulated in response to cellular stresses remains unclear. In particular, the dynamic regulation of the deacetylase activities responsible for p53 deacetylation during cellular stress is unknown. In the current study, we investigated the dynamic regulation of HDAC1 (histone deacetylase 1) and SIRT1 (sirtuin 1), two major enzymes for p53 deacetylation, during cell stress. We found that various cell stress events induce HDAC1 acetylation. The increased level of HDAC1 acetylation correlates with the level of p53 acetylation. Acetylated HDAC1 loses the ability to deacetylate p53. Cellular stresses also promote the decline of the SIRT1 protein in a proteasome-dependent pathway, which also results in the increase of p53 acetylation. Importantly, the decreased level of SIRT1 also contributes to the accumulation of HDAC1 acetylation as SIRT1 deacetylates HDAC1. Therefore, the increase of HDAC1 acetylation and reduced level of SIRT1 protein during cellular stress directly link to the induction of p53 acetylation. These results unveil the mechanism underlying the dynamic regulation of p53 acetylation during cell stress. PMID:25950477

  13. Determination of urinary 4,4'-methylenedianiline and its acetylated metabolites by solid-phase extraction and HPLC analysis with UV and electrochemical detection.

    PubMed

    Robert, A; Ducos, P; Francin, J M

    1995-01-01

    An analytical procedure based on solid-phase extraction and high-performance liquid chromatography using both ultraviolet and electrochemical detection was developed to determine, without derivatization, stable urinary forms of 4,4'-methylenedianiline (MDA) and of its acetylated metabolites at the micrograms/l level, in post-shift urine from 63 exposed workers. The determination of MDA, N-acetyl MDA (MAMDA) and N,N'-diacetyl MDA (DAMDA) was achieved on the non-hydrolysed urine samples, and that of total MDA on urine samples after alkaline hydrolysis. It was necessary to protect urine samples with sulfamic acid in order to stabilize amines and to improve the precision and accuracy of the analyses. Under these conditions, unstable labile conjugates were determined as their parent stable amine. The distribution of total MDA, MDA, MAMDA and DAMDA was assessed in 116 urine samples. Their relative concentrations (arithmetic means) were found to be in the following order: total MDA > MAMDA > MDA > DAMDA. While MAMDA represented more than 50% of total MDA, MDA and DAMDA were lower than 15% and 3% respectively. Acetylation of MDA, described as a possible pathway of detoxication, is confirmed to be an important metabolization route in humans, essentially through the monoacetylated metabolite. However, the individual ratio MAMDA/total MDA was found to vary widely (roughly from 0% to 100%). PMID:8847112

  14. Expression of Brassica juncea 3-hydroxy-3-methylglutaryl CoA synthase is developmentally regulated and stress-responsive.

    PubMed

    Alex, D; Bach, T J; Chye, M L

    2000-06-01

    3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) is an enzyme in mevalonate biosynthesis. In plants, investigations have focused on HMG CoA reductase (HMGR) and less is known of the preceding enzyme, HMGS. To understand the regulation of HMGS, we have isolated a Brassica juncea cDNA encoding HMGS, BjHMGS1, for use as a hybridization probe in Northern blot analyses. BjHMGS is expressed in all plant organs and shows developmental regulation in flower, seed and seedling, with highest expression in early development. In seedlings, expression is highest in young hypocotyls and is induced during the greening of etiolated cotyledons. BjHMGS is down-regulated by abscisic acid, osmotic stress and dehydration, the effects of which arrested seedling growth. Thus BjHMGS expression shows correlation with rapid cell division and growth, like HMGR. This is not unexpected, as mevalonate is the precursor to many essential isoprenoid compounds, including sterols for membrane biogenesis. Wounding, methyl jasmonate or salicylic acid induce BjHMGS expression, suggesting that, like HMGR, HMGS is involved in defence. As in animals, coordinated regulation of HMGS with HMGR occurred in B. juncea upon germination and in response to salicylic acid. HMGS assays confirmed that Escherichia coli-expressed recombinant BjHMGS1 shows HMGS activity that is inhibited by F244, a specific inhibitor of HMGS. Southern blot analysis revealed gene families encoding HMGS in Brassica species and a summation of homologous genes in the fusion amphidiploid genome of B. juncea, a bi-parental species derived from diploids B. nigra and B. campestris. PMID:10849357

  15. Expression of Brassica juncea 3-hydroxy-3-methylglutaryl CoA synthase is developmentally regulated and stress-responsive.

    TOXLINE Toxicology Bibliographic Information

    Alex D; Bach TJ; Chye ML

    2000-06-01

    3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) is an enzyme in mevalonate biosynthesis. In plants, investigations have focused on HMG CoA reductase (HMGR) and less is known of the preceding enzyme, HMGS. To understand the regulation of HMGS, we have isolated a Brassica juncea cDNA encoding HMGS, BjHMGS1, for use as a hybridization probe in Northern blot analyses. BjHMGS is expressed in all plant organs and shows developmental regulation in flower, seed and seedling, with highest expression in early development. In seedlings, expression is highest in young hypocotyls and is induced during the greening of etiolated cotyledons. BjHMGS is down-regulated by abscisic acid, osmotic stress and dehydration, the effects of which arrested seedling growth. Thus BjHMGS expression shows correlation with rapid cell division and growth, like HMGR. This is not unexpected, as mevalonate is the precursor to many essential isoprenoid compounds, including sterols for membrane biogenesis. Wounding, methyl jasmonate or salicylic acid induce BjHMGS expression, suggesting that, like HMGR, HMGS is involved in defence. As in animals, coordinated regulation of HMGS with HMGR occurred in B. juncea upon germination and in response to salicylic acid. HMGS assays confirmed that Escherichia coli-expressed recombinant BjHMGS1 shows HMGS activity that is inhibited by F244, a specific inhibitor of HMGS. Southern blot analysis revealed gene families encoding HMGS in Brassica species and a summation of homologous genes in the fusion amphidiploid genome of B. juncea, a bi-parental species derived from diploids B. nigra and B. campestris.

  16. Identification and preliminary characterization of AcsF, a putative Ni-insertase used in the biosynthesis of acetyl-CoA synthase from Clostridium thermoaceticum.

    PubMed

    Loke, Huay-Keng; Lindahl, Paul A

    2003-01-01

    The acsABCDE genes in the Clostridium thermoaceticum genome are used for autotrophic acetyl-CoA synthesis using the Wood-Ljungdahl pathway. A 2.8-kb region between acsC and acsD was cloned and sequenced. Two open reading frames, orf7 (approximately 1.9 kb) and acsF (approximately 0.7 kb) were identified. orf7 appears to encode an Fe-S protein, in that it contains five conserved cysteine residues, three of which are present in a motif (CGGXXXCGXC) commonly used to coordinate Fe-S clusters. However, Orf7 is probably not involved in autotrophic acetyl-CoA synthesis, as homologous genes are present in organisms that do not utilize this pathway and are absent in many that do. In contrast, acsF is probably involved in this pathway. Sequence alignment of AcsF and eleven homologs reveals a number of conserved regions, including a P-loop that binds nucleoside triphosphates and catalyzes their hydrolysis. One homolog is CooC, an ATPase/GTPase that inserts Ni into a precursor form of the C-cluster of the carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum. Purified AcsF lacked Ni and Fe, and slowly catalyzed the hydrolysis of ATP. Such similarities to CooC suggest that AcsF may function to insert Ni into a Ni-deficient form of the bifunctional acetyl-CoA synthase/CODH from C. thermoaceticum (ACS(Ct)). However, this could not be established, as expression of acsF did not effect activation of recombinant AcsAB expressed in E. coli. Also, E. coli cells defective in hypB retained the ability to synthesize active recombinant AcsAB. Rather, the concentration of extracellular Ni(2+) ions was critical to activation. PMID:12538050

  17. Identification and preliminary characterization of acsF, a Putative Ni-insertase used in the biosynthesis of acetyl-CoA synthase from Clostridium thermoaceticum

    SciTech Connect

    Huay-Keng Loke; Paul A. Lindahl

    2003-01-01

    OAK-B135 The acsABCDE genes in the Clostridium thermoaceticum genome are used for autotrophic acetyl-CoA synthesis using the Wood/Ljungdahl pathway. A 2.8 kb region between acsC and acsD was cloned and sequenced. Two open reading frames, orf7 ({approx} 1.9 kb) and acsF ({approx} 0.7 kb) were identified. orf7 appears to encode an Fe-S protein, in that it contains 5 conserved cysteine residues, 3 of which are present in a motif (CXXXXXCXXC) commonly used to coordinate Fe-S clusters. However, Orf7 is probably not involved in autotrophic acetyl-CoA synthesis, as homologous genes are present in organisms that do not utilize this pathway and are absent in many that do. In contrast, acsF is probably involved in this pathway. Sequence alignment of AcsF and 11 homologs reveals a number of conserved regions, including a P-loop that binds nucleoside triphosphates and catalyzes their hydrolysis. One homolog is CooC, an ATPase/GTPase that inserts Ni into a precursor form of the C-cluster of the carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum. Purified AcsF lacked Ni and Fe, and slowly catalyzed the hydrolysis of ATP. Such similarities to CooC suggest that AcsF may function to insert Ni into a Ni-deficient form of the bifunctional acetyl-CoA synthase/CODH from C. thermoaceticum (ACSCt). However, this could not be established, as expression of acsF did not effect activation of recombinant AcsAB expressed in E. coli. Also, E. coli cells defective in hypB retained the ability to synthesize active recombinant AcsAB. Rather, the concentration of extracellular Ni2+ ions was critical to activation.

  18. Acid-functionalized nanoparticles for biomass hydrolysis

    NASA Astrophysics Data System (ADS)

    Pena Duque, Leidy Eugenia

    Cellulosic ethanol is a renewable source of energy. Lignocellulosic biomass is a complex material composed mainly of cellulose, hemicellulose, and lignin. Biomass pretreatment is a required step to make sugar polymers liable to hydrolysis. Mineral acids are commonly used for biomass pretreatment. Using acid catalysts that can be recovered and reused could make the process economically more attractive. The overall goal of this dissertation is the development of a recyclable nanocatalyst for the hydrolysis of biomass sugars. Cobalt iron oxide nanoparticles (CoFe2O4) were synthesized to provide a magnetic core that could be separated from reaction using a magnetic field and modified to carry acid functional groups. X-ray diffraction (XRD) confirmed the crystal structure was that of cobalt spinel ferrite. CoFe2O4 were covered with silica which served as linker for the acid functions. Silica-coated nanoparticles were functionalized with three different acid functions: perfluoropropyl-sulfonic acid, carboxylic acid, and propyl-sulfonic acid. Transmission electron microscope (TEM) images were analyzed to obtain particle size distributions of the nanoparticles. Total carbon, nitrogen, and sulfur were quantified using an elemental analyzer. Fourier transform infra-red spectra confirmed the presence of sulfonic and carboxylic acid functions and ion-exchange titrations accounted for the total amount of catalytic acid sites per nanoparticle mass. These nanoparticles were evaluated for their performance to hydrolyze the beta-1,4 glycosidic bond of the cellobiose molecule. Propyl-sulfonic (PS) and perfluoropropyl-sulfonic (PFS) acid functionalized nanoparticles catalyzed the hydrolysis of cellobiose significantly better than the control. PS and PFS were also evaluated for their capacity to solubilize wheat straw hemicelluloses and performed better than the control. Although PFS nanoparticles were stronger acid catalysts, the acid functions leached out of the nanoparticle during the catalytic reactions. PS nanoparticles were further evaluated for the pretreatment of corn stover in order to increase digestibility of the biomass. The pretreatment was carried out at three different catalyst load and temperature levels. At 180C, the total glucose yield was linearly correlated to the catalyst load. A maximum glucose yield of 90% and 58% of the hemicellulose sugars were obtained at this temperature.

  19. RNAi knockdown of acetyl-CoA carboxylase gene eliminates jinggangmycin-enhanced reproduction and population growth in the brown planthopper, Nilaparvata lugens.

    PubMed

    Zhang, Yi-Xin; Ge, Lin-Quan; Jiang, Yi-Ping; Lu, Xiu-Li; Li, Xin; Stanley, David; Song, Qi-Sheng; Wu, Jin-Cai

    2015-01-01

    A major challenge in ecology lies in understanding the coexistence of intraguild species, well documented at the organismal level, but not at the molecular level. This study focused on the effects of the antibiotic, jinggangmycin (JGM), a fungicide widely used in Asian rice agroecosystems, on reproduction of insects within the planthopper guild, including the brown planthopper (BPH) Nilaparvata lugens and the white-backed planthopper (WBPH) Sogatella furcifera, both serious resurgence rice pests. JGM exposure significantly increased BPH fecundity and population growth, but suppressed both parameters in laboratory and field WBPH populations. We used digital gene expression and transcriptomic analyses to identify a panel of differentially expressed genes, including a set of up-regulated genes in JGM-treated BPH, which were down-regulated in JGM-treated WBPH. RNAi silencing of Acetyl Co-A carboxylase (ACC), highly expressed in JGM-treated BPH, reduced ACC expression (by >?60%) and eliminated JGM-induced fecundity increases in BPH. These findings support our hypothesis that differences in ACC expression separates intraguild species at the molecular level. PMID:26482193

  20. RNAi knockdown of acetyl-CoA carboxylase gene eliminates jinggangmycin-enhanced reproduction and population growth in the brown planthopper, Nilaparvata lugens

    PubMed Central

    Zhang, Yi-Xin; Ge, Lin-Quan; Jiang, Yi-Ping; Lu, Xiu-Li; Li, Xin; Stanley, David; Song, Qi-Sheng; Wu, Jin-Cai

    2015-01-01

    A major challenge in ecology lies in understanding the coexistence of intraguild species, well documented at the organismal level, but not at the molecular level. This study focused on the effects of the antibiotic, jinggangmycin (JGM), a fungicide widely used in Asian rice agroecosystems, on reproduction of insects within the planthopper guild, including the brown planthopper (BPH) Nilaparvata lugens and the white-backed planthopper (WBPH) Sogatella furcifera, both serious resurgence rice pests. JGM exposure significantly increased BPH fecundity and population growth, but suppressed both parameters in laboratory and field WBPH populations. We used digital gene expression and transcriptomic analyses to identify a panel of differentially expressed genes, including a set of up-regulated genes in JGM-treated BPH, which were down-regulated in JGM-treated WBPH. RNAi silencing of Acetyl Co-A carboxylase (ACC), highly expressed in JGM-treated BPH, reduced ACC expression (by > 60%) and eliminated JGM-induced fecundity increases in BPH. These findings support our hypothesis that differences in ACC expression separates intraguild species at the molecular level. PMID:26482193

  1. Multiple mass isotopomer tracing of acetyl-CoA metabolism in Langendorff-perfused rat hearts: channeling of acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase.

    PubMed

    Li, Qingling; Deng, Shuang; Ibarra, Rafael A; Anderson, Vernon E; Brunengraber, Henri; Zhang, Guo-Fang

    2015-03-27

    We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (?citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [(13)C2,(2)H3]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [(13)C6]glucose or [1,2-(13)C2]palmitate) or/and M1 acetyl-CoA (e.g. [1-(13)C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose palmitate insulin dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA. PMID:25645937

  2. The influence of the primary and secondary xanthan structure on the enzymatic hydrolysis of the xanthan backbone.

    PubMed

    Kool, Marijn M; Schols, Henk A; Delahaije, Roy J B M; Sworn, Graham; Wierenga, Peter A; Gruppen, Harry

    2013-09-12

    Differently modified xanthans, varying in degree of acetylation and/or pyruvylation were incubated with the experimental cellulase mixture C1-G1 from Myceliophthora thermophila C1. The ionic strength and/or temperature of the xanthan solutions were varied, to obtain different xanthan conformations. The exact conformation at the selected incubation conditions was determined by circular dichroism. The xanthan degradation was analyzed by size exclusion chromatography. It was shown that at a fixed xanthan conformation, the backbone degradation by cellulases is equal for each type of xanthan. Complete backbone degradation is only obtained at a fully disordered conformation, indicating that only the secondary xanthan structure influences the final degree of hydrolysis by cellulases. It is thereby shown that, independently on the degree of substitution, xanthan can be completely hydrolyzed to oligosaccharides. These oligosaccharides can be used to further investigate the primary structure of different xanthans and to correlate the molecular structure to the xanthan functionalities. PMID:23911459

  3. H4K44 Acetylation Facilitates Chromatin Accessibility during Meiosis.

    PubMed

    Hu, Jialei; Donahue, Greg; Dorsey, Jean; Govin, Jrme; Yuan, Zuofei; Garcia, Benjamin A; Shah, Parisha P; Berger, Shelley L

    2015-12-01

    Meiotic recombination hotspots are associated with histone post-translational modifications and open chromatin. However, it remains unclear how histonemodifications and chromatin structure regulate meiotic recombination. Here, we identify acetylation of histone H4 at Lys44 (H4K44ac) occurring on the nucleosomal lateral surface. We show that H4K44 is acetylated at pre-meiosis and meiosis and displays genome-wide enrichment at recombination hotspots in meiosis. Acetylation at H4K44 is required for normal meiotic recombination, normal levels of double-strand breaks (DSBs) during meiosis, and optimal sporulation. Non-modifiable H4K44R results in increased nucleosomal occupancy around DSB hotspots. Our results indicate that H4K44ac functions to facilitate chromatin accessibility favorable for normal DSB formation and meiotic recombination. PMID:26628362

  4. An acetylation rheostat for the control of muscle energy homeostasis.

    PubMed

    Menzies, Keir; Auwerx, Johan

    2013-12-01

    In recent years, the role of acetylation has gained ground as an essential modulator of intermediary metabolism in skeletal muscle. Imbalance in energy homeostasis or chronic cellular stress, due to diet, aging, or disease, translate into alterations in the acetylation levels of key proteins which govern bioenergetics, cellular substrate use, and/or changes in mitochondrial content and function. For example, cellular stress induced by exercise or caloric restriction can alter the coordinated activity of acetyltransferases and deacetylases to increase mitochondrial biogenesis and function in order to adapt to low energetic levels. The natural duality of these enzymes, as metabolic sensors and effector proteins, has helped biologists to understand how the body can integrate seemingly distinct signaling pathways to control mitochondrial biogenesis, insulin sensitivity, glucose transport, reactive oxygen species handling, angiogenesis, and muscle satellite cell proliferation/differentiation. Our review will summarize the recent developments related to acetylation-dependent responses following metabolic stress in skeletal muscle. PMID:23999889

  5. An acetylation rheostat for the control of muscle energy homeostasis

    PubMed Central

    Menzies, Keir; Auwerx, Johan

    2013-01-01

    In recent years the role of acetylation has gained ground as an essential modulator of intermediary metabolism in skeletal muscle. Imbalance in energy homeostasis or chronic cellular stress, due to diet, aging or disease, translate into alterations in the acetylation levels of key proteins which governs bioenergetics, cellular substrate use and/or changes in mitochondrial content and function. For example, cellular stress induced by exercise or caloric restriction can alter the coordinated activity of acetyltransferases and deacetylases to increase mitochondrial biogenesis and function in order to adapt to low energetic levels. The natural duality of these enzymes, as metabolic sensors and effector proteins, have helped biologists understand how the body can integrate seemingly distinct signaling pathways to control mitochondrial biogenesis, insulin sensitivity, glucose transport, reactive oxygen species handling, angiogenesis and muscle satellite cell proliferation/differentiation. Our review will summarize the recent developments related to acetylation dependent responses following metabolic stress in skeletal muscle. PMID:23999889

  6. Exploring the accessible conformations of N-terminal acetylated ?-synuclein

    PubMed Central

    Moriarty, Gina M.; Janowska, Maria K.; Kang, Lijuan; Baum, Jean

    2014-01-01

    Alpha synuclein (?syn) fibrils are found in the Lewy Bodies of patients with Parkinsons disease (PD). The aggregation of the ?syn monomer to soluble oligomers and insoluble fibril aggregates is believed to be one of the causes of PD. Recently, the view of the native state of ?syn as a monomeric ensemble was challenged by a report suggesting that ?syn exists in its native state as a helical tetramer. This review reports on our current understanding of ?syn within the context of these recent developments and describes the work performed by a number of groups to address the monomer/tetramer debate. A number of in depth studies have subsequently shown that both non-acetylated and acetylated ?syn purified under mild conditions are primarily monomer. A description of the accessible states of acetylated ?syn monomer and the ability of ?syn to self-associate is explored. PMID:23499431

  7. Synthetic Biology for Engineering Acetyl Coenzyme A Metabolism in Yeast

    PubMed Central

    2014-01-01

    ABSTRACT The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels, chemicals, and pharmaceuticals. The use of this cell factory for cost-efficient production of novel fuels and chemicals requires high yields and low by-product production. Many industrially interesting chemicals are biosynthesized from acetyl coenzyme A (acetyl-CoA), which serves as a central precursor metabolite in yeast. To ensure high yields in production of these chemicals, it is necessary to engineer the central carbon metabolism so that ethanol production is minimized (or eliminated) and acetyl-CoA can be formed from glucose in high yield. Here the perspective of generating yeast platform strains that have such properties is discussed in the context of a major breakthrough with expression of a functional pyruvate dehydrogenase complex in the cytosol. PMID:25370498

  8. Dynamic changes in histone acetylation regulate origins of DNA replication

    PubMed Central

    Unnikrishnan, Ashwin; Gafken, Philip R.; Tsukiyama, Toshio

    2011-01-01

    While histone modifications have been implicated in many DNA-dependent processes, their precise role in DNA replication remains largely unknown. Here, we describe a very efficient, single-step method to specifically purify histones located around an origin of replication from S. cerevisiae. Using high-resolution mass spectrometry, we have obtained a comprehensive view of the histone modifications surrounding the origin of replication throughout the cell cycle. We have discovered that histone H3 and H4 acetylation is dynamically regulated around an origin of replication, at the level of multiply-acetylated histones. Furthermore, we find that this acetylation is required for efficient origin activation during S-phase. PMID:20228802

  9. Pretreatment and enzymatic hydrolysis of corn fiber

    SciTech Connect

    Grohmann, K.; Bothast, R.J.

    1996-10-01

    Corn fiber is a co-product of the corn wet milling industry which is usually marketed as a low value animal feed ingredient. Approximately 1.2 x 10{sup 6} dry tons of this material are produced annually in the United States. The fiber is composed of kernel cell wall fractions and a residual starch which can all be potentially hydrolyzed to a mixture of glucose, xylose, arabinose and galactose. We have investigated a sequential saccharification of polysaccharides in corn fiber by a treatment with dilute sulfuric acid at 100 to 160{degrees}C followed by partial neutralization and enzymatic hydrolysis with mixed cellulose and amyloglucosidase enzymes at 45{degrees}C. The sequential treatment achieved a high (approximately 85%) conversion of all polysaccharides in the corn fiber to monomeric sugars, which were in most cases fermentable to ethanol by the recombinant bacterium Escherichia coli KOll.

  10. Kinetics of the enzymatic hydrolysis of cellulose

    SciTech Connect

    Wald, S.; Wilke, C.R.; Blanch, H.W.

    1984-01-01

    Enzymatic hydrolysis of cellulose for sugar production offers advantages of higher conversion, minimal by-product formation, low energy requirements, and mild operating conditions over other chemical conversions. The development of a kinetic model, based on observable, macroscopic properties of the overall system, is helpful in design and economic evaluation of processes for sugar conversion and ethanol production. A kinetic model is presented, incorporating enzyme adsorption, product inhibition, and considers a multiple enzyme and substrate system. This model was capable of simulating saccharification of a lignocellulosic material, rice straw, at high substrate (up to 333 g/L) and enzyme concentrations (up to 9.2 FPU/mL) that are common to proposed process designs. (Refs. 37).

  11. Investigation of the acetylation mechanism by GCN5 histone acetyltransferase.

    PubMed

    Jiang, Junfeng; Lu, Junyan; Lu, Dan; Liang, Zhongjie; Li, Lianchun; Ouyang, Sisheng; Kong, Xiangqian; Jiang, Hualiang; Shen, Bairong; Luo, Cheng

    2012-01-01

    The histone acetylation of post-translational modification can be highly dynamic and play a crucial role in regulating cellular proliferation, survival, differentiation and motility. Of the enzymes that mediate post-translation modifications, the GCN5 of the histone acetyltransferase (HAT) proteins family that add acetyl groups to target lysine residues within histones, has been most extensively studied. According to the mechanism studies of GCN5 related proteins, two key processes, deprotonation and acetylation, must be involved. However, as a fundamental issue, the structure of hGCN5/AcCoA/pH3 remains elusive. Although biological experiments have proved that GCN5 mediates the acetylation process through the sequential mechanism pathway, a dynamic view of the catalytic process and the molecular basis for hGCN5/AcCoA/pH3 are still not available and none of theoretical studies has been reported to other related enzymes in HAT family. To explore the molecular basis for the catalytic mechanism, computational approaches including molecular modeling, molecular dynamic (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) simulation were carried out. The initial hGCN5/AcCoA/pH3 complex structure was modeled and a reasonable snapshot was extracted from the trajectory of a 20 ns MD simulation, with considering post-MD analysis and reported experimental results. Those residues playing crucial roles in binding affinity and acetylation reaction were comprehensively investigated. It demonstrated Glu80 acted as the general base for deprotonation of Lys171 from H3. Furthermore, the two-dimensional QM/MM potential energy surface was employed to study the sequential pathway acetylation mechanism. Energy barriers of addition-elimination reaction in acetylation obtained from QM/MM calculation indicated the point of the intermediate ternary complex. Our study may provide insights into the detailed mechanism for acetylation reaction of GCN5, and has important implications for the discovery of regulators against GCN5 enzymes and related HAT family enzymes. PMID:22574209

  12. Interaction of RNA polymerase II with acetylated nucleosomal core particles

    SciTech Connect

    Pineiro, M.; Gonzalez, P.J.; Hernandez, F.; Palacian, E. )

    1991-05-31

    Chemical acetylation of nucleosomal cores is accompanied by an increase in their efficiency as in vitro transcription templates. Low amounts of acetic anhydride cause preferential modification of the amino-terminal tails of core histones. Modification of these domains, which causes moderate structural effects, is apparently correlated with the observed stimulation of RNA synthesis. In contrast, extensive modification of the globular regions of core histones, which is accompanied by a large structural relaxation of the particle, causes little additional effect on transcription. Acetylation of the amino-terminal domains of histones might stimulate transcription by changing the interaction of the histone tails with components of the transcriptional machinery.

  13. Class Projects in Physical Organic Chemistry: The Hydrolysis of Aspirin

    ERIC Educational Resources Information Center

    Marrs, Peter S.

    2004-01-01

    An exercise that provides a hands-on demonstration of the hydrolysis of aspirin is presented. The key to understanding the hydrolysis is recognizing that all six process may occur simultaneously and that the observed rate constant is the sum of the rate constants that one rate constant dominates the overall process.

  14. Continuous acid hydrolysis of waste cellulose for ethanol production

    SciTech Connect

    Rugg, B.; Brenner, W.

    1980-01-01

    The continuous dilute acid hydrolysis of waste cellulose materials (paper pulp and wood sawdust at 10 and 95% solids, respectively) to glucose in a twin screw reactor gave high conversion yields and good energy efficiencies. A scheme for a scaled-up acid hydrolysis-fermentation distillation facility based on a waste cellulose feedstock to produce fuel-grade ethanol is proposed.

  15. Enzymatic hydrolysis of steryl ferulates and steryl glycosides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Steryl ferulates and steryl glycosides are phytosterol conjugates found characteristically in cereals. Their properties in enzymatic hydrolysis are, however, not yet well known. Steryl ferulates and steryl glycosides were extracted and purified from rye and wheat bran. Their rates of hydrolysis with...

  16. The hydrolysis of phosphate diesters in cyclohexane and acetone

    PubMed Central

    Stockbridge, Randy B.

    2010-01-01

    The hydrolysis of phosphate diesters is one of the most difficult reactions known. Here we show that in acetone or cyclohexane, at 25 C, phosphodiesters undergo hydrolysis 5 105 and 2 109-fold more rapidly than in water, respectively, and that this rate enhancement is achieved by lowering the enthalpy of activation. PMID:20448876

  17. Ultrasound Enhancement of Enzymatic Hydrolysis of Cellulose Plant Matter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The work reported here is based on acceleration of enzymatic hydrolysis of plant biomass substrate by introduction of low intensity, uniform ultrasound field into a reaction chamber (bio-reactor). This method may serve as improvement of rates in the hydrolysis of cellulosic materials to sugars, whi...

  18. Simultaneous determination of hydrolysis and mutarotation rates during the enzymatic hydrolysis of lactose.

    PubMed

    Jenkins, Daniel M; Teruel, Michael A; Reyes-de-Corcuera, Jos I; Young, Owen

    2008-09-24

    An experiment is described in which a custom-made glucose electrode is used to directly monitor the enzymatic hydrolysis of lactose to glucose. The transient profile of beta- d-glucose can be used to simultaneously determine the rate constants for mutarotation and for enzymatic hydrolysis by applying a dynamic nonlinear regression routine. Due to differences in the mutarotation rate constants between lactose and glucose, the beta- d-glucose concentration "overshoots" equilibrium under certain conditions, which can be modeled mathematically. This overshoot can be observed reliably and used to quantify the differences in mutarotational equilibria between glucose and lactose. These observations may be important for the analysis of dairy products and commercial lactase preparations and illustrate an unusual kinetic phenomenon caused by intramolecular forces. This approach may also be important for the accurate determination of a variety of oligosaccharides such as glycogen, which tend to be composed primarily of one stereoisomer. PMID:18712880

  19. Trihalomethane hydrolysis in drinking water at elevated temperatures.

    PubMed

    Zhang, Xiao-Lu; Yang, Hong-Wei; Wang, Xiao-Mao; Karanfil, Tanju; Xie, Yuefeng F

    2015-07-01

    Hydrolysis could contribute to the loss of trihalomethanes (THMs) in the drinking water at elevated temperatures. This study was aimed at investigating THM hydrolysis pertaining to the storage of hot boiled water in enclosed containers. The water pH value was in the range of 6.1-8.2 and the water temperature was varied from 65 to 95 °C. The effects of halide ions, natural organic matter, and drinking water matrix were investigated. Results showed that the hydrolysis rates declined in the order following CHBrCl2 > CHBr2Cl > CHBr3 > CHCl3. THM hydrolysis was primarily through the alkaline pathway, except for CHCl3 in water at relatively low pH value. The activation energies for the alkaline hydrolysis of CHCl3, CHBrCl2, CHBr2Cl and CHBr3 were 109, 113, 115 and 116 kJ/mol, respectively. No hydrolysis intermediates could accumulate in the water. The natural organic matter, and probably other constituents, in drinking water could substantially decrease THM hydrolysis rates by more than 50%. When a drinking water was at 90 °C or above, the first order rate constants for THM hydrolysis were in the magnitude of 10(-2)‒10(-1) 1/h. When the boiled real tap water was stored in an enclosed container, THMs continued increasing during the first few hours and then kept decreasing later on due to the competition between hydrolysis and further formation. The removal of THMs, especially brominated THMs, by hydrolysis would greatly reduce one's exposure to disinfection by-products by consuming the boiled water stored in enclosed containers. PMID:25898249

  20. CRP Is an Activator of Yersinia pestis Biofilm Formation that Operates via a Mechanism Involving gmhA and waaAE-coaD

    PubMed Central

    Liu, Lei; Fang, Haihong; Yang, Huiying; Zhang, Yiquan; Han, Yanping; Zhou, Dongsheng; Yang, Ruifu

    2016-01-01

    gmhA encodes a phosphoheptose isomerase that catalyzes the biosynthesis of heptose, a conserved component of lipopolysaccharide (LPS). GmhA plays an important role in Yersinia pestis biofilm blockage in the flea gut. waaA, waaE, and coaD constitute a three-gene operon waaAE-coaD in Y. pestis. waaA encodes a transferase that is responsible for binding lipid-A to the core oligosaccharide of LPS. WaaA is a key determinant in Y. pestis biofilm formation, and the waaA expression is positively regulated by the two-component regulatory system PhoP/PhoQ. WaaE is involved in LPS modification and is necessary for Y. pestis biofilm production. In this study, the biofilm-related phenotypic assays indicate that the global regulator CRP stimulates Y. pestis biofilm formation in vitro and on nematodes, while it has no regulatory effect on the biosynthesis of the biofilm-signaling molecular 3′,5′-cyclic diguanosine monophosphate. Further gene regulation experiments disclose that CRP does not regulate the hms genes at the transcriptional level but directly promotes the gmhA transcription and indirectly activates the waaAE-coaD transcription through directly acting on phoPQ-YPO1632. Thus, it is speculated that CRP-mediated carbon catabolite regulation of Y. pestis biofilm formation depends on the CRP-dependent carbon source metabolic pathways of the biosynthesis, modification, and transportation of biofilm exopolysaccharide. PMID:27014218

  1. Effect of Genistein and L-Carnitine and Their Combination on Gene Expression of Hepatocyte HMG-COA Reductase and LDL Receptor in Experimental Nephrotic Syndrome

    PubMed Central

    YOUSEFINEJAD, Abbas; SIASSI, Fereydoon; MIRSHAFIEY, Abbas; ESHRAGHIAN, Mohammad-Reza; KOOHDANI, Fariba; JAVANBAKHT, Mohammad Hassan; SEDAGHAT, Reza; RAMEZANI, Atena; ZAREI, Mahnaz; DJALALI, Mahmoud

    2015-01-01

    Background: Nephrotic syndrome is a disorder that leads to hyperlipidemia. L-carnitine and genistein can effect on lipid metabolism and the syndrome. In the present study, we have delved into the separate and the twin-effects of L-carnitine and genistein on the gene expressions of HMG-COA reductase and LDL receptor in experimental nephrotic syndrome. Methods: In this controlled experimental study, 50 male Sprague–Dawley rats were randomly divided into five groups: NC (normal-control), PC (patient-control), LC (L-carnitine), G (genistein), LCG (L-carnitine-genistein). Adriamycin was used for inducing nephrotic syndrome and the spot urine samples and urine protein-to-creatinine ratio were measured. Hepatocytic RNA was extracted and real-time PCR was used for HMG-COA Reductase and LDL receptor gene Expression measurement. Results: The final weight of the patients groups were lower than the NC group (P=0.001), and weight gain of the NC group was higher than the other groups (P<0.001). The proteinuria and urine protein-to-creatinine ratio showed significant differences between PC group and LC, G and LCG groups at week 7 (P<0.001). The expression of HMGCOA Reductase mRNA down regulated in LC, G and LCG groups in comparison with PC group (P<0.001). ΔCT of LDLr mRNA showed significant differences between the PC group and the other patient groups (P<0.001). Conclusion: This study shows a significant decreasing (P<0.001) and non-significant increasing trend in HMG-COA Reductase and LDLr gene expression, respectively, and synergistic effect of L-carnitine and genistein on these genes in experimental nephrotic syndrome. PMID:26576346

  2. A colorimetric assay for the determination of acetyl xylan esterase or cephalosporin C acetyl esterase activities using 7-amino cephalosporanic acid, cephalosporin C, or acetylated xylan as substrate.

    PubMed

    Martnez-Martnez, Irene; Montoro-Garca, Silvia; Lozada-Ramrez, Jos Daniel; Snchez-Ferrer, Alvaro; Garca-Carmona, Francisco

    2007-10-15

    A bromothymol blue-based colorimetric assay has been devised to screen for acetyl xylan esterase or cephalosporin C (CPC) deacetylase activities using 7-amino cephalosporanic acid (7-ACA), CPC, or acetylated xylan as substrate. These enzymes are not screened with their natural substrates because of the tedious procedures available previously. Acetyl xylan esterase from Bacillus pumilus CECT 5072 was cloned, expressed in Escherichia coli Rosetta (DE3), and characterized using this assay. Similar K(M) values for 7-ACA and CPC were obtained when compared with those described using HPLC methods. The assay is easy to perform and can be carried out in robotic high-throughput colorimetric devices normally used in directed evolution experiments. The assay allowed us to detect improvements in activity at a minimum of twofold with a very low coefficient of variance in 96-well plates. This method is significantly faster and more convenient to use than are known HPLC and pH-stat procedures. PMID:17651681

  3. Carnitine Acetyltransferase: Candidate for the Transfer of Acetyl Groups Through the Mitochondrial Membrane of Yeast1

    PubMed Central

    Kohlhaw, Gunter B.; Tan-Wilson, Anna

    1977-01-01

    On the basis of its specific activity and its affinity for acetyl-coenzyme A, carnitine acetyltransferase appears to be the most likely candidate for acetyl group transfer out of yeast mitochondria. PMID:320182

  4. COA User's Guide

    SciTech Connect

    Fox, B.; Pautz, J.; Sellers, C.

    1999-01-28

    The Department of Energy (DOE) has one of the largest and most complete collections of information on crude oil composition that is available to the public. The computer program that manages this database of crude oil analyses has recently been rewritten to allow easier access to this information. This report describes how the new system can be accessed and how the information contained in the Crude Oil Analysis Data Bank can be obtained.

  5. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in...

  6. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...-Acetyl-L-methionine (Chemical Abstracts Service Registry No. 65-82-7) is the derivative of the amino acid... provide a total of 3.1 percent L- and DL-methionine (expressed as the free amino acid) by weight of the... contained therein. (2) The amounts of additive and each amino acid contained in any mixture. (3)...

  7. Tubulin acetylation: responsible enzymes, biological functions and human diseases.

    PubMed

    Li, Lin; Yang, Xiang-Jiao

    2015-11-01

    Microtubules have important functions ranging from maintenance of cell morphology to subcellular transport, cellular signaling, cell migration, and formation of cell polarity. At the organismal level, microtubules are crucial for various biological processes, such as viral entry, inflammation, immunity, learning and memory in mammals. Microtubules are subject to various covalent modifications. One such modification is tubulin acetylation, which is associated with stable microtubules and conserved from protists to humans. In the past three decades, this reversible modification has been studied extensively. In mammals, its level is mainly governed by opposing actions of α-tubulin acetyltransferase 1 (ATAT1) and histone deacetylase 6 (HDAC6). Knockout studies of the mouse enzymes have yielded new insights into biological functions of tubulin acetylation. Abnormal levels of this modification are linked to neurological disorders, cancer, heart diseases and other pathological conditions, thereby yielding important therapeutic implications. This review summarizes related studies and concludes that tubulin acetylation is important for regulating microtubule architecture and maintaining microtubule integrity. Together with detyrosination, glutamylation and other modifications, tubulin acetylation may form a unique 'language' to regulate microtubule structure and function. PMID:26227334

  8. Mass spectrometry-based detection of protein acetylation

    PubMed Central

    Li, Yu; Silva, Jeffrey C.; Skinner, Mary E.; Lombard, David B.

    2014-01-01

    Summary Improved sample preparation techniques and increasingly sensitive mass spectrometry (MS) analysis have revolutionized the study of protein post-translational modifications (PTMs). Here, we describe a general approach for immunopurification and MS-based identification of acetylated proteins in biological samples. This approach is useful characterizing changes in the acetylome in response to biological interventions (1). PMID:24014401

  9. The Tale of Protein Lysine Acetylation in the Cytoplasm

    PubMed Central

    Sadoul, Karin; Wang, Jin; Diagouraga, Boubou; Khochbin, Saadi

    2011-01-01

    Reversible posttranslational modification of internal lysines in many cellular or viral proteins is now emerging as part of critical signalling processes controlling a variety of cellular functions beyond chromatin and transcription. This paper aims at demonstrating the role of lysine acetylation in the cytoplasm driving and coordinating key events such as cytoskeleton dynamics, intracellular trafficking, vesicle fusion, metabolism, and stress response. PMID:21151618

  10. SCANDIUM TRIFLATE CATALYZED ACETYLATION OF STARCH UNDER MILD CONDITIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Scandium (III) trifluoromethan sulfonate (Sc(OTf)3) was investigated as a catalyst for the acetylation of starch in order to determine the potential for preparing new types of starch esters under mild conditions. At room temperature, dry granular corn starch reacts with acetic anhydride in the pres...

  11. Genetic Control of Differential Acetylation in Diabetic Rats

    PubMed Central

    Kaisaki, Pamela J.; Otto, Georg W.; McGouran, Joanna F.; Toubal, Amine; Argoud, Karne; Waller-Evans, Helen; Finlay, Clare; Caldrari, Sophie; Bihoreau, Marie-Thrse; Kessler, Benedikt M.; Gauguier, Dominique; Mott, Richard

    2014-01-01

    Post-translational protein modifications such as acetylation have significant regulatory roles in metabolic processes, but their relationship to both variation in gene expression and DNA sequence is unclear. We address this question in the Goto-Kakizaki (GK) rat inbred strain, a model of polygenic type 2 diabetes. Expression of the NAD-dependent deacetylase Sirtuin-3 is down-regulated in GK rats compared to normoglycemic Brown Norway (BN) rats. We show first that a promoter SNP causes down-regulation of Sirtuin-3 expression in GK rats. We then use mass-spectrometry to identify proteome-wide differential lysine acetylation of putative Sirtuin-3 protein targets in livers of GK and BN rats. These include many proteins in pathways connected to diabetes and metabolic syndrome. We finally sequence GK and BN liver transcriptomes and find that mRNA expression of these targets does not differ significantly between GK and BN rats, in contrast to other components of the same pathways. We conclude that physiological differences between GK and BN rats are mediated by a combination of differential protein acetylation and gene transcription and that genetic variation can modulate acetylation independently of expression. PMID:24743600

  12. Synthesis of tri-O-acetyl-D-allal from levoglucosenone.

    PubMed

    Giordano, Enrique D V; Frinchaboy, Agustina; Suárez, Alejandra G; Spanevello, Rolando A

    2012-09-01

    Tri-O-acetyl-D-allal has been enantiospecifically synthesized in six steps from levoglucosenone in 55% overall yield. A key step in the synthesis is the anhydro bridge ring-opening with concomitant formation of a 1,3-oxathiolane-2-thione ring. PMID:22920651

  13. Acetylation mediates Cx43 reduction caused by electrical stimulation.

    PubMed

    Meraviglia, Viviana; Azzimato, Valerio; Colussi, Claudia; Florio, Maria Cristina; Binda, Anna; Panariti, Alice; Qanud, Khaled; Suffredini, Silvia; Gennaccaro, Laura; Miragoli, Michele; Barbuti, Andrea; Lampe, Paul D; Gaetano, Carlo; Pramstaller, Peter P; Capogrossi, Maurizio C; Recchia, Fabio A; Pompilio, Giulio; Rivolta, Ilaria; Rossini, Alessandra

    2015-10-01

    Communication between cardiomyocytes depends upon gap junctions (GJ). Previous studies have demonstrated that electrical stimulation induces GJ remodeling and modifies histone acetylase (HAT) and deacetylase (HDAC) activities, although these two results have not been linked. The aim of this work was to establish whether electrical stimulation modulates GJ-mediated cardiac cell-cell communication by acetylation-dependent mechanisms. Field stimulation of HL-1 cardiomyocytes at 0.5 Hz for 24 h significantly reduced connexin43 (Cx43) expression and cell-cell communication. HDAC activity was down-regulated whereas HAT activity was not modified resulting in increased acetylation of Cx43. Consistent with a post-translational mechanism, we did not observe a reduction in Cx43 mRNA in electrically stimulated cells, while the proteasomal inhibitor MG132 maintained Cx43 expression. Further, the treatment of paced cells with the HAT inhibitor Anacardic Acid maintained both the levels of Cx43 and cell-cell communication. Finally, we observed increased acetylation of Cx43 in the left ventricles of dogs subjected to chronic tachypacing as a model of abnormal ventricular activation. In conclusion, our findings suggest that altered electrical activity can regulate cardiomyocyte communication by influencing the acetylation status of Cx43. PMID:26264759

  14. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in...

  15. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... amino acid methionine formed by addition of an acetyl group to the alpha-amino group of methionine. It... amino acid) by weight of the total protein of the finished food, including the amount naturally present... of the additive contained therein. (2) The amounts of additive and each amino acid contained in...

  16. Prebiotically plausible oligoribonucleotide ligation facilitated by chemoselective acetylation

    NASA Astrophysics Data System (ADS)

    Bowler, Frank R.; Chan, Christopher K. W.; Duffy, Colm D.; Gerland, Batrice; Islam, Saidul; Powner, Matthew W.; Sutherland, John D.; Xu, Jianfeng

    2013-05-01

    The recent synthesis of pyrimidine ribonucleoside-2?,3?-cyclic phosphates under prebiotically plausible conditions has strengthened the case for the involvement of ribonucleic acid (RNA) at an early stage in the origin of life. However, a prebiotic conversion of these weakly activated monomers, and their purine counterparts, to the 3?,5?-linked RNA polymers of extant biochemistry has been lacking (previous attempts led only to short oligomers with mixed linkages). Here we show that the 2?-hydroxyl group of oligoribonucleotide-3?-phosphates can be chemoselectively acetylated in water under prebiotically credible conditions, which allows rapid and efficient template-directed ligation. The 2?-O-acetyl group at the ligation junction of the product RNA strand can be removed under conditions that leave the internucleotide bonds intact. Remarkably, acetylation of mixed oligomers that possess either 2?- or 3?-terminal phosphates is selective for the 2?-hydroxyl group of the latter. This newly discovered chemistry thus suggests a prebiotic route from ribonucleoside-2?,3?-cyclic phosphates to predominantly 3?,5?-linked RNA via partially 2?-O-acetylated RNA.

  17. Acetylation in hormone signaling and the cell cycle.

    PubMed

    Fu, Maofu; Wang, Chenguang; Wang, Jian; Zafonte, Brian T; Lisanti, Michael P; Pestell, Richard G

    2002-06-01

    The last decade has seen a substantial change in thinking about the role of acetylation in regulating diverse cellular processes. The correlation between histone acetylation and gene transcription has been known for many years. The cloning and biochemical characterization of the enzymes that regulate this post-translational modification has led to an understanding of the diverse role histone acetyltransferases (HATs) play in cellular function. Histone acetylases modify histones, transcription factors, co-activators, nuclear transport proteins, structural proteins and components of the cell cycle. This review focuses on the role of histone acetylases in coordinating hormone signaling and the cell cycle. Transition through the cell cycle is regulated by a family of protein kinase holoenzymes, the cyclin-dependent kinases (Cdks) and their heterodimeric cyclin partners. Recent studies have identified important cross-talk between the cell cycle regulatory apparatus and proteins regulating histone acetylation. The evidence for a dynamic interplay between components regulating the cell cycle and acetylation of target substrates provides an important new level of complexity in the mechanisms governing hormone signaling. PMID:12486878

  18. Ultrafast hydrolysis of a Lewis photoacid.

    PubMed

    Henrich, Joseph D; Suchyta, Scott; Kohler, Bern

    2015-02-12

    This study explores the concept that electronic excitation can dramatically enhance Lewis acidity. Specifically, it is shown that photoexcitation transforms an electron-deficient organic compound of negligible Lewis acidity in its electronic ground state into a potent excited-state Lewis acid that releases a proton from a nearby water molecule in 3.1 ps. It was shown previously (Peon et al. J. Phys. Chem. A 2001, 105, 5768) that the excited state of methyl viologen (MV(2+)) is quenched rapidly in aqueous solution with the formation of an unidentified photoproduct. In this study, the quenching mechanism and the identity of the photoproduct were investigated by the femtosecond transient absorption and fluorescence upconversion techniques. Transient absorption signals at UV probe wavelengths reveal a long-lived species with a pH-dependent lifetime due to reaction with hydronium ions at a bimolecular rate of 3.1 10(9) M(-1) s(-1). This species is revealed to be a charge-transfer complex consisting of a ground-state MV(2+) ion and a hydroxide ion formed when a water molecule transfers a proton to the bulk solvent. Formation of a contact ion pair between MV(2+) and hydroxide shifts the absorption spectrum of the former ion by a few nm to longer wavelengths, yielding a transient absorption spectrum with a distinctive triangle wave appearance. The slight shift of this spectrum, which is in excellent agreement with steady-state difference spectra recorded for MV(2+) at high pH, is consistent with an ion pair but not with a covalent adduct (pseudobase). The long lifetime of the ion pair at neutral pH indicates that dissociation occurs many orders of magnitude more slowly than predicted by the Smoluchowski-Debye equation. Remarkably, there is no evidence of geminate recombination, suggesting that the proton that is transferred to the solvent is conducted at least several water shells away. Although the hydrolysis mechanism has yet to be fully established, evidence suggests that the strongly oxidizing excited state of MV(2+) triggers the proton-coupled oxidation of a water molecule. The observed kinetic isotope effect of 1.7 seen in D2O vs H2O is of the magnitude expected for an ultrafast concerted proton-electron transfer reaction. The ultrafast hydrolysis seen here may be a general excited-state quenching mechanism for electronically excited Lewis acids and other powerful photooxidants in aqueous solution. PMID:25510461

  19. Nucleosome competition reveals processive acetylation by the SAGA HAT module

    PubMed Central

    Ringel, Alison E.; Cieniewicz, Anne M.; Taverna, Sean D.; Wolberger, Cynthia

    2015-01-01

    The Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex hyperacetylates histone tails in vivo in a manner that depends upon histone 3 lysine 4 trimethylation (H3K4me3), a histone mark enriched at promoters of actively transcribed genes. SAGA contains a separable subcomplex known as the histone acetyltransferase (HAT) module that contains the HAT, Gcn5, bound to Sgf29, Ada2, and Ada3. Sgf29 contains a tandem Tudor domain that recognizes H3K4me3-containing peptides and is required for histone hyperacetylation in vivo. However, the mechanism by which H3K4me3 recognition leads to lysine hyperacetylation is unknown, as in vitro studies show no effect of the H3K4me3 modification on histone peptide acetylation by Gcn5. To determine how H3K4me3 binding by Sgf29 leads to histone hyperacetylation by Gcn5, we used differential fluorescent labeling of histones to monitor acetylation of individual subpopulations of methylated and unmodified nucleosomes in a mixture. We find that the SAGA HAT module preferentially acetylates H3K4me3 nucleosomes in a mixture containing excess unmodified nucleosomes and that this effect requires the Tudor domain of Sgf29. The H3K4me3 mark promotes processive, multisite acetylation of histone H3 by Gcn5 that can account for the different acetylation patterns established by SAGA at promoters versus coding regions. Our results establish a model for Sgf29 function at gene promoters and define a mechanism governing crosstalk between histone modifications. PMID:26401015

  20. Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila

    PubMed Central

    1995-01-01

    In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta- tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of alpha- tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications. PMID:7775576

  1. Hydrolysis kinetics of secoisolariciresinol diglucoside oligomers from flaxseed.

    PubMed

    Yuan, Jian-Ping; Li, Xin; Xu, Shi-Ping; Wang, Jiang-Hai; Liu, Xin

    2008-11-12

    Flaxseed is the richest dietary source of the lignan secoisolariciresinol diglucoside (SDG) and contains the largest amount of SDG oligomers, which are often hydrolyzed to break the ester linkages for the release of SDG and the glycosidic bonds for the release of secoisolariciresinol (SECO). The alkaline hydrolysis reaction kinetics of SDG oligomers from flaxseed and the acid hydrolysis process of SDG and other glucosides were investigated. For the kinetic modeling, a pseudo-first-order reaction was assumed. The results showed that the alkaline hydrolysis of SDG oligomers followed first-order reaction kinetics under mild alkaline hydrolytic conditions and that the concentration of sodium hydroxide had a strong influence on the activation energy of the alkaline hydrolysis of SDG oligomers. The results also indicated that the main acid hydrolysates of SDG included secoisolariciresinol monoglucoside (SMG), SECO, and anhydrosecoisolariciresinol (anhydro-SECO) and that the extent and the main hydrolysates of the acid hydrolysis reaction depended on the acid concentration, hydrolysis temperature, and time. In addition, the production and change of p-coumaric acid glucoside, ferulic acid glucoside and their methyl esters and p-coumaric acid, ferulic acid, and their methyl esters during the process of hydrolysis was also investigated. PMID:18925741

  2. Effect of hydrolysis on identifying prenatal cannabis exposure.

    PubMed

    Gray, Teresa R; Barnes, Allan J; Huestis, Marilyn A

    2010-07-01

    Identification of prenatal cannabis exposure is important due to potential cognitive and behavioral consequences. A two-dimensional gas chromatography-mass spectrometry method for cannabinol, Delta(9)-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), 8beta,11-dihydroxy-THC, and 11-nor-9-carboxy-THC (THCCOOH) quantification in human meconium was developed and validated. Alkaline, enzymatic, and enzyme-alkaline tandem hydrolysis conditions were optimized with THC- and THCCOOH-glucuronide reference standards. Limits of quantification ranged from 10 to 15 ng/g, and calibration curves were linear to 500 ng/g. Bias and intra-day and inter-day imprecision were <12.3%. Hydrolysis efficiencies were analyte-dependent; THC-glucuronide was effectively cleaved by enzyme, but not base. Conversely, THCCOOH-glucuronide was most sensitive to alkaline hydrolysis. Enzyme-alkaline tandem hydrolysis maximized efficiency for both glucuronides. Identification of cannabinoid-positive meconium specimens nearly doubled following alkaline and enzyme-alkaline hydrolysis. Although no 11-OH-THC glucuronide standard is available, enzymatic hydrolysis improved 11-OH-THC detection in authentic specimens. Maximal identification of cannabis-exposed neonates and the widest range of cannabis biomarkers are achieved with enzyme-alkaline tandem hydrolysis. PMID:20517601

  3. N-Acetyl-d-Glucosamine-6-Phosphate Deacetylase: Substrate Activation via a Single Divalent Metal Ion

    PubMed Central

    Hall, Richard S.; Xiang, Dao Feng; Xu, Chengfu; Raushel, Frank M.

    2008-01-01

    NagA is a member of the amidohydrolase superfamily and catalyzes the deacetylation of N-acetyl-d-glucosamine-6-phosphate. The catalytic mechanism of this enzyme was addressed by the characterization of the catalytic properties of metal-substituted derivatives of NagA from Escherichia coli with a variety of substrate analogs. The reaction mechanism is of interest since NagA from bacterial sources is found with either one or two divalent metal ions in the active site. This observation indicates that there has been a divergence in the evolution of NagA and suggests that there are fundamental differences in the mechanistic details for substrate activation and hydrolysis. NagA from E. coli was inactivated by the removal of the zinc bound to the active site and the apo-enzyme reactivated upon incubation with one equivalent of Zn2+, Cd2+, Co2+, Mn2+, Ni2+ or Fe2+. In the proposed catalytic mechanism the reaction is initiated by the polarization of the carbonyl group of the substrate via a direct interaction with the divalent metal ion and His-143. The invariant aspartate (Asp-273) found at the end of ?-strand 8 in all members of the amidohydrolase superfamily abstracts a proton from the metal-bound water molecule (or hydroxide) to promote the hydrolytic attack on the carbonyl group of the substrate. A tetrahedral intermediate is formed and then collapses with cleavage of the C-N bond after proton transfer to the leaving group amine by Asp-273. The lack of a solvent isotope effect by D2O and the absence of any changes to the kinetic constants with increases in solvent viscosity indicate that net product formation is not limited to any significant extent by proton transfer steps or the release of products. N-trifluoroacetyl-d-glucosamine-6-phosphate is hydrolyzed by NagA 26-fold faster than the corresponding N-acetyl derivative. This result is consistent with the formation or collapse of the tetrahedral intermediate as the rate limiting step in the catalytic mechanism of NagA. PMID:17567047

  4. Overexpression of a bacterial chymotrypsin: application for L-amino acid ester hydrolysis.

    PubMed

    Volont, Federica; Pisanelli, Ines; D'Arrigo, Paola; Viani, Fiorenza; Molla, Gianluca; Servi, Stefano; Pollegioni, Loredano

    2011-12-10

    In this work, a reliable protocol was designed to rapidly express and purify a microbial chymotrypsin(ogen) as a useful alternative to using animal proteases. The cDNA encoding for chymotrypsinogen from the deuteromycete Metarhizium anisopliae (chy1) was overexpressed in an Origami2(DE3) E. coli strain deficient in thioredoxin reductase and glutathione reductase activities, thus possibly allowing disulfide exchange. By using a quick purification protocol, in which the hexahistidine tag was added at the C-terminal end of the protease, the recombinant CHY1 protein could be purified in a single step on an Ni-NTA column as a mixture of 19.5- and 15-kDa mature active forms and did not require further activation/maturation steps. This expression and purification procedure offers an easier and faster means of producing recombinant CHY1 chymotrypsin than that previously described for Pichia pastoris. The kinetic properties could be characterized and CHY1 chymotrypsin was demonstrated to efficiently catalyze N-acetylated L-phenylalanine and L-tyrosine methyl ester hydrolysis. PMID:22142732

  5. The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-induced Lysine Acetylation of Mitochondrial Proteins

    PubMed Central

    Davies, Michael N.; Kjalarsdottir, Lilja; Thompson, J. Will; Dubois, Laura G.; Stevens, Robert D.; Ilkayeva, Olga R.; Brosnan, M. Julia; Rolph, Timothy P.; Grimsrud, Paul A.; Muoio, Deborah M.

    2016-01-01

    Lysine acetylation (AcK), a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT), an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK. PMID:26748706

  6. The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins.

    PubMed

    Davies, Michael N; Kjalarsdottir, Lilja; Thompson, J Will; Dubois, Laura G; Stevens, Robert D; Ilkayeva, Olga R; Brosnan, M Julia; Rolph, Timothy P; Grimsrud, Paul A; Muoio, Deborah M

    2016-01-12

    Lysine acetylation (AcK), a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT), an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK. PMID:26748706

  7. General lysosomal hydrolysis can process prorenin accurately.

    PubMed

    Xa, Lucie K; Lacombe, Marie-Jose; Mercure, Chantal; Lazure, Claude; Reudelhuber, Timothy L

    2014-09-01

    Renin, an aspartyl protease that catalyzes the rate-limiting step of the renin-angiotensin system, is first synthesized as an inactive precursor, prorenin. Prorenin is activated by the proteolytic removal of an amino terminal prosegment in the dense granules of the juxtaglomerular (JG) cells of the kidney by one or more proteases whose identity is uncertain but commonly referred to as the prorenin-processing enzyme (PPE). Because several extrarenal tissues secrete only prorenin, we tested the hypothesis that the unique ability of JG cells to produce active renin might be explained by the existence of a PPE whose expression is restricted to JG cells. We found that inducing renin production by the mouse kidney by up to 20-fold was not associated with the concomitant induction of candidate PPEs. Because the renin-containing granules of JG cells also contain several lysosomal hydrolases, we engineered mouse Ren1 prorenin to be targeted to the classical vesicular lysosomes of cultured HEK-293 cells, where it was accurately processed and stored. Furthermore, we found that HEK cell lysosomes hydrolyzed any artificial extensions placed on the protein and that active renin was extraordinarily resistant to proteolytic degradation. Altogether, our results demonstrate that accurate processing of prorenin is not restricted to JG cells but can occur in classical vesicular lysosomes of heterologous cells. The implication is that renin production may not require a specific PPE but rather can be achieved by general hydrolysis in the lysosome-like granules of JG cells. PMID:24965790

  8. Kinetics of iodine hydrolysis in unbuffered solutions

    SciTech Connect

    Palmer, D.A.; Lyons, L.J.

    1988-01-01

    The kinetics of hydrolysis or disproportionation of hypoiodite were studied spectrophotometrically in basic solution at an ionic strength of 0.2 M as a function of pH, iodide and total iodine concentration, and temperature. The existence of three independent pathways for this second-order process was confirmed. The pH-stat method was used to monitor the corresponding reaction of hypoiodous acid in weakly alkaline solution. The generalized rate law for the disproportionation is: /minus/d((HOI) + (OI/sup /minus//))dt = k /sub a/(HOI)/sup 2/ + k/sub b/(HOI) (OI/sup /minus//) + k/sub c/(OI/sup /minus//)/sup 2/ + k/sub d/(I/sub 2/OH/sup /minus//) (OI/sup /minus//). The values of k/sub a/ and k/sub b/ are substantially smaller than previously reported. However, an unexplained contribution to the rate law resulting from the pH-stat measurements was also obtained. The rapid recombination of iodide and iodate in HClO/sub 4/ solutions was followed by stopped-flow spectrophotometry at three ionic strengths, and over a range of iodide and hydrogen ion concentrations, and at eight temperatures. Fifth-order kinetics were observed with no detectable induction period. 14 refs., 4 figs., 1 tab.

  9. Synthesis, hydrolysis and stability of psilocin glucuronide.

    PubMed

    Martin, Rafaela; Schürenkamp, Jennifer; Pfeiffer, Heidi; Lehr, Matthias; Köhler, Helga

    2014-04-01

    A two-step synthesis of psilocin glucuronide (PCG), the main metabolite of psilocin, with methyl 2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-α-d-glucopyranuronate is reported. With the synthesized PCG, hydrolysis conditions in serum and urine were optimized. Escherichia coli proved to be a better enzyme source for β-glucuronidase than Helix pomatia. It was essential to add ascorbic acid to serum samples to protect psilocin during incubation. Furthermore the stability of PCG and psilocin was compared as stability data are the basis for forensic interpretation of measurements. PCG showed a greater long-term stability after six months in deep frozen serum and urine samples than psilocin. The short-term stability of PCG for one week in whole blood at room temperature and in deep frozen samples was also better than that of psilocin. Therefore, PCG can be considered to be more stable than the labile psilocin and should always be included if psilocin is analyzed in samples. PMID:24513688

  10. Enzymatic hydrolysis of biomass from wood.

    PubMed

    Álvarez, Consolación; Reyes-Sosa, Francisco Manuel; Díez, Bruno

    2016-03-01

    Current research and development in cellulosic ethanol production has been focused mainly on agricultural residues and dedicated energy crops such as corn stover and switchgrass; however, woody biomass remains a very important feedstock for ethanol production. The precise composition of hemicellulose in the wood is strongly dependent on the plant species, therefore different types of enzymes are needed based on hemicellulose complexity and type of pretreatment. In general, hardwood species have much lower recalcitrance to enzymes than softwood. For hardwood, xylanases, beta-xylosidases and xyloglucanases are the main hemicellulases involved in degradation of the hemicellulose backbone, while for softwood the effect of mannanases and beta-mannosidases is more relevant. Furthermore, there are different key accessory enzymes involved in removing the hemicellulosic fraction and increasing accessibility of cellulases to the cellulose fibres improving the hydrolysis process. A diversity of enzymatic cocktails has been tested using from low to high densities of biomass (2-20% total solids) and a broad range of results has been obtained. The performance of recently developed commercial cocktails on hardwoods and softwoods will enable a further step for the commercialization of fuel ethanol from wood. PMID:26833542

  11. Hydrolysis and Partial Recycling of a Chloroaluminate Ionic Liquid

    PubMed Central

    Fang, Ming-Hong; Wang, Li-Sheng

    2007-01-01

    Hydrolysis of the ionic liquid Et3NHCl-2AlCl3 and a process for recycling the triethylamine were studied. When the hydrolysis was carried out at a relatively high temperature, the released HCl could be absorbed more easily. With addition of sodium hydroxide to the aqueous hydrolysis solution, a feasible process for recycling triethylamine was developed, involving first distillation of triethylamine, followed by filtration of the aluminium hydroxide. The yield of recovered triethylamine was about 95%. The triethylhydrogenammonium chloride prepared from the recycled triethylamine was of good purity and could be reused to synthesize new chloroaluminate ionic liquids.

  12. Formation of artifactual metabolites of doxylamine following acid hydrolysis.

    PubMed

    Holder, C L; Korfmacher, W A; Rushing, L G; Thompson, H C; Slikker, W; Gosnell, A B

    1987-08-01

    This study describes the use of gas chromatographic-mass spectrometric, high-performance liquid chromatographic and capillary column gas chromatographic separation techniques in demonstrating the production of several artifactual compounds reported in the literature as metabolites of doxylamine. Rhesus monkey urinary extracts which contained doxylamine and doxylamine metabolites were examined with and without acid hydrolysis. The production of 1-phenyl-1-(2-pyridinyl)ethanol and 1-phenyl-1-(2-pyridinyl)ethylene under acid hydrolysis conditions was demonstrated. These artifactual products were shown to originate from the acid hydrolysis of 2-[1-phenyl-1-(2-pyridinyl)ethoxy] acetic acid and not from doxylamine. PMID:3667771

  13. Purification, physicochemical, and kinetic properties of liver acetyl-CoA:arylamine N-acetyltransferase from rapid acetylator rabbits.

    PubMed

    Andres, H H; Vogel, R S; Tarr, G E; Johnson, L; Weber, W W

    1987-04-01

    Cytosolic liver acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from homozygous rapid acetylator rabbits (strain III/J) was purified to homogeneity as judged by gel filtration sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis and isoelectrofocusing. The isoelectric point was estimated to be 5.2. The molecular weight was determined to be 33,500 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis and 33,000 by Sephacryl S-200 gel filtration. The amino acid composition is reported and 16 tryptic peptides were sequenced by Edmann degradation, including a peptide from which a very specific oligonucleotide probe can be synthesized. The enzyme contained neither amino sugars nor cofactors. A broad pH optimum from pH 5.9 to 8.6 was observed. N-Acetyltransferase activity showed a strong dependency on the salt concentration. From the influence of the basicity of the acceptor amine on the maximum velocity, it was concluded that the formation of the covalent acetyl-enzyme intermediate is the rate-limiting step in the N-acetyltransferase-catalyzed acetylation of amines. The covalent intermediate reacts, then, in a fast step with the acceptor amine, when using aniline derivatives with pKa values ranging from 5.65 to 1.74. However, with the weakly basic 4-nitroaniline, the acetyltransfer from the catalytic intermediate to the amine seems to be rate-limiting. A structure-activity study of 30 aniline derivatives that differ in hydrophobicity, position, size, charge, and number of substituents showed that some ortho-substituted derivatives were not acetylated. PMID:3574290

  14. Interaction between succinyl CoA synthetase and the heme-biosynthetic enzyme ALAS-E is disrupted in sideroblastic anemia.

    PubMed

    Furuyama, K; Sassa, S

    2000-03-01

    The first and the rate-limiting enzyme of heme biosynthesis is delta-aminolevulinate synthase (ALAS), which is localized in mitochondria. There are 2 tissue-specific isoforms of ALAS, erythroid-specific (ALAS-E) and nonspecific ALAS (ALAS-N). To identify possible mitochondrial factors that modulate ALAS-E function, we screened a human bone marrow cDNA library, using the mitochondrial form of human ALAS-E as a bait protein in the yeast 2-hybrid system. Our screening led to the isolation of the beta subunit of human ATP-specific succinyl CoA synthetase (SCS-betaA). Using transient expression and coimmunoprecipitation, we verified that mitochodrially expressed SCS-betaA associates specifically with ALAS-E and not with ALAS-N. Furthermore, the ALAS-E mutants R411C and M426V associated with SCS-betaA, but the D190V mutant did not. Because the D190V mutant was identified in a patient with pyridoxine-refractory X-linked sideroblastic anemia, our findings suggest that appropriate association of SCS-betaA and ALAS-E promotes efficient use of succinyl CoA by ALAS-E or helps translocate ALAS-E into mitochondria. PMID:10727444

  15. Correlation of changes in rate of sterol synthesis with changes in HMG CoA reductase activity in cultured lens epithelial cells

    SciTech Connect

    Cenedella, R.J.; Hitchener, W.R.

    1986-05-01

    In the present study, the authors correlated changes in HMG CoA reductase activity with changes in relative rates of sterol synthesis measured from either /sup 3/H/sub 2/O or 1-/sup 14/C-acetate for bovine lens epithelial cells cultured in the presence or absence of lipoproteins. Enzyme activity and rates of incorporation of /sup 3/H/sub 2/O or 1-/sup 14/C-acetate into digitonin precipitable sterols were measured in cells on the 4th day of subculture in DMEM containing 9% whole calf serum (WM) or 9% lipoprotein deficient serum (LDM). In three experiments, HMG CoA reductase activity (U/10/sup 6/ cells) averaged 2.2 +/- 0.1 times greater for cells grown in LDM than WM. Sterol synthesis averaged 3.0 +/- 0.4 times greater when measured with /sup 3/H/sub 2/O and 4.0 +/- 1.1 times greater when measured with /sup 14/C-acetate. Thus, /sup 3/H/sub 2/O and /sup 14/C-acetate appear to be comparable substrates for estimating changes in relative rates of sterol synthesis by cultured cells. The larger increases in rates of sterol synthesis than in reductase activity in response to decreased cholesterol could reflect stimulation at additional metabolic steps in the cholesterol pathway beyond mevalonic acid.

  16. 1200 nt rat liver mRNA identified by differential hybridization exhibits coordinate regulation with 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase

    SciTech Connect

    Tanaka, R.D.; Clarke, C.F.; Fogelman, A.M.; Edwards, P.A.

    1986-05-01

    Differential hybridization has been used to identify genes in rat liver that encode transcripts which are increased by the drugs cholestyramine and mevinolin and are decreased by dietary cholesterol. This approach should prove useful in isolating and identifying coordinately regulated genes involved in the isoprene biosynthetic pathway. Rat liver poly (A)/sup +/ RNA was isolated from animals fed diets supplemented with either cholestyramine and mevinolin or with cholesterol. Radiolabeled cDNAs generated from these two RNA preparations were used to screen a rat cDNAs library. A preliminary screen of 10,000 recombinants has led to the identification of a clone with an insert of 1200 bp that hybridizes to a mRNA species of about 1200 nt. The level of this RNA species in rat liver is elevated by the drugs cholestyramine and mevinolin and is decreased by cholesterol feeding. This RNA species is also decreased by mevalonate administration to rats. The regulation of this 1200 nt mRNA species mirrors that of HMG CoA reductase and HMG CoA synthase. It seems very likely that this 1200 nt mRNA encodes a polypeptide which is involved in the isoprene biosynthetic pathway.

  17. Proteome-wide lysine acetylation profiling of the human pathogen Mycobacterium tuberculosis.

    PubMed

    Xie, Longxiang; Wang, Xiaobo; Zeng, Jie; Zhou, Mingliang; Duan, Xiangke; Li, Qiming; Zhang, Zhen; Luo, Hongping; Pang, Lei; Li, Wu; Liao, Guojian; Yu, Xia; Li, Yunxu; Huang, Hairong; Xie, Jianping

    2015-02-01

    N(ɛ)-Acetylation of lysine residues represents a pivotal post-translational modification used by both eukaryotes and prokaryotes to modulate diverse biological processes. Mycobacterium tuberculosis is the causative agent of tuberculosis, one of the most formidable public health threats. Many aspects of the biology of M. tuberculosis remain elusive, in particular the extent and function of N(ɛ)-lysine acetylation. With a combination of anti-acetyllysine antibody-based immunoaffinity enrichment with high-resolution mass spectrometry, we identified 1128 acetylation sites on 658 acetylated M. tuberculosis proteins. GO analysis of the acetylome showed that acetylated proteins are involved in the regulation of diverse cellular processes including metabolism and protein synthesis. Six types of acetylated peptide sequence motif were revealed from the acetylome. Twenty lysine-acetylated proteins showed homology with acetylated proteins previously identified from Escherichia coli, Salmonella enterica, Bacillus subtilis and Streptomyces roseosporus, with several acetylation sites highly conserved among four or five bacteria, suggesting that acetylated proteins are more conserved. Notably, several proteins including isocitrate lyase involved in the persistence, virulence and antibiotic resistance are acetylated, and site-directed mutagenesis of isocitrate lyase acetylation site to glutamine led to a decrease of the enzyme activity, indicating major roles of KAc in these proteins engaged cellular processes. Our data firstly provides a global survey of M. tuberculosis acetylation, and implicates extensive regulatory role of acetylation in this pathogen. This may serve as an important basis to address the roles of lysine acetylation in M. tuberculosis metabolism, persistence and virulence. PMID:25456444

  18. Role of Carnitine Acetyltransferases in Acetyl Coenzyme A Metabolism in Aspergillus nidulans ▿

    PubMed Central

    Hynes, Michael J.; Murray, Sandra L.; Andrianopoulos, Alex; Davis, Meryl A.

    2011-01-01

    The flow of carbon metabolites between cellular compartments is an essential feature of fungal metabolism. During growth on ethanol, acetate, or fatty acids, acetyl units must enter the mitochondrion for metabolism via the tricarboxylic acid cycle, and acetyl coenzyme A (acetyl-CoA) in the cytoplasm is essential for the biosynthetic reactions and for protein acetylation. Acetyl-CoA is produced in the cytoplasm by acetyl-CoA synthetase during growth on acetate and ethanol while β-oxidation of fatty acids generates acetyl-CoA in peroxisomes. The acetyl-carnitine shuttle in which acetyl-CoA is reversibly converted to acetyl-carnitine by carnitine acetyltransferase (CAT) enzymes is important for intracellular transport of acetyl units. In the filamentous ascomycete Aspergillus nidulans, a cytoplasmic CAT, encoded by facC, is essential for growth on sources of cytoplasmic acetyl-CoA while a second CAT, encoded by the acuJ gene, is essential for growth on fatty acids as well as acetate. We have shown that AcuJ contains an N-terminal mitochondrial targeting sequence and a C-terminal peroxisomal targeting sequence (PTS) and is localized to both peroxisomes and mitochondria, independent of the carbon source. Mislocalization of AcuJ to the cytoplasm does not result in loss of growth on acetate but prevents growth on fatty acids. Therefore, while mitochondrial AcuJ is essential for the transfer of acetyl units to mitochondria, peroxisomal localization is required only for transfer from peroxisomes to mitochondria. Peroxisomal AcuJ was not required for the import of acetyl-CoA into peroxisomes for conversion to malate by malate synthase (MLS), and export of acetyl-CoA from peroxisomes to the cytoplasm was found to be independent of FacC when MLS was mislocalized to the cytoplasm. PMID:21296915

  19. Coupled Ferredoxin and Crotonyl Coenzyme A (CoA) Reduction with NADH Catalyzed by the Butyryl-CoA Dehydrogenase/Etf Complex from Clostridium kluyveri?

    PubMed Central

    Li, Fuli; Hinderberger, Julia; Seedorf, Henning; Zhang, Jin; Buckel, Wolfgang; Thauer, Rudolf K.

    2008-01-01

    Cell extracts of butyrate-forming clostridia have been shown to catalyze acetyl-coenzyme A (acetyl-CoA)- and ferredoxin-dependent formation of H2 from NADH. It has been proposed that these bacteria contain an NADH:ferredoxin oxidoreductase which is allosterically regulated by acetyl-CoA. We report here that ferredoxin reduction with NADH in cell extracts from Clostridium kluyveri is catalyzed by the butyryl-CoA dehydrogenase/Etf complex and that the acetyl-CoA dependence previously observed is due to the fact that the cell extracts catalyze the reduction of acetyl-CoA with NADH via crotonyl-CoA to butyryl-CoA. The cytoplasmic butyryl-CoA dehydrogenase complex was purified and is shown to couple the endergonic reduction of ferredoxin (E0? = ?410 mV) with NADH (E0? = ?320 mV) to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0? = ?10 mV) with NADH. The stoichiometry of the fully coupled reaction is extrapolated to be as follows: 2 NADH + 1 oxidized ferredoxin + 1 crotonyl-CoA = 2 NAD+ + 1 ferredoxin reduced by two electrons + 1 butyryl-CoA. The implications of this finding for the energy metabolism of butyrate-forming anaerobes are discussed in the accompanying paper. PMID:17993531

  20. A General Approach for Teaching Hydrolysis of Salts.

    ERIC Educational Resources Information Center

    Aguirre-Ode, Fernando

    1987-01-01

    Presented is a general approach and equation for teaching the hydrolysis of salts. This general equation covers many more sets of conditions than those currently in textbooks. The simplifying assumptions leading to the known limiting equations are straightforward. (RH)

  1. Kinetics of the hydrolysis of guanosine 5'-phospho-2-methylimidazolide

    NASA Technical Reports Server (NTRS)

    Kanavarioti, Anastassia

    1986-01-01

    The hydrolysis kinetics of guanosine 5'-phospho-2-methylimidazolide (2-MeImpG) in aqueous buffered solutions of various pH's was studied at 75 and 37 C, using spectrophotometric and HPLC techniques. The hydrolysis was found to be very slow even at low pH. At 75 C and pH at or below l.0, two kinetic processes were observed: the more rapid one was attributed to the hydrolysis of the phosphoimidazolide P-N bond; the second, much slower one, was attributed to the cleavage of the glycosidic bond. It is noted that the P-N hydrolysis in phosphoimidazolides is very slow compared to other phosphoramidates, and that this might be one of the reasons why the phosphoimidazolides showed an extraordinary ability to form long oligomers under template-directed conditions.

  2. Enzymatic hydrolysis of steryl glycosides for their analysis in foods.

    PubMed

    Münger, Linda H; Nyström, Laura

    2014-11-15

    Steryl glycosides (SG) contribute significantly to the total intake of phytosterols. The standard analytical procedure involving acid hydrolysis fails to reflect the correct sterol profile of SG due to isomerization of some of the labile sterols. Therefore, various glycosylases were evaluated for their ability to hydrolyse SG under milder conditions. Using a pure SG mixture in aqueous solution, the highest glycolytic activity, as demonstrated by the decrease in SG and increase in free sterols was achieved using inulinase preparations (decrease of >95%). High glycolytic activity was also demonstrated using hemicellulase (63%). The applicability of enzymatic hydrolysis using inulinase preparations was further verified on SG extracted from foods. For example in potato peel Δ(5)-avenasteryl glucoside, a labile SG, was well preserved and contributed 26.9% of the total SG. Therefore, enzymatic hydrolysis is suitable for replacing acid hydrolysis of SG in food lipid extracts to accurately determine the sterol profile of SG. PMID:24912717

  3. Hydrolysis of fMet-tRNA by Peptidyl Transferase

    PubMed Central

    Caskey, C. T.; Beaudet, A. L.; Scolnick, E. M.; Rosman, M.

    1971-01-01

    Escherichia coli and rabbit reticulocyte (f[3H]Met-tRNAAUGribosome) intermediates undergo hydrolysis, with release of f[3H]methionine, upon addition of tRNA or CpCpA in the presence of acetone. This ribosomal catalyzed reaction has similar requirements, pH optimum, and antibiotic sensitivity to those of peptidyl transferase. Two antibiotics, lincomycin with E. coli ribosomes and anisomycin with reticulocyte ribosomes, inhibit peptide-bond formation and transesterification activities of peptidyl transferase, but stimulate hydrolysis of f[3H]Met-tRNA. Earlier studies have suggested peptidyl transferase activity is essential for R factor-dependent hydrolysis of f(3H)Met-tRNA. These studies indicate that peptidyl transferase has the capacity for f(3H)Met-tRNA hydrolysis and, therefore, may be responsible for peptidyl-tRNA cleavage during peptide chain termination. PMID:4943558

  4. ESTIMATION OF CARBOXYLIC ACID ESTER HYDROLYSIS RATE CONSTANTS

    EPA Science Inventory

    SPARC chemical reactivity models were extended to calculate hydrolysis rate constants for carboxylic acid esters from molecular structure. The energy differences between the initial state and the transition state for a molecule of interest are factored into internal and external...

  5. Optimization of enzymatic hydrolysis of cassava to obtain fermentable sugars*

    PubMed Central

    Collares, Renata M.; Miklasevicius, Luiza V. S.; Bassaco, Mariana M.; Salau, Nina P. G.; Mazutti, Marcio A.; Bisognin, Dilson A.; Terra, Lisiane M.

    2012-01-01

    This work evaluates the enzymatic hydrolysis of starch from cassava using pectinase, ?-amylase, and amyloglucosidase. A central composite rotational design (CCRD) was carried out to evaluate the effects of amyloglucosidase, pectinase, reaction time, and solid to liquid ratio. All the experiments were carried out in a bioreactor with working volume of 2 L. Approximately 98% efficiency hydrolysis was obtained, resulting in a concentration of total reducing sugar released of 160 g/L. It was concluded that pectinase improved the hydrolysis of starch from cassava. Reaction time was found to be significant until 7 h of reaction. A solid to liquid ratio of 1.0 was considered suitable for hydrolysis of starch from cassava. Amyloglucosidase was a significant variable in the process: after its addition to the reaction media, a 30%50% increase in the amount of total reducing sugar released was observed. At optimal conditions the maximum productivity obtained was 22.9 g/(Lh). PMID:22761249

  6. Energetic approach of biomass hydrolysis in supercritical water.

    PubMed

    Cantero, Danilo A; Vaquerizo, Luis; Mato, Fidel; Bermejo, M Dolores; Cocero, M José

    2015-03-01

    Cellulose hydrolysis can be performed in supercritical water with a high selectivity of soluble sugars. The process produces high-pressure steam that can be integrated, from an energy point of view, with the whole biomass treating process. This work investigates the integration of biomass hydrolysis reactors with commercial combined heat and power (CHP) schemes, with special attention to reactor outlet streams. The innovation developed in this work allows adequate energy integration possibilities for heating and compression by using high temperature of the flue gases and direct shaft work from the turbine. The integration of biomass hydrolysis with a CHP process allows the selective conversion of biomass into sugars with low heat requirements. Integrating these two processes, the CHP scheme yield is enhanced around 10% by injecting water in the gas turbine. Furthermore, the hydrolysis reactor can be held at 400°C and 23 MPa using only the gas turbine outlet streams. PMID:25536511

  7. 6A-O-[(4-biphenylyl)acetyl]-alpha-, -beta-, and -gamma-cyclodextrins and 6A-deoxy-6A-[[(4-biphenylyl)acetyl]amino]-alpha-, -beta-, and -gamma-cyclodextrins: potential prodrugs for colon-specific delivery.

    PubMed

    Uekama, K; Minami, K; Hirayama, F

    1997-08-15

    Cyclodextrins (CyDs) are known to be fermented to small saccharides by colonic microflora, whereas they are only slightly hydrolyzable and thus are not easily absorbed in the stomach and small intestine. This property of CyDs is particularly useful for colon-specific delivery of drugs. In this study, an antiinflammatory 4-biphenylylacetic acid (BPAA) was selectively conjugated onto one of the primary hydroxyl groups of alpha-, beta-, and gamma-CyDs through an ester or amide linkage, 6A-O-[(4-biphenylyl)acetyl[-alpha-, -beta-, and -gamma-CyDs (1-3) and 6A-deoxy-6A-[[(4-biphenylyl)acetyl]amino]-alpha-, -beta-, and -gamma-CyDs (4-6). In rat cecal and colonic contents (10%, w/v), 1 and 3 released more than 95% of BPAA within 1-2 h, and 2 released about 50% of the drug within 12 h. The amide prodrugs, 4-6, did not release BPAA in the cecal contents, but gave BPAA/maltose or BPAA/triose conjugates linked through an amide bond. On the other hand, these prodrugs were found to be stable in the contents of rat stomachs and small intestines, in intestinal or liver homogenates, and in rat blood. The serum levels of BPAA increased about 3 h after oral administration of 1 and 3 to rats, accompanying a marked increase in the serum levels, whereas 2 and 4-6 resulted in little increase of the serum levels. These facts suggest that BPAA is released after the ring opening of CyDs followed by the ester hydrolysis, and the BPAA activation takes place site-specifically in the cecum and colon. Therefore, the present CyD prodrug approach provides a versatile means of constructing a novel colon-specific drug delivery system. PMID:9276021

  8. [Changes in chemistry component structure and microstructure characterization of acetylated wood before and after UV radiation].

    PubMed

    Fu, Zhan; Liu, Yi; Xing, Fang-Ru; Guo, Hong-Wu

    2014-11-01

    The poplar powder was acetylated with different duration as sample, processed ray radiation by using ultraviolet test box, contrasting the influences to lightfastness of wood with different acetylation degree, analyzing changing rules of characteristic peaks' intensity which belonged to the chemistry components of samples based on FTIR spectra, and the relationship between duration of acetylation and changes of chemistry components was established, The results showed that: Before UV radiation, the characteristic peaks' intensity of acetylated poplar powder at 1 739 cm(-1) which belonged to C = O in saturated esters compounds and 1 385 cm(-1) which belonged to C-H in acetate were higher than untreated ones', the poplar powder with 40 min's acetylation has the highest characteristic peaks' intensity, highest weight gain rate, remarkable acetylation effect; After UV radiation, characteristic peaks' intensity of Benzene at 1 504 cm(-1) which belonged to lignin of poplar powder was obviously higher than untreated ones', and characteristic peaks' intensity of poplar powder with 40 min's acetylation was the highest, this showed that acetylation could effectively reduce the light degradation of wood chemistry components, in order to improve the lightfastness, especially the poplar powder with 40 min's acetylation; SEM photos showed that, the fibrous surface of acetylated poplar powder was more smooth and had more narrow particle size than untreated ones', so acetylation can effectively improve the stability of wood. PMID:25752036

  9. Ubiquitination of Notch1 is regulated by MAML1-mediated p300 acetylation of Notch1

    SciTech Connect

    Popko-Scibor, Anita E.; Lindberg, Mikael J.; Hansson, Magnus L.; Holmlund, Teresa; Wallberg, Annika E.

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer p300 acetylates conserved lysines within Notch1 C-terminal nuclear localization signal. Black-Right-Pointing-Pointer MAML1 and CSL, components of Notch transcription complex, increase Notch acetylation. Black-Right-Pointing-Pointer MAML1-dependent acetylation of Notch1 by p300 decreases the ubiquitination of Notch1. Black-Right-Pointing-Pointer CDK8 inhibits Notch acetylation and Notch transcription enhanced by p300. -- Abstract: Earlier studies demonstrated the involvement of the p300 histone acetyltransferase in Notch signaling but the precise mechanisms by which p300 might modulate Notch function remains to be investigated. In this study, we show that p300 acetylates Notch1 ICD in cell culture assay and in vitro, and conserved lysines located within the Notch C-terminal nuclear localization signal are essential for Notch acetylation. MAML1 and CSL, which are components of the Notch transcription complex, enhance Notch acetylation and we suggest that MAML1 increases Notch acetylation by potentiating p300 autoacetylation. Furthermore, MAML1-dependent acetylation of Notch1 ICD by p300 decreases the ubiquitination of Notch1 ICD in cellular assays. CDK8 has been shown to target Notch1 for ubiquitination and proteosomal degradation. We show that CDK8 inhibits Notch acetylation and Notch transcription enhanced by p300. Therefore, we speculate that acetylation of Notch1 might be a mechanism to regulate Notch activity by interfering with ubiquitin-dependent pathways.

  10. Multiple Mass Isotopomer Tracing of Acetyl-CoA Metabolism in Langendorff-perfused Rat Hearts

    PubMed Central

    Li, Qingling; Deng, Shuang; Ibarra, Rafael A.; Anderson, Vernon E.; Brunengraber, Henri; Zhang, Guo-Fang

    2015-01-01

    We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [13C2,2H3]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [13C6]glucose or [1,2-13C2]palmitate) or/and M1 acetyl-CoA (e.g. [1-13C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA. PMID:25645937

  11. N-Terminal Acetylation Acts as an Avidity Enhancer Within an Interconnected Multiprotein Complex

    SciTech Connect

    Scott, Daniel C.; Monda, Julie K.; Bennett, Eric J.; Harper, J. Wade; Schulman, Brenda A.

    2012-10-25

    Although many eukaryotic proteins are amino (N)-terminally acetylated, structural mechanisms by which N-terminal acetylation mediates protein interactions are largely unknown. Here, we found that N-terminal acetylation of the E2 enzyme, Ubc12, dictates distinctive E3-dependent ligation of the ubiquitin-like protein Nedd8 to Cul1. Structural, biochemical, biophysical, and genetic analyses revealed how complete burial of Ubc12's N-acetyl-methionine in a hydrophobic pocket in the E3, Dcn1, promotes cullin neddylation. The results suggest that the N-terminal acetyl both directs Ubc12's interactions with Dcn1 and prevents repulsion of a charged N terminus. Our data provide a link between acetylation and ubiquitin-like protein conjugation and define a mechanism for N-terminal acetylation-dependent recognition.

  12. Infrared and 13C MAS nuclear magnetic resonance spectroscopic study of acetylation of cotton

    NASA Astrophysics Data System (ADS)

    Adebajo, Moses O.; Frost, Ray L.

    2004-01-01

    The acetylation of commercial cotton samples with acetic anhydride without solvents in the presence of about 5% 4-dimethylaminopyridine (DMAP) catalyst was followed using Fourier transform infrared (FTIR) and 13C MAS NMR spectroscopy. This preliminary investigation was conducted in an effort to develop hydrophobic, biodegradable, cellulosic materials for subsequent application in oil spill cleanup. The FTIR results provide clear evidence for successful acetylation though the NMR results indicate that the level of acetylation is low. Nevertheless, the overall results indicate that cotton fibres are potential candidates suitable for further development via acetylation into hydrophobic sorbent materials for subsequent oil spill cleanup application. The results also indicate that de-acetylation, the reverse of the equilibrium acetylation reaction, occurred when the acetylation reaction was prolonged beyond 3 h.

  13. Hydrolysis of ammonia borane and metal amidoboranes: A comparative study.

    PubMed

    Banu, Tahamida; Debnath, Tanay; Ash, Tamalika; Das, Abhijit K

    2015-11-21

    A gas phase mechanistic investigation has been carried out theoretically to explore the hydrolysis pathway of ammonia borane (NH3BH3) and metal amidoboranes (MNH2BH3, M = Li,Na). The Solvation Model based on Density (SMD) has been employed to show the effect of bulk water on the reaction mechanism. Gibbs free energy of solvation has also been computed to evaluate the stabilization of the participating systems in water medium which directly affects the barrier heights in the potential energy surface of hydrolysis reaction. To validate the experimentally observed kinetics studies, we have carried out transition state theory calculations on these hydrolysis reactions. Our result shows that the hydrolysis of both the metal amidoboranes exhibits greatly improved kinetics over the neat NH3BH3 hydrolysis which corroborates well with the experimental observation. Between the two amidoboranes, hydrolysis of LiNH2BH3 is found to be kinetically favored over that of NaNH2BH3, making it a better candidate for releasing molecular hydrogen. PMID:26590535

  14. Hydrolysis of ammonia borane and metal amidoboranes: A comparative study

    NASA Astrophysics Data System (ADS)

    Banu, Tahamida; Debnath, Tanay; Ash, Tamalika; Das, Abhijit K.

    2015-11-01

    A gas phase mechanistic investigation has been carried out theoretically to explore the hydrolysis pathway of ammonia borane (NH3BH3) and metal amidoboranes (MNH2BH3, M = Li,Na). The Solvation Model based on Density (SMD) has been employed to show the effect of bulk water on the reaction mechanism. Gibbs free energy of solvation has also been computed to evaluate the stabilization of the participating systems in water medium which directly affects the barrier heights in the potential energy surface of hydrolysis reaction. To validate the experimentally observed kinetics studies, we have carried out transition state theory calculations on these hydrolysis reactions. Our result shows that the hydrolysis of both the metal amidoboranes exhibits greatly improved kinetics over the neat NH3BH3 hydrolysis which corroborates well with the experimental observation. Between the two amidoboranes, hydrolysis of LiNH2BH3 is found to be kinetically favored over that of NaNH2BH3, making it a better candidate for releasing molecular hydrogen.

  15. Development of complete hydrolysis of pectins from apple pomace.

    PubMed

    Wikiera, Agnieszka; Mika, Magdalena; Starzyńska-Janiszewska, Anna; Stodolak, Bożena

    2015-04-01

    Enzymatically extracted pectins have a more complex structure than those obtained by conventional methods. As a result, they are less susceptible to hydrolysis, which makes the precise determination of their composition difficult. The aim of the study was to develop a method of complete hydrolysis of enzymatically extracted apple pectins. Substrates were pectins isolated from apple pomace by the use of xylanase and multicatalytic preparation Celluclast and apple pomace. Hydrolysis was performed by a chemical method with 2M TFA at 100 °C and 120 °C and a combined acidic/enzymatic method. After hydrolysis, the contents of galacturonic acid and neutral sugars were measured by HPLC. Complete hydrolysis of polygalacturonic acid occurred after 2.5h incubation with 2M TFA at 120 °C. The efficient hydrolysis of neutral sugars in pectins was performed with 2M TFA at 100 °C for 2.5h. Monomers most susceptible to concentrated acid were rhamnose, mannose and arabinose. PMID:25442606

  16. Electron transfer precedes ATP hydrolysis during nitrogenase catalysis.

    PubMed

    Duval, Simon; Danyal, Karamatullah; Shaw, Sudipta; Lytle, Anna K; Dean, Dennis R; Hoffman, Brian M; Antony, Edwin; Seefeldt, Lance C

    2013-10-01

    The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s(-1), 25 C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s(-1), 25 C), (ii) ATP hydrolysis (kATP = 70 s(-1), 25 C), (iii) Phosphate release (kPi = 16 s(-1), 25 C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s(-1), 25 C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein-protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Fe(ox)(ADP)2 protein and the reduced MoFe protein. PMID:24062462

  17. Study of microwave effects on the lipase-catalyzed hydrolysis.

    PubMed

    Chen, Chia-Chen; Reddy, P Muralidhar; Devi, C Shobha; Chang, Po-Chi; Ho, Yen-Peng

    2016-01-01

    The effect of microwave heating on lipase-catalyzed reaction remains controversial. It is not clear whether the reaction rate enhancements are purely due to thermal/heating effects or to non-thermal effects. Therefore, quantitative mass spectrometry was used to conduct accurate kinetic analysis of lipase-catalyzed hydrolysis of triolein by microwave and conventional heating. Commercial lipases from Candida rugosa (CRL), Porcine Pancreas (PPL), and Burkholderia cepacia (BCL) were used. Hydrolysis reactions were performed at various temperatures and pH levels, along with various amounts of buffer and enzymes. Hydrolysis product yields at each time point using an internal-standard method showed no significant difference between microwave and conventional heating conditions when the reaction was carried out at the same temperature. CRL showed optimum catalytic activity at 37C, while PPL and BCL had better activities at 50C. The phosphate buffer was found to give a better hydrolysis yield than the Tris-HCl buffer. Overall results prove that a non-thermal effect does not exist in microwave-assisted lipase hydrolysis of triolein. Therefore, conventional heating at high temperatures (e.g., 50C) can be also used to accelerate hydrolysis reactions. PMID:26672464

  18. Evaluation of abalone ?-glucuronidase substitution in current urine hydrolysis procedures.

    PubMed

    Malik-Wolf, Brittany; Vorce, Shawn; Holler, Justin; Bosy, Thomas

    2014-04-01

    This study examined the potential of abalone ?-glucuronidase as a viable and cost effective alternative to current hydrolysis procedures using acid, Helix pomatia ?-glucuronidase and Escherichia coli ?-glucuronidase. Abalone ?-glucuronidase successfully hydrolyzed oxazepam-glucuronide and lorazepam-glucuronide within 5% of the spiked control concentration. Benzodiazepines present in authentic urine specimens were within 20% of the concentrations obtained with the current hydrolysis procedure using H. pomatia ?-glucuronidase. JWH 018 N-(5-hydroxypentyl) ?-d-glucuronide was hydrolyzed within 10% of the control concentration. Authentic urine specimens showed improved glucuronide cleavage using abalone ?-glucuronidase with up to an 85% increase of drug concentration, compared with the results obtained using E. coli ?-glucuronidase. The JWH 018 and JWH 073 carboxylic acid metabolites also showed increased drug concentrations of up to 24%. Abalone ?-glucuronidase was able to completely hydrolyze a morphine-3-glucuronide control, but only 82% of total morphine was hydrolyzed in authentic urine specimens compared with acid hydrolysis results. Hydrolysis of codeine and hydromorphone varied between specimens, suggesting that abalone ?-glucuronidase may not be as efficient in hydrolyzing the glucuronide linkages in opioid compounds compared with acid hydrolysis. Abalone ?-glucuronidase demonstrates effectiveness as a low cost option for enzyme hydrolysis of benzodiazepines and synthetic cannabinoids. PMID:24488113

  19. ARDent about acetylation and deacetylation in hypoxia signalling.

    PubMed

    Bilton, Rebecca; Trottier, Eric; Pouyssgur, Jacques; Brahimi-Horn, M Christiane

    2006-12-01

    Given the key role that the alpha subunit of the alphabeta heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1) has in tumourigenesis, and in particular in angiogenesis, a full understanding of its regulation is crucial to the development of cancer therapeutics. Posttranslational acetylation and deacetylation of this subunit by an acetyltransferase called Arrest-defective-1 (ARD1) and by different histone deacetylases (HDACs), respectively, has been suggested as a mechanism. However, conflicting data bring into question the foundations of this mechanism and at present it is not clear what the precise role of these proteins is with respect to HIF. Nonetheless, the observation that small-molecule inhibitors of HDACs have anti-angiogenic activity suggests that acetylation and deacetylation of HIF or HIF modifiers represents a potential target in cancer therapy. PMID:17070052

  20. Tissue distribution of N-acetyltransferase 1 and 2 catalyzing the N-acetylation of 4-aminobiphenyl and O-acetylation of N-hydroxy-4-aminobiphenyl in the congenic rapid and slow acetylator Syrian hamster.

    PubMed

    Hein, David W; Doll, Mark A; Nerland, Donald E; Fretland, Adrian J

    2006-04-01

    N-acetyltransferase 1 (NAT1) and 2 (NAT2) enzymes catalyzing both deactivation (N-acetylation) and activation (O-acetylation) of arylamine carcinogens such as 4-aminobiphenyl (ABP) were investigated in a Syrian hamster model congenic at the NAT2 locus. NAT2 catalytic activities (measured with p-aminobenzoic acid) were significantly (P < 0.001) higher in rapid than slow acetylators in all tissues (except heart and prostate where activity was undetectable in slow acetylators). NAT1 catalytic activities (measured with sulfamethazine) were low but detectable in most tissues tested and did not differ significantly between rapid and slow acetylators. ABP N-acetyltransferase activity was detected in all tissues of rapid acetylators but was below the limit of detection in all tissues of slow acetylators except liver where it was about 15-fold lower than rapid acetylators. ABP N-acetyltransferase activities correlated with NAT2 activities (r2 = 0.871; P < 0.0001) but not with NAT1 activities (r2 = 0.132; P > 0.05). Levels of N-hydroxy-ABP O-acetyltransferase activities were significantly (P < 0.05) higher in rapid than slow acetylator cytosols for many but not all tissues. The N-hydroxy-ABP O-acetyltransferase activities correlated with ABP N-acetyltransferase activities (r2 = 0.695; P < 0.0001) and NAT2 activities (r2 = 0.521, P < 0.0001) but not with NAT1 activities (r2 = 0.115; P > 0.05). The results suggest widespread tissue distribution of both NAT1 and NAT2, which catalyzes both N- and O-acetylation. These conclusions are important for interpretation of molecular epidemiological investigations into the role of N-acetyltransferase polymorphisms in various diseases including cancer. PMID:16482518

  1. Givinostat inhibition of hepatic stellate cell proliferation and protein acetylation

    PubMed Central

    Wang, Yu-Gang; Xu, Ling; Wang, Ting; Wei, Jue; Meng, Wen-Ying; Wang, Na; Shi, Min

    2015-01-01

    AIM: To explore the effect of the histone deacetylase inhibitor givinostat on proteins related to regulation of hepatic stellate cell proliferation. METHODS: The cell counting kit-8 assay and flow cytometry were used to observe changes in proliferation, apoptosis, and cell cycle in hepatic stellate cells treated with givinostat. Western blot was used to observe expression changes in p21, p57, CDK4, CDK6, cyclinD1, caspase-3, and caspase-9 in hepatic stellate cells exposed to givinostat. The scratch assay was used to analyze the effect of givinostat on cell migration. Effects of givinostat on the reactive oxygen species profile, mitochondrial membrane potential, and mitochondrial permeability transition pore opening in JS-1 cells were observed by laser confocal microscopy. RESULTS: Givinostat significantly inhibited JS-1 cell proliferation and promoted cell apoptosis, leading to cell cycle arrest in G0/G1 phases. Treatment with givinostat downregulated protein expression of CDK4, CDK6, and cyclin D1, whereas expression of p21 and p57 was significantly increased. The givinostat-induced apoptosis of hepatic stellate cells was mainly mediated through p38 and extracellular signal-regulated kinase 1/2. Givinostat treatment increased intracellular reactive oxygen species production, decreased mitochondrial membrane potential, and promoted mitochondrial permeability transition pore opening. Acetylation of superoxide dismutase (acetyl K68) and nuclear factor-κB p65 (acetyl K310) was upregulated, while there was no change in protein expression. Moreover, the notable beneficial effect of givinostat on liver fibrosis was also confirmed in the mouse models. CONCLUSION: Givinostat has antifibrotic activities via regulating the acetylation of nuclear factor-κB and superoxide dismutase 2, thus inhibiting hepatic stellate cell proliferation and inducing apoptosis. PMID:26217084

  2. Bis(acetyl-acetonato-?O,O')(pyridine-?N)zinc(II).

    PubMed

    Brahma, Sanjaya; Srinidhi, M; Shivashankar, S A; Narasimhamurthy, T; Rathore, R S

    2011-06-01

    In the title compound, [Zn(C(5)H(7)O(2))(2)(C(5)H(5)N)], the metal atom has square-pyramidal coordination geometry with the basal plane defined by the four O atoms of the chelating acetyl-acetonate ligands and with the axial position occupied by the pyridine N atom. The crystal packing is characterized by a C-H?O hydrogen-bonded ribbon structure approximately parallel to [10[Formula: see text

  3. The asymmetric total synthesis of (+)-N-acetyl norloline.

    PubMed

    Ye, Jian-Liang; Liu, Yang; Yang, Zhi-Ping; Huang, Pei-Qiang

    2016-01-11

    The asymmetric total synthesis of (+)-N-acetyl norloline, the putative biogenic precursor of all known loline alkaloids, has been achieved in 12 steps from commercially available (R)-glyceraldehyde acetonide. The synthesis relies on the Rassu/Casiraghi's vinylogous aldol reaction, an intramolecular oxa-heteroconjugate addition and a reductive amination to establish the four contiguous stereogenic centers and construct the strained oxygen-bridge under mild conditions. PMID:26538080

  4. Ethyl 2-acetylhydrazono-2-phenylacetate

    PubMed Central

    Xu, Liang-Zhong; Yi, Xu; An, Guang-Wei; Zhang, Gong-Sheng; Li, Chun-Fang

    2008-01-01

    The title compound, C12H14N2O3, was synthesized as an intermediate for the synthesis of metamitron. The benzene ring forms dihedral angles of 86.3?(2) and 10.0?(3) with the ethyl group and the acetylimino plane, respectively. The crystal structure involves intermolecular CH?O and NH?O hydrogen bonds. PMID:21200890

  5. 2-Acetylhydrazono-2-phenylacetohydrazide

    PubMed Central

    Feng, Bai-Cheng; Yang, Zhi; Yi, Xu

    2008-01-01

    The title compound, C10H12N4O2, was prepared as an intermediate for the synthesis of metamitron. The benzene ring plane forms dihedral angles of 66.0?(1) and 3.5?(5) with the hydrazine plane and the acetylimino plane, respectively. The crystal structure involves intermolecular NH?O hydrogen bonds. PMID:21201803

  6. Histone acetylation modifiers in the pathogenesis of malignant disease.

    PubMed

    Mahlknecht, U; Hoelzer, D

    2000-08-01

    Chromatin structure is gaining increasing attention as a potential target in the treatment of cancer. Relaxation of the chromatin fiber facilitates transcription and is regulated by two competing enzymatic activities, histone acetyltransferases (HATs) and histone deacetylases (HDACs), which modify the acetylation state of histone proteins and other promoter-bound transcription factors. While HATs, which are frequently part of multisubunit coactivator complexes, lead to the relaxation of chromatin structure and transcriptional activation, HDACs tend to associate with multisubunit core-pressor complexes, which result in chromatin condensation and transcriptional repression of specific target genes. HATs and HDACs are known to be involved both in the pathogenesis as well as in the suppression of cancer. Some of the genes encoding these enzymes have been shown to be rearranged in the context of chromosomal translocations in human acute leukemias and solid tumors, where fusions of regulatory and coding regions of a variety of transcription factor genes result in completely new gene products that may interfere with regulatory cascades controlling cell growth and differentiation. On the other hand, some histone acetylation-modifying enzymes have been located within chromosomal regions that are particularly prone to chromosomal breaks. In these cases gains and losses of chromosomal material may affect the availability of functionally active HATs and HDACs, which in turn disturbs the tightly controlled equilibrium of histone acetylation. We review herein the recent achievements, which further help to elucidate the biological role of histone acetylation modifying enzymes and their potential impact on our current understanding of the molecular changes involved in the development of solid tumors and leukemias. PMID:11055583

  7. Regulation of Histone Acetylation by Autophagy in Parkinson Disease.

    PubMed

    Park, Goonho; Tan, Jieqiong; Garcia, Guillermina; Kang, Yunyi; Salvesen, Guy; Zhang, Zhuohua

    2016-02-12

    Parkinson disease (PD) is the most common age-dependent neurodegenerative movement disorder. Accumulated evidence indicates both environmental and genetic factors play important roles in PD pathogenesis, but the potential interaction between environment and genetics in PD etiology remains largely elusive. Here, we report that PD-related neurotoxins induce both expression and acetylation of multiple sites of histones in cultured human cells and mouse midbrain dopaminergic (DA) neurons. Consistently, levels of histone acetylation are markedly higher in midbrain DA neurons of PD patients compared to those of their matched control individuals. Further analysis reveals that multiple histone deacetylases (HDACs) are concurrently decreased in 1-methyl-4-phenylpyridinium (MPP(+))-treated cells and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse brains, as well as midbrain tissues of human PD patients. Finally, inhibition of histone acetyltransferase (HAT) protects, whereas inhibition of HDAC1 and HDAC2 potentiates, MPP(+)-induced cell death. Pharmacological and genetic inhibition of autophagy suppresses MPP(+)-induced HDACs degradation. The study reveals that PD environmental factors induce HDACs degradation and histone acetylation increase in DA neurons via autophagy and identifies an epigenetic mechanism in PD pathogenesis. PMID:26699403

  8. Regulation of bacterial physiology by lysine acetylation of proteins.

    PubMed

    Bernal, Vicente; Castaño-Cerezo, Sara; Gallego-Jara, Julia; Écija-Conesa, Ana; de Diego, Teresa; Iborra, José Luis; Cánovas, Manuel

    2014-12-25

    Post-translational modification of proteins is a reversible mechanism of cellular adaptation to changing environmental conditions. In eukaryotes, the physiological relevance of N-ɛ-lysine protein acetylation is well demonstrated. In recent times, important roles in the regulation of metabolic processes in bacteria are being uncovered, adding complexity to cellular regulatory networks. The aim of this mini-review is to sum up the current state-of-the-art in the regulation of bacterial physiology by protein acetylation. Current knowledge on the molecular biology aspects of known bacterial protein acetyltransferases and deacetylases will be summarized. Protein acetylation in Escherichia coli, Salmonella enterica, Bacillus subtilis, Rhodopseudomonas palustris and Mycobacterium tuberculosis, will be explained in the light of their physiological relevance. Progress in the elucidation of bacterial acetylomes and the emerging understanding of chemical acylation mechanisms will be discussed together with their regulatory and evolutionary implications. Fundamental molecular studies detailing this recently discovered regulatory mechanism pave the way for their prospective application for the construction of synthetic regulation networks. PMID:24636882

  9. Determination of NAT2 acetylation status in the Greenlandic population.

    PubMed

    Geller, Frank; Soborg, Bolette; Koch, Anders; Michelsen, Sascha Wilk; Bjorn-Mortensen, Karen; Carstensen, Lisbeth; Birch, Emilie; Nordholm, Anne Christine; Johansen, Marie Mila Broby; Børresen, Malene Landbo; Feenstra, Bjarke; Melbye, Mads

    2016-04-01

    N-acetyltransferase 2 (NAT2) is a well-studied phase II xenobiotic metabolizing enzyme relevant in drug metabolism and cancerogenesis. NAT2 activity is largely determined by genetic polymorphisms in the coding region of the corresponding gene. We investigated NAT2 acetylation status in 1556 individuals from Greenland based on four different single nucleotide polymorphism (SNP) panels and the tagging SNP rs1495741. There was good concordance between the NAT2 status inferred by the different SNP combinations. Overall, the fraction of slow acetylators was low with 17.5 % and varied depending on the degree of Inuit ancestry; in individuals with <50 % Inuit ancestry, we observed more than 25 % slow acetylators reflecting European ancestry. Greenland has a high incidence of tuberculosis, and individual dosing of isoniazid according to NAT2 status has been shown to improve treatment and reduce side effects. Our findings could be a first step in pharmacogenetics-based tuberculosis therapy in Greenland. PMID:25794903

  10. Regulation of acetyl coenzyme A synthetase in Escherichia coli.

    PubMed

    Kumari, S; Beatty, C M; Browning, D F; Busby, S J; Simel, E J; Hovel-Miner, G; Wolfe, A J

    2000-08-01

    Cells of Escherichia coli growing on sugars that result in catabolite repression or amino acids that feed into glycolysis undergo a metabolic switch associated with the production and utilization of acetate. As they divide exponentially, these cells excrete acetate via the phosphotransacetylase-acetate kinase pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively. Here, we present evidence that this switch occurs primarily through the induction of acs and that the timing and magnitude of this induction depend, in part, on the direct action of the carbon regulator cyclic AMP receptor protein (CRP) and the oxygen regulator FNR. It also depends, probably indirectly, upon the glyoxylate shunt repressor IclR, its activator FadR, and many enzymes involved in acetate metabolism. On the basis of these results, we propose that cells induce acs, and thus their ability to assimilate acetate, in response to rising cyclic AMP levels, falling oxygen partial pressure, and the flux of carbon through acetate-associated pathways. PMID:10894724

  11. Poly(ADP-Ribosyl)ation Affects Histone Acetylation and Transcription

    PubMed Central

    Verdone, Loredana; La Fortezza, Marco; Ciccarone, Fabio; Caiafa, Paola; Zampieri, Michele; Caserta, Micaela

    2015-01-01

    Poly(ADP-ribosyl)ation (PARylation) is a posttranslational protein modification catalyzed by members of the poly(ADP-ribose) polymerase (PARP) enzyme family. PARylation regulates a wide variety of biological processes in most eukaryotic cells including energy metabolism and cell death, maintenance of genomic stability, chromatin structure and transcription. Inside the nucleus, cross-talk between PARylation and other epigenetic modifications, such as DNA and histone methylation, was already described. In the present work, using PJ34 or ABT888 to inhibit PARP activity or over-expressing poly(ADP-ribose) glycohydrolase (PARG), we show decrease of global histone H3 and H4 acetylation. This effect is accompanied by a reduction of the steady state mRNA level of p300, Pcaf, and Tnf?, but not of Dnmt1. Chromatin immunoprecipitation (ChIP) analyses, performed at the level of the Transcription Start Site (TSS) of these four genes, reveal that changes in histone acetylation are specific for each promoter. Finally, we demonstrate an increase of global deacetylase activity in nuclear extracts from cells treated with PJ34, whereas global acetyltransferase activity is not affected, suggesting a role for PARP in the inhibition of histone deacetylases. Taken together, these results show an important link between PARylation and histone acetylation regulated transcription. PMID:26636673

  12. Investigation of acetylated chitosan microspheres as potential chemoembolic agents.

    PubMed

    Zhou, Xuan; Kong, Ming; Cheng, Xiaojie; Li, Jingjing; Li, Jing; Chen, Xiguang

    2014-11-01

    The aim was to investigate the potential of chitosan microspheres (CMs) with different acetylation using as a chemoembolic agent. Chitosan microspheres (CMs) were prepared via water-in-oil (W/O) emulsification cross-linking method, and acetylated chitosan microspheres (ACMs) were obtained by acetylation of CMs. Next, we characterized the morphology, size, composition and degrees of deacetylation using scanning electron microscopy (TEM), dynamic laser light scattering (DLS), and Fourier transform infrared spectrometer (FTIR). All microspheres had smooth surfaces and good mechanical flexibility, and all could pass through a 5F catheter. The swelling rate (SR) of CMs decreased significantly with the increase of pH (4.0-10.0) but ACMs did not change under the same conditions. Protein absorption assays suggested that albumin was more greatly adsorbed on CMs than on ACMs. Furthermore, CMs caused more blood clots than ACMs. ACMs caused hemolysis less than CMs (<5% of the time). Data indicated that ACMs had more hemocompatibility. Cytotoxicity tests indicated that ACMs initially had less cell attached proliferation but increased with incubation. In contrast, the relative growth rate of mouse embryo fibroblasts (MEFs) on CMs decreased gradually. The results suggested that ACMs could stimulate the growth of MEFs, and CMs were not cytotoxic to MEFs. Thus, ACMs were more biocompatible with greater potential to be used as chemoembolic material. PMID:25311962

  13. Rodent models of the human isoniazid-acetylator polymorphism.

    PubMed

    Tannen, R H; Weber, W W

    1979-01-01

    Inbred strains and subpopulations of rats, laboratory mice, and deer mice were examined for individual variation in the ability to metabolize several arylamines (p-aminobenzoic acid, sulfamethazine, aniline, alpha-naphthylamine, and aminofluorene) by N-acetylation. Individual differences within species were found to be dependent upon the tissue source of N-acetyltransferase activity and the acetyl acceptor employed. Long-Evans rats possessed about 2-fold more p-aminobenzoic acid N-acetyltransferase activity in blood and liver than Sprague-Dawley rats; no strain differences could be found with sulfamethazine. Nine strains of laboratory mice (Mus musculus) were found to have considerable liver p-aminobenzoic acid N-acetyltransferase activity but only slight activity towards sulfamethazine. No strain differences were apparent in regard to liver N-acetyltransferase activity. Blood p-aminobenzoic acid N-acetyltransferase activity was distinctly polymorphic in laboratory mice; of the nine strains tested, only A/J mice did not have this activity. Partially inbred deer mice (Peromyscus maniculatus) showed a narrower phenotypic range than random-bred stock from which they were obtained, which suggests the existence of distinct subpopulations with respect to N-acetylation capacity. Presumptive evidence for multiple forms of N-acetyltransferase in liver and blood was obtained through a study of substrate specificity. PMID:40765

  14. Impacts of microalgae pre-treatments for improved anaerobic digestion: thermal treatment, thermal hydrolysis, ultrasound and enzymatic hydrolysis.

    PubMed

    Ometto, Francesco; Quiroga, Gerardo; Peni?ka, Pavel; Whitton, Rachel; Jefferson, Bruce; Villa, Raffaella

    2014-11-15

    Anaerobic digestion (AD) of microalgae is primarily inhibited by the chemical composition of their cell walls containing biopolymers able to resist bacterial degradation. Adoption of pre-treatments such as thermal, thermal hydrolysis, ultrasound and enzymatic hydrolysis have the potential to remove these inhibitory compounds and enhance biogas yields by degrading the cell wall, and releasing the intracellular algogenic organic matter (AOM). This work investigated the effect of four pre-treatments on three microalgae species, and their impact on the quantity of soluble biomass released in the media and thus on the digestion process yields. The analysis of the composition of the soluble COD released and of the TEM images of the cells showed two main degradation actions associated with the processes: (1) cell wall damage with the release of intracellular AOM (thermal, thermal hydrolysis and ultrasound) and (2) degradation of the cell wall constituents with the release of intracellular AOM and the solubilisation of the cell wall biopolymers (enzymatic hydrolysis). As a result of this, enzymatic hydrolysis showed the greatest biogas yield increments (>270%) followed by thermal hydrolysis (60-100%) and ultrasounds (30-60%). PMID:25150520

  15. Hydrolysis and fractionation of lignocellulosic biomass

    DOEpatents

    Torget, Robert W. (Littleton, CO); Padukone, Nandan (Denver, CO); Hatzis, Christos (Denver, CO); Wyman, Charles E. (Lakewood, CO)

    2000-01-01

    A multi-function process is described for the hydrolysis and fractionation of lignocellulosic biomass to separate hemicellulosic sugars from other biomass components such as extractives and proteins; a portion of the solubilized lignin; cellulose; glucose derived from cellulose; and insoluble lignin from said biomass comprising one or more of the following: optionally, as function 1, introducing a dilute acid of pH 1.0-5.0 into a continual shrinking bed reactor containing a lignocellulosic biomass material at a temperature of about 94 to about 160.degree. C. for a period of about 10 to about 120 minutes at a volumetric flow rate of about 1 to about 5 reactor volumes to effect solubilization of extractives, lignin, and protein by keeping the solid to liquid ratio constant throughout the solubilization process; as function 2, introducing a dilute acid of pH 1.0-5.0, either as virgin acid or an acidic stream from another function, into a continual shrinking bed reactor containing either fresh biomass or the partially fractionated lignocellulosic biomass material from function 1 at a temperature of about 94-220.degree. C. for a period of about 10 to about 60 minutes at a volumetric flow rate of about 1 to about 5 reactor volumes to effect solubilization of hemicellulosic sugars, semisoluble sugars and other compounds, and amorphous glucans by keeping the solid to liquid ratio constant throughout the solubilization process; as function 3, optionally, introducing a dilute acid of pH 1.0-5.0 either as virgin acid or an acidic stream from another function, into a continual shrinking bed reactor containing the partially fractionated lignocellulosic biomass material from function 2 at a temperature of about 180-280.degree. C. for a period of about 10 to about 60 minutes at a volumetric flow rate of 1 to about 5 reactor volumes to effect solubilization of cellulosic sugars by keeping the solid to liquid ratio constant throughout the solubilization process; and as function 4, optionally, introducing a dilute acid of pH 1.0-5.0 either as virgin acid or an acidic stream from another function, into a continual shrinking bed reactor containing the partially fractionated lignocellulosic biomass material from function 3 at a temperature of about 180-280.degree. C. for a period of about 10 to about 60 minutes at a volumetric flow rate of about 1 to about 5 reactor volumes to effect solubilization of cellulosic sugars by keeping the solid to liquid ratio constant throughout the solubilization process.

  16. Acetylation of Werner syndrome protein (WRN): relationships with DNA damage, DNA replication and DNA metabolic activities

    PubMed Central

    Lozada, Enerlyn; Yi, Jingjie; Luo, Jianyuan; Orren, David K.

    2014-01-01

    Loss of WRN function causes Werner Syndrome, characterized by increased genomic instability, elevated cancer susceptibility and premature aging. Although WRN is subject to acetylation, phosphorylation and sumoylation, the impact of these modifications on WRNs DNA metabolic function remains unclear. Here, we examined in further depth the relationship between WRN acetylation and its role in DNA metabolism, particularly in response to induced DNA damage. Our results demonstrate that endogenous WRN is acetylated somewhat under unperturbed conditions. However, levels of acetylated WRN significantly increase after treatment with certain DNA damaging agents or the replication inhibitor hydroxyurea. Use of DNA repair-deficient cells or repair pathway inhibitors further increase levels of acetylated WRN, indicating that induced DNA lesions and their persistence are at least partly responsible for increased acetylation. Notably, acetylation of WRN correlates with inhibition of DNA synthesis, suggesting that replication blockage might underlie this effect. Moreover, WRN acetylation modulates its affinity for and activity on certain DNA structures, in a manner that may enhance its relative specificity for physiological substrates. Our results also show that acetylation and deacetylation of endogenous WRN is a dynamic process, with sirtuins and other histone deacetylases contributing to WRN deacetylation. These findings advance our understanding of the dynamics of WRN acetylation under unperturbed conditions and following DNA damage induction, linking this modification not only to DNA damage persistence but also potentially to replication stalling caused by specific DNA lesions. Our results are consistent with proposed metabolic roles for WRN and genomic instability phenotypes associated with WRN deficiency. PMID:24965941

  17. Acetylation mimic of lysine 280 exacerbates human Tau neurotoxicity in vivo.

    PubMed

    Gorsky, Marianna Karina; Burnouf, Sylvie; Dols, Jacqueline; Mandelkow, Eckhard; Partridge, Linda

    2016-01-01

    Dysfunction and accumulation of the microtubule-associated human Tau (hTau) protein into intraneuronal aggregates is observed in many neurodegenerative disorders including Alzheimer's disease (AD). Reversible lysine acetylation has recently emerged as a post-translational modification that may play an important role in the modulation of hTau pathology. Acetylated hTau species have been observed within hTau aggregates in human AD brains and multi-acetylation of hTau in vitro regulates its propensity to aggregate. However, whether lysine acetylation at position 280 (K280) modulates hTau-induced toxicity in vivo is unknown. We generated new Drosophila transgenic models of hTau pathology to evaluate the contribution of K280 acetylation to hTau toxicity, by analysing the respective toxicity of pseudo-acetylated (K280Q) and pseudo-de-acetylated (K280R) mutant forms of hTau. We observed that mis-expression of pseudo-acetylated K280Q-hTau in the adult fly nervous system potently exacerbated fly locomotion defects and photoreceptor neurodegeneration. In addition, modulation of K280 influenced total hTau levels and phosphorylation without changing hTau solubility. Altogether, our results indicate that pseudo-acetylation of the single K280 residue is sufficient to exacerbate hTau neurotoxicity in vivo, suggesting that acetylated K280-hTau species contribute to the pathological events leading to neurodegeneration in AD. PMID:26940749

  18. Proteome-wide analysis reveals widespread lysine acetylation of major protein complexes in the malaria parasite

    PubMed Central

    Cobbold, Simon A.; Santos, Joana M.; Ochoa, Alejandro; Perlman, David H.; Llinás, Manuel

    2016-01-01

    Lysine acetylation is a ubiquitous post-translational modification in many organisms including the malaria parasite Plasmodium falciparum, yet the full extent of acetylation across the parasite proteome remains unresolved. Moreover, the functional significance of acetylation or how specific acetyl-lysine sites are regulated is largely unknown. Here we report a seven-fold expansion of the known parasite ‘acetylome’, characterizing 2,876 acetylation sites on 1,146 proteins. We observe that lysine acetylation targets a diverse range of protein complexes and is particularly enriched within the Apicomplexan AP2 (ApiAP2) DNA-binding protein family. Using quantitative proteomics we determined that artificial perturbation of the acetate/acetyl-CoA balance alters the acetyl-lysine occupancy of several ApiAP2 DNA-binding proteins and related transcriptional proteins. This metabolic signaling could mediate significant downstream transcriptional responses, as we show that acetylation of an ApiAP2 DNA-binding domain ablates its DNA-binding propensity. Lastly, we investigated the acetyl-lysine targets of each class of lysine deacetylase in order to begin to explore how each class of enzyme contributes to regulating the P. falciparum acetylome. PMID:26813983

  19. Proteome-wide analysis reveals widespread lysine acetylation of major protein complexes in the malaria parasite.

    PubMed

    Cobbold, Simon A; Santos, Joana M; Ochoa, Alejandro; Perlman, David H; Llins, Manuel

    2016-01-01

    Lysine acetylation is a ubiquitous post-translational modification in many organisms including the malaria parasite Plasmodium falciparum, yet the full extent of acetylation across the parasite proteome remains unresolved. Moreover, the functional significance of acetylation or how specific acetyl-lysine sites are regulated is largely unknown. Here we report a seven-fold expansion of the known parasite 'acetylome', characterizing 2,876 acetylation sites on 1,146 proteins. We observe that lysine acetylation targets a diverse range of protein complexes and is particularly enriched within the Apicomplexan AP2 (ApiAP2) DNA-binding protein family. Using quantitative proteomics we determined that artificial perturbation of the acetate/acetyl-CoA balance alters the acetyl-lysine occupancy of several ApiAP2 DNA-binding proteins and related transcriptional proteins. This metabolic signaling could mediate significant downstream transcriptional responses, as we show that acetylation of an ApiAP2 DNA-binding domain ablates its DNA-binding propensity. Lastly, we investigated the acetyl-lysine targets of each class of lysine deacetylase in order to begin to explore how each class of enzyme contributes to regulating the P. falciparum acetylome. PMID:26813983

  20. Cell differentiation along multiple pathways accompanied by changes in histone acetylation status.

    PubMed

    Legartov, So?a; Kozubek, Stanislav; Franek, Michal; Zdrhal, Zbyn?k; Lochmanov, Gabriela; Martinet, Nadine; Brtov, Eva

    2014-04-01

    Post-translational modification of histones is fundamental to the regulation of basic nuclear processes and subsequent cellular events, including differentiation. In this study, we analyzed acetylated forms of histones H2A, H2B, and H4 during induced differentiation in mouse (mESCs) and human (hESCs) embryonic stem cells and during induced enterocytic differentiation of colon cancer cells in vitro. Endoderm-like differentiation of mESCs induced by retinoic acid and enterocytic differentiation induced by histone deacetylase inhibitor sodium butyrate were accompanied by increased mono-, di-, and tri-acetylation of histone H2B and a pronounced increase in di- and tri-acetylation of histone H4. In enterocytes, mono-acetylation of histone H2A also increased and tetra-acetylation of histone H4 appeared only after induction of this differentiation pathway. During differentiation of hESCs, we observed increased mono-acetylation and decreased tri-acetylation of H2B. Mono-, di-, and tri-acetylation of H4 were reduced, manifested by a significant increase in nonacetylated H4 histones. Levels of acetylated histones increased during induced differentiation in mESCs and during histone deacetylase (HDAC) inhibitor-induced enterocytic differentiation, whereas differentiation of human ESCs was associated with reduced acetylation of histones H2B and H4. PMID:24697692

  1. Acetylation mimic of lysine 280 exacerbates human Tau neurotoxicity in vivo

    PubMed Central

    Gorsky, Marianna Karina; Burnouf, Sylvie; Dols, Jacqueline; Mandelkow, Eckhard; Partridge, Linda

    2016-01-01

    Dysfunction and accumulation of the microtubule-associated human Tau (hTau) protein into intraneuronal aggregates is observed in many neurodegenerative disorders including Alzheimer’s disease (AD). Reversible lysine acetylation has recently emerged as a post-translational modification that may play an important role in the modulation of hTau pathology. Acetylated hTau species have been observed within hTau aggregates in human AD brains and multi-acetylation of hTau in vitro regulates its propensity to aggregate. However, whether lysine acetylation at position 280 (K280) modulates hTau-induced toxicity in vivo is unknown. We generated new Drosophila transgenic models of hTau pathology to evaluate the contribution of K280 acetylation to hTau toxicity, by analysing the respective toxicity of pseudo-acetylated (K280Q) and pseudo-de-acetylated (K280R) mutant forms of hTau. We observed that mis-expression of pseudo-acetylated K280Q-hTau in the adult fly nervous system potently exacerbated fly locomotion defects and photoreceptor neurodegeneration. In addition, modulation of K280 influenced total hTau levels and phosphorylation without changing hTau solubility. Altogether, our results indicate that pseudo-acetylation of the single K280 residue is sufficient to exacerbate hTau neurotoxicity in vivo, suggesting that acetylated K280-hTau species contribute to the pathological events leading to neurodegeneration in AD. PMID:26940749

  2. Kinetics of catalyzed hydrolysis of 4-methylumbelliferyl caprylate (MUCAP) salmonella reagent

    NASA Astrophysics Data System (ADS)

    Al-Kady, Ahmed S.; Ahmed, El-Sadat I.; Gaber, M.; Hussein, Mohamed M.; Ebeid, El-Zeiny M.

    2011-09-01

    The kinetics of chemical hydrolysis including neutral, acid- and base-catalyzed hydrolysis of 4-methylumbelliferyl caprylate (MUCAP) salmonella reagent were studied at different temperatures. The rate constants and activation parameters were determined by following the build-up of fluorescence peak of the hydrolysis product 4-methylumbelliferone (4-MU). The time scale of esterase enzyme hydrolysis caused by salmonella was compared with chemical hydrolysis as a background process.

  3. Kinetics of catalyzed hydrolysis of 4-methylumbelliferyl caprylate (MUCAP) salmonella reagent.

    PubMed

    Al-Kady, Ahmed S; Ahmed, El-Sadat I; Gaber, M; Hussein, Mohamed M; Ebeid, El-Zeiny M

    2011-09-01

    The kinetics of chemical hydrolysis including neutral, acid- and base-catalyzed hydrolysis of 4-methylumbelliferyl caprylate (MUCAP) salmonella reagent were studied at different temperatures. The rate constants and activation parameters were determined by following the build-up of fluorescence peak of the hydrolysis product 4-methylumbelliferone (4-MU). The time scale of esterase enzyme hydrolysis caused by salmonella was compared with chemical hydrolysis as a background process. PMID:21715222

  4. Endogenous apolipoprotein E modulates cholesterol efflux and cholesteryl ester hydrolysis mediated by high-density lipoprotein-3 and lipid-free apolipoproteins in mouse peritoneal macrophages.

    PubMed

    Langer, C; Huang, Y; Cullen, P; Wiesenhtter, B; Mahley, R W; Assmann, G; von Eckardstein, A

    2000-01-01

    We investigated the effect of endogenous apolipoprotein (apo) E synthesis in mouse peritoneal macrophages on cholesterol efflux and intracellular cholesteryl ester hydrolysis mediated by high-density lipoprotein-3 (HDL3) and lipid-free apolipoproteins (apo). After loading with acetylated LDL (acLDL) peritoneal macrophages from wild-type (apoE(+/+)) and apoE-deficient (apoE(-/-)) mice were incubated with medium alone or with liposomes, HDL3, lipid-free apoA-I, or lipid-free apoE3. Cholesterol and cholesteryl esters in the cells and culture media were quantified by HPLC. Incubation of apoE(+/+) or apoE(-/-) macrophages for 18 h with medium alone or with liposomes did not cause significant changes in cellular cholesterol. Addition of HDL3, apoA-I, or apoE3 to the medium led to significant cholesterol efflux, which was less efficient in apoE(-/-) macrophages than in apoE(+/+) macrophages. HDL and lipid-free apolipoproteins were more effective in reducing the cellular content of cholesteryl esters of apoE(+/+) macrophages than of apoE(-/-) macrophages, suggesting that endogenous apoE stimulates cholesteryl ester hydrolysis. The difference in the mass of cholesteryl esters was more pronounced for cholesteryl arachidonate and linoleate than for cholesteryl oleate or palmitate. Furthermore, in [(14)C]arachidonate labeling experiments cholesterol arachidonate hydrolysis was higher in apoE(+/+) macrophages than in apoE(-/-) macrophages in the presence of cholesterol efflux mediated by HDL3 or apoA-I. In contrast, in the absence of cholesterol efflux cholesterol arachidonate synthesis was higher in apoE(+/+) macrophages than in apoE-/- macrophages. Taken together, our data suggest that endogenous apoE stimulates cholesterol efflux and intracellular cholesteryl ester hydrolysis mediated by HDL3 and lipid-free apolipoproteins in mouse peritoneal macrophages. This may contribute to the antiatherogenic effect of apoE. PMID:10933584

  5. Fluoride incorporation into apatite crystals delays amelogenin hydrolysis

    PubMed Central

    DenBesten, Pamela; Zhu, Li; Li, Wu; Tanimoto, Kotaro; Liu, Haichuan; Witkowska, Halina Ewa

    2012-01-01

    Enamel fluorosis has been related to an increase in the amount of amelogenin in fluorosed enamel as compared to normal enamel in the maturation stage. In this study we tested the hypothesis that fluoride incorporated into carbonated apatite alters amelogenin hydrolysis. Recombinant human amelogenin (rh174) was allowed to bind to 0.15 mg of carbonated hydroxyapatite (CAP) or fluoride-containing carbonated hydroxyapatite (F-CAP) synthesized to contain 100, 1000 or 4000 ppm F-. After 3 h digestion with recombinant human MMP20 or KLK4, bound protein was characterized by reverse-phase HPLC. Proteolytic fragments formed after 24 h digestion of amelogenin, were identified by LC tandem mass spectrometry (LCMS/MS). The hydrolysis of amelogenin bound to F100-CAP by both MMP20 and KLK4 was significantly reduced in a dose dependent manner as compared to CAP. After 24 h hydrolysis, the number of cleavage sites in bound amelogenin by MMP20 were similar in CAP and F100-CAP, whereas there were 24 fewer cleavage sites identified for the KLK4 hydrolysis on F100-CAP as compared to CAP. These results suggest that the reduced hydrolysis of amelogenins in fluorosed enamel may be partially due to the increased fluoride content in fluoride containing apatite, contributing to the hypomineralized enamel matrix phenotype observed in fluorosed enamel. PMID:22243219

  6. Enzymatic hydrolysis of fractionated products from oil thermally oxidated

    SciTech Connect

    Yashida, H.; Alexander, J.C.

    1983-01-01

    Enzymatic hydrolysis of the acylglycerol products obtained from thermally oxidized vegetable oils was studied. Corn, sunflower and soybean oils were heated in the laboratory at 180/sup 0/C for 50, 70 and 100 hr with aeration and directly fractionated by silicic acid column chromatography. By successive elution with 20%, then 60% isopropyl ether in n-hexane, and diethyl ether, the thermally oxidized oils were separated into three fractions: the nonpolar fraction (monomeric compounds), slightly polar fraction (dimeric compounds), and polar fraction comprising oligomeric compounds. Enzymatic hydrolysis with pancreatic lipase showed that the monomers were hydrolyzed as rapidly as the corresponding unheated oils, the dimers much more slowly, and the oligomeric compounds barely at all. Overall, the hydrolysis of the dimers was less than 23% of that for the monomers, with small differences among the oils. Longer heating periods resulted in greater reductions in hydrolysis of the dimeric compounds. These results suggest that the degree of enzymatic hydrolysis of the fractionated acylglycerol compounds is related to differences in the thermal oxidative deterioration, and amounts of polar compounds in the products. (33 Refs.)

  7. Acid hydrolysis of corn stover in a concentrated slurry system

    SciTech Connect

    Teng, K.F.

    1984-01-01

    The objective of this research was to characterize the hydrolysis kinetics of corn stover using sulfuric acid as the catalyst in a concentrated slurry. The kinetic data were obtained in a specially constructed continuous flow reactor with an in-line sampling system. The scope and range of experimental variables included temperature, acid concentration, liquid-to-solid ratio and reaction time. It is found that glucan hydrolysis follows a zero order kinetic in the range of experimental variables investigated. Both acid concentration and temperature increase the rate of glucan hydrolysis. A rate expression for glucan hydrolysis is developed both on the basis of adsorbed acid and bulk acid concentration. A kinetic model is proposed for the reaction. An integral approach is used to determine the parameters in the kinetic model and their numerical values are found to be comparable to those reported in the literature. It is found that at a given temperature, acid concentration and glucan conversion, there is an optimum liquid-to-solid ratio which maximizes glucose concentration. It is shown experimentally that glucan hydrolysis is not mass transfer limited at as low a liquid-to-solid ratio as 2:5:1.

  8. Starch hydrolysis modeling: application to fuel ethanol production.

    PubMed

    Murthy, Ganti S; Johnston, David B; Rausch, Kent D; Tumbleson, M E; Singh, Vijay

    2011-09-01

    Efficiency of the starch hydrolysis in the dry grind corn process is a determining factor for overall conversion of starch to ethanol. A model, based on a molecular approach, was developed to simulate structure and hydrolysis of starch. Starch structure was modeled based on a cluster model of amylopectin. Enzymatic hydrolysis of amylose and amylopectin was modeled using a Monte Carlo simulation method. The model included the effects of process variables such as temperature, pH, enzyme activity and enzyme dose. Pure starches from wet milled waxy and high-amylose corn hybrids and ground yellow dent corn were hydrolyzed to validate the model. Standard deviations in the model predictions for glucose concentration and DE values after saccharification were less than 0.15% (w/v) and 0.35%, respectively. Correlation coefficients for model predictions and experimental values were 0.60 and 0.91 for liquefaction and 0.84 and 0.71 for saccharification of amylose and amylopectin, respectively. Model predictions for glucose (R2 = 0.69-0.79) and DP4+ (R2 = 0.8-0.68) were more accurate than the maltotriose and maltose for hydrolysis of high-amylose and waxy corn starch. For yellow dent corn, simulation predictions for glucose were accurate (R2 > 0.73) indicating that the model can be used to predict the glucose concentrations during starch hydrolysis. PMID:21487699

  9. Enzymatic hydrolysis of organophosphate insecticides, a possible pesticide disposal method.

    PubMed Central

    Munnecke, D M

    1976-01-01

    A crude cell extract from a mixed bacterial culture growing on parathion, an organophosphate insecticide, hydrolyzed parathion (21 C) at a rate of 416 nmol/min per mg of protein. This rate of enzymatic hydrolysis, when compared with chemical hydrolysis by 0.1 N sodium hydroxide at 40 C, was 2, 450 times faster. Eight of 12 commonly used organophosphate insecticides were enzymatically hydrolyzed with this enzyme preparation at rates ranging from 12 to 1,360 nmol/min per mg of protein. Seven pesticides were hydrolyzed at rates significantly higher (40 to 1,005 times faster) than chemical hydrolysis. The pH optimum for enzymatic hydrolysis of the eight pesticides ranged from 8.5 to 9.5, with less than 50% of maximal activity expressed at pH 7.0. Maximal enzyme activity occurred at 35 C. The crude extract lost its activity at the rate of only 0.75%/day when stored at 6 C. Eight organic solvents, ranging from methanol to hexane, at low concentrations stimulated enzymatic hydrolysis by 3 to 20%, whereas at higher concentrations (1,000 mg/liter) they inhibited the reaction (9 to 50%). Parathion metabolites p-nitrophenol, hydroquinone, and diethylthiophosphoric acid, at up to 100-mg/liter concentrations, did not significantly influence enzyme activity. PMID:9901

  10. Factors affecting the rate of hydrolysis of starch in food.

    PubMed

    Snow, P; O'Dea, K

    1981-12-01

    After accurate determination of the content of available carbohydrate in a wide variety of cereals, as in vitro method was used to study factors that influence hydrolysis rates of starch in foods. Fiber, physical form, cooking, and the possible presence of a natural amylase inhibitor were all shown to affect hydrolysis rates of starch. Fiber only exerted an inhibiting effect on the rate of hydrolysis when it formed a physical barrier to limit access of the hydrolytic enzymes to the starch (as in whole brown rice, for example). Particle size played an important role in determining the rate of hydrolysis. Cooking made the starch much more readily available for enzymic hydrolysis presumably by gelatinizing it. Stoneground wholemeal flour was hydrolyzed more slowly than white flour. This is consistent with the presence of a natural amylase inhibitor that has been isolated from wheat germ in the whole grain. Our results suggest that such amylase inhibitor activity is destroyed by passage through the roller mill, since the starch in wheat germ and standard wholemeal flour (i.e., not stoneground but reconstituted after passage through the roller mill) was hydrolyzed at a rate identical to white flour. PMID:6172034

  11. QM/MM investigation of ATP hydrolysis in aqueous solution.

    PubMed

    Wang, Cui; Huang, Wenting; Liao, Jie-Lou

    2015-03-01

    Adenosine-5'-triphosphate (ATP) hydrolysis represents a most important reaction in biology. Despite extensive research efforts, the mechanism for ATP hydrolysis in aqueous solution still remains under debate. Previous theoretical studies often predefined reaction coordinates to characterize the mechanism for ATP hydrolysis in water with Mg(2+) by evaluating free energy profiles through these preassumed reaction paths. In the present work, a nudged elastic band method is applied to identify the minimum energy path calculated with a hybrid quantum mechanics and molecular mechanics approach. Along the reaction path, the free energy profile was obtained to have a single transition state and the activation energy of 32.5 kcal/mol. This transition state bears a four-centered structure that describes a concerted nature of the reaction. In the More-O'Ferrall-Jencks diagram, the results show that the reaction proceeds through a concerted path before the system reaches the transition state and along an associative path after the transition state. In addition, the calculated reaction free energy is -7.0 kcal/mol, in good agreement with experiment, capturing the exothermic feature of MgATP(2-) hydrolysis in aqueous solution, whereas the reaction was often shown to be endothermic in the previous theoretical studies. As Mg(2+) is required for ATP hydrolysis in cells, its role in the reaction is also elucidated. PMID:25658024

  12. Lactose Hydrolysis in Milk and Dairy Whey Using Microbial ?-Galactosidases.

    PubMed

    Dutra Rosolen, Michele; Gennari, Adriano; Volpato, Giandra; Volken de Souza, Claucia Fernanda

    2015-01-01

    This work aimed at evaluating the influence of enzyme concentration, temperature, and reaction time in the lactose hydrolysis process in milk, cheese whey, and whey permeate, using two commercial ?-galactosidases of microbial origins. We used Aspergillus oryzae (at temperatures of 10 and 55C) and Kluyveromyces lactis (at temperatures of 10 and 37C) ?-galactosidases, both in 3, 6, and 9?U/mL concentrations. In the temperature of 10C, the K. lactis ?-galactosidase enzyme is more efficient in the milk, cheese whey, and whey permeate lactose hydrolysis when compared to A. oryzae. However, in the enzyme reaction time and concentration conditions evaluated, 100% lactose hydrolysis was not reached using the K. lactis ?-galactosidase. The total lactose hydrolysis in whey and permeate was obtained with the A. oryzae enzyme, when using its optimum temperature (55C), at the end of a 12?h reaction, regardless of the enzyme concentration used. For the lactose present in milk, this result occurred in the concentrations of 6 and 9?U/mL, with the same time and temperature conditions. The studied parameters in the lactose enzymatic hydrolysis are critical for enabling the application of ?-galactosidases in the food industry. PMID:26587283

  13. Granular starch hydrolysis for fuel ethanol production

    NASA Astrophysics Data System (ADS)

    Wang, Ping

    Granular starch hydrolyzing enzymes (GSHE) convert starch into fermentable sugars at low temperatures (≤48°C). Use of GSHE in dry grind process can eliminate high temperature requirements during cooking and liquefaction (≥90°C). In this study, GSHE was compared with two combinations of commercial alpha-amylase and glucoamylase (DG1 and DG2, respectively). All three enzyme treatments resulted in comparable ethanol concentrations (between 14.1 to 14.2% v/v at 72 hr), ethanol conversion efficiencies and ethanol and DDGS yields. Sugar profiles for the GSHE treatment were different from DG1 and DG2 treatments, especially for glucose. During simultaneous saccharification and fermentation (SSF), the highest glucose concentration for the GSHE treatment was 7% (w/v); for DG1 and DG2 treatments, maximum glucose concentration was 19% (w/v). GSHE was used in one of the fractionation technologies (enzymatic dry grind) to improve recovery of germ and pericarp fiber prior to fermentation. The enzymatic dry grind process with GSHE was compared with the conventional dry grind process using GSHE with the same process parameters of dry solids content, pH, temperature, time, enzyme and yeast usages. Ethanol concentration (at 72 hr) of the enzymatic process was 15.5% (v/v), which was 9.2% higher than the conventional process (14.2% v/v). Distillers dried grains with solubles (DDGS) generated from the enzymatic process (9.8% db) was 66% less than conventional process (28.3% db). Three additional coproducts, germ 8.0% (db), pericarp fiber 7.7% (db) and endosperm fiber 5.2% (db) were produced. Costs and amounts of GSHE used is an important factor affecting dry grind process economics. Proteases can weaken protein matrix to aid starch release and may reduce GSHE doses. Proteases also can hydrolyze protein into free amino nitrogen (FAN), which can be used as a yeast nutrient during fermentation. Two types of proteases, exoprotease and endoprotease, were studied; protease and urea addition were evaluated in the dry grind process using GSHE (GSH process). Addition of proteases resulted in higher ethanol concentrations (15.2 to 18.0% v/v) and lower (DDGS) yields (32.9 to 45.8% db) compared to the control (no protease addition). As level of proteases and GSHE increased, ethanol concentrations increased and DDGS yields decreased. Proteases addition reduced required GSHE dose. Ethanol concentrations with protease addition alone were higher than with urea or with addition of both protease and urea. Corn endosperm consists of soft and hard endosperm. More exposed starch granules and rough surfaces produced from soft endosperm compared to hard endosperm will create more surface area which will benefit the solid phase hydrolysis as used in GSH process. In this study, the effects of protease, urea, endosperm hardness and GSHE levels on the GSH process were evaluated. Soft and hard endosperm materials were obtained by grinding and sifting flaking grits from dry milling pilot plant. Soft endosperm resulted in higher ethanol concentrations (at 72 hr) compared to ground corn or hard endosperm. Addition of urea increased ethanol concentrations (at 72 hr) for soft and hard endosperm. The effect of protease addition on increasing ethanol concentrations and fermentation rates was more predominant for soft endosperm, less for hard endosperm and least for ground corn. The GSH process with protease resulted in higher ethanol concentration than that with urea. For fermentation of soft endosperm, GSHE dose can be reduced. Ground corn fermented faster at the beginning than hard and soft endosperm due to the presence of inherent nutrients which enhanced yeast growth.

  14. Distinct and predictive histone lysine acetylation patterns at promoters, enhancers, and gene bodies.

    PubMed

    Rajagopal, Nisha; Ernst, Jason; Ray, Pradipta; Wu, Jie; Zhang, Michael; Kellis, Manolis; Ren, Bing

    2014-11-01

    In eukaryotic cells, histone lysines are frequently acetylated. However, unlike modifications such as methylations, histone acetylation modifications are often considered redundant. As such, the functional roles of distinct histone acetylations are largely unexplored. We previously developed an algorithm RFECS to discover the most informative modifications associated with the classification or prediction of mammalian enhancers. Here, we used this tool to identify the modifications most predictive of promoters, enhancers, and gene bodies. Unexpectedly, we found that histone acetylation alone performs well in distinguishing these unique genomic regions. Further, we found the association of characteristic acetylation patterns with genic regions and association of chromatin state with splicing. Taken together, our work underscores the diverse functional roles of histone acetylation in gene regulation and provides several testable hypotheses to dissect these roles. PMID:25122670

  15. In silico analysis of protein Lys-N𝜀-acetylation in plants

    PubMed Central

    Rao, R. Shyama Prasad; Thelen, Jay J.; Miernyk, Ján A.

    2014-01-01

    Among post-translational modifications, there are some conceptual similarities between Lys-N𝜀-acetylation and Ser/Thr/Tyr O-phosphorylation. Herein we present a bioinformatics-based overview of reversible protein Lys-acetylation, including some comparisons with reversible protein phosphorylation. The study of Lys-acetylation of plant proteins has lagged behind studies of mammalian and microbial cells; 1000s of acetylation sites have been identified in mammalian proteins compared with only hundreds of sites in plant proteins. While most previous emphasis was focused on post-translational modifications of histones, more recent studies have addressed metabolic regulation. Being directly coupled with cellular CoA/acetyl-CoA and NAD/NADH, reversible Lys-N𝜀-acetylation has the potential to control, or contribute to control, of primary metabolism, signaling, and growth and development. PMID:25136347

  16. Acetylation regulates gluconeogenesis by promoting PEPCK1 degradation via recruiting the UBR5 ubiquitin ligase

    PubMed Central

    Jiang, Wenqing; Wang, Shiwen; Xiao, Mengtao; Lin, Yan; Zhou, Lisha; Lei, Qunying; Xiong, Yue; Guan, Kun-Liang; Zhao, Shimin

    2014-01-01

    SUMMARY Protein acetylation has emerged as a major mechanism in regulating cellular metabolism. Whereas most glycolytic steps are reversible, the reaction catalyzed by pyruvate kinase is irreversible and the reverse reaction requires phosphoenolpyruvate carboxykinase (PEPCK1) to commit for gluconeogenesis. Here we show that acetylation regulates the stability of the gluconeogenic rate limiting enzyme PEPCK1, thereby modulating cellular response to glucose. High glucose destabilizes PEPCK1 by stimulating its acetylation. PEPCK1 is acetylated by the P300 acetyltransferase and this acetylation stimulates the interaction between PEPCK1 and UBR5, a HECT domain containing E3 ubiquitin ligase, therefore promoting PEPCK1 ubiquitinylation and degradation. Conversely, SIRT2 deacetylates and stabilizes PEPCK1. These observations represent an example that acetylation targets a metabolic enzyme to a specific E3 ligase in response to metabolic condition changes. Given that increased levels of PEPCK is linked with type II diabetes, this study also identifies potential therapeutic targets for diabetes. PMID:21726808

  17. Monoolein production by triglycerides hydrolysis using immobilized Rhizopus oryzae lipase.

    PubMed

    Ghattas, Nesrine; Abidi, Ferid; Galai, Said; Marzouki, M Nejib; Salah, Abderraouf Ben

    2014-07-01

    Lipase extracted from Rhizopus oryzae was immobilized in alginate gel beads. The effects of the immobilization conditions, such as, alginate concentration, CaCl2 concentration and amount of initial enzyme on retained activity (specific activity ratio of entrapped active lipase to free lipase) were investigated. The optimal conditions for lipase entrapment were determined: 2% (w/v) alginate concentration, 100mM CaCl2 and enzyme ratio of 2000IU/mL.In such conditions, immobilized lipase by inclusion in alginate showed a highest stability and activity, on olive oil hydrolysis reaction where it could be reused for 10 cycles. After 15min of hydrolysis reaction, the mass composition of monoolein, diolein and triolein were about 78%, 10% and 12%. Hydrolysis' products purification by column chromatography lead to a successful separation of reaction compounds and provide a pure fraction of monoolein which is considered as the widest used emulsifier in food and pharmaceutical industries. PMID:24755261

  18. Designer xylanosomes: protein nanostructures for enhanced xylan hydrolysis.

    PubMed

    McClendon, Shara D; Mao, Zichao; Shin, Hyun-Dong; Wagschal, Kurt; Chen, Rachel R

    2012-06-01

    This work reports the successful design, construction, and application of multi-functional, self-assembling protein complex, termed xylanosomes. Using the architecture of cellulosomes as template, these structures were designed specifically for hemicellulose hydrolysis. Four different xylanosomes were developed, with up to three different hemicellulase activities combined into a single structure. Each xylanosome was composed of two native or chimeric hemicellulases and tested on wheat arabinoxylan or destarched corn bran for enzymatic hydrolysis. After 24-h incubation, soluble sugars released from arabinoxylan increased up to 30 % with xylanosomes containing a xylanase and bi-functional arabinofuranosidase/xylosidase over the corresponding free, unstructured enzymes. Additionally, xylanosomes with a xylanase and a ferulic acid esterase removed between 15 and 20 % more ferulic acid from wheat arabinoxylan than free enzymes. Furthermore, xylanosomes exhibited synergy with cellulases on destarched corn bran, suggesting a possible use of these nanostructures in cellulose hydrolysis. PMID:22555497

  19. In vitro hydrolysis of monofluorophosphate by dental plaque microorganisms.

    PubMed

    Jackson, L R

    1982-07-01

    Enzymic hydrolysis of sodium monofluorophosphate by suspensions of dental microorganisms has been demonstrated at pH 5.1, pH 7.0, and pH 8.4, using a fluoride-selective electrode. The extracellular medium from viable Streptococcus mutans K1R cells contained low MFPase and paranitrophenyl phosphatase activity. It is hypothesized that the enzymes responsible for MFP hydrolysis by S. mutans K1R are intracellular, and that cell disruption is necessary for hydrolysis to be manifested; this question requires further study. In vitro MFPase activity was of a magnitude consistent with the hypothesis that it may significantly raise the fluoride ion concentration of plaque within the several minutes MFP would be in the mouth during toothbrushing. PMID:6282948

  20. Base hydrolysis and supercritical water oxidation of PBX-9404

    SciTech Connect

    Flesner, R.L.; Spontarelli, T.; Dell`Orco, P.C.; Kramer, J.F.; Sanchez, J.A.

    1994-11-09

    Base hydrolysis in combination with hydrothermal processing has been proposed as an environmentally acceptable alternative to open burning/open detonation for degradation and destruction of high explosives. In this report, the authors examine gaseous and aqueous products of base hydrolysis of the HMX-based plastic bonded explosive, PBX-9404. The authors also examine products from the subsequent hydrothermal treatment of the base hydrolysate. The gases produced from hydrolysis of PBX-9404 are ammonia, nitrous oxide, and nitrogen. Major aqueous products are sodium formate, acetate, nitrate, and nitrite, but not all carbon products have been identified. Hydrothermal processing of base hydrolysate destroyed up to 98% of the organic carbon in solution, and higher destruction efficiencies are possible. Major gas products detected from hydrothermal processing were nitrogen and nitrous oxide.

  1. Autoimmune regulator is acetylated by transcription coactivator CBP/p300

    SciTech Connect

    Saare, Mario; Rebane, Ana; SIAF, Swiss Institute of Allergy and Asthma Research, University of Zuerich, Davos ; Rajashekar, Balaji; Vilo, Jaak; Peterson, Paert

    2012-08-15

    The Autoimmune Regulator (AIRE) is a regulator of transcription in the thymic medulla, where it controls the expression of a large set of peripheral-tissue specific genes. AIRE interacts with the transcriptional coactivator and acetyltransferase CBP and synergistically cooperates with it in transcriptional activation. Here, we aimed to study a possible role of AIRE acetylation in the modulation of its activity. We found that AIRE is acetylated in tissue culture cells and this acetylation is enhanced by overexpression of CBP and the CBP paralog p300. The acetylated lysines were located within nuclear localization signal and SAND domain. AIRE with mutations that mimicked acetylated K243 and K253 in the SAND domain had reduced transactivation activity and accumulated into fewer and larger nuclear bodies, whereas mutations that mimicked the unacetylated lysines were functionally similar to wild-type AIRE. Analogously to CBP, p300 localized to AIRE-containing nuclear bodies, however, the overexpression of p300 did not enhance the transcriptional activation of AIRE-regulated genes. Further studies showed that overexpression of p300 stabilized the AIRE protein. Interestingly, gene expression profiling revealed that AIRE, with mutations mimicking K243/K253 acetylation in SAND, was able to activate gene expression, although the affected genes were different and the activation level was lower from those regulated by wild-type AIRE. Our results suggest that the AIRE acetylation can influence the selection of AIRE activated genes. -- Highlights: Black-Right-Pointing-Pointer AIRE is acetylated by the acetyltransferases p300 and CBP. Black-Right-Pointing-Pointer Acetylation occurs between CARD and SAND domains and within the SAND domain. Black-Right-Pointing-Pointer Acetylation increases the size of AIRE nuclear dots. Black-Right-Pointing-Pointer Acetylation increases AIRE protein stability. Black-Right-Pointing-Pointer AIRE acetylation mimic regulates a different set of AIRE target genes.

  2. Sulfation of deoxynivalenol, its acetylated derivatives, and T2-toxin?

    PubMed Central

    Fruhmann, Philipp; Skrinjar, Philipp; Weber, Julia; Mikula, Hannes; Warth, Benedikt; Sulyok, Michael; Krska, Rudolf; Adam, Gerhard; Rosenberg, Erwin; Hametner, Christian; Frhlich, Johannes

    2014-01-01

    The synthesis of several sulfates of trichothecene mycotoxins is presented. Deoxynivalenol (DON) and its acetylated derivatives were synthesized from 3-acetyldeoxynivalenol (3ADON) and used as substrate for sulfation in order to reach a series of five different DON-based sulfates as well as T2-toxin-3-sulfate. These substances are suspected to be formed during phase-II metabolism in plants and humans. The sulfation was performed using a sulfuryl imidazolium salt, which was synthesized prior to use. All protected intermediates and final products were characterized via NMR and will serve as reference materials for further investigations in the fields of toxicology and bioanalytics of mycotoxins. PMID:25170180

  3. Tetra-ethyl-ammonium (acetyl-acetonato)bromidotricarbonyl-rhenate(I).

    PubMed

    Brink, Alice; Visser, Hendrik G; Roodt, Andreas

    2010-01-01

    In the title compound, (C(8)H(20)N)[ReBr(C(5)H(7)O(2))(CO)(3)], the Re(I) atom in the rhenate anion is surrounded by three carbonyl ligands orientated in a facial arrangement, a bromide ligand and an acetyl-acetonate ligand, leading to a distorted octa-hedral ReC(3)BrO(2) coordination with a O-Re-O bite angle of 85.66?(7). An array of C-H?O and C-H?Br hydrogen-bonding inter-actions between the cations and the surrounding rhenate anions stabilize the crystal structure. PMID:21522557

  4. Tetraethylammonium (acetylacetonato)bromidotricarbonylrhenate(I)

    PubMed Central

    Brink, Alice; Visser, Hendrik G.; Roodt, Andreas

    2011-01-01

    In the title compound, (C8H20N)[ReBr(C5H7O2)(CO)3], the ReI atom in the rhenate anion is surrounded by three carbonyl ligands orientated in a facial arrangement, a bromide ligand and an acetylacetonate ligand, leading to a distorted octahedral ReC3BrO2 coordination with a OReO bite angle of 85.66?(7). An array of CH?O and CH?Br hydrogen-bonding interactions between the cations and the surrounding rhenate anions stabilize the crystal structure. PMID:21522557

  5. Therapeutics Targeting Protein Acetylation Perturb Latency of Human Viruses.

    PubMed

    Conrad, Ryan J; Ott, Melanie

    2016-03-18

    Persistent viral infections are widespread and represent significant public health burdens. Some viruses endure in a latent state by co-opting the host epigenetic machinery to manipulate viral gene expression. Small molecules targeting epigenetic pathways are now in the clinic for certain cancers and are considered as potential treatment strategies to reverse latency in HIV-infected individuals. In this review, we discuss how drugs interfering with one epigenetic pathway, protein acetylation, perturb latency of three families of pathogenic human viruses-retroviruses, herpesviruses, and papillomaviruses. PMID:26845514

  6. Impact of ?-amylase combined with hydrochloric acid hydrolysis on structure and digestion of waxy rice starch.

    PubMed

    Li, Hongyan; Zhu, Yanqiao; Jiao, Aiquan; Zhao, Jianwei; Chen, Xiaoming; Wei, Benxi; Hu, Xiuting; Wu, Chunsen; Jin, Zhengyu; Tian, Yaoqi

    2013-04-01

    The structure and in vitro digestibility of native waxy rice starch by the combined hydrolysis of ?-amylase and hydrochloric acid were investigated in this study. The combined hydrolysis technique generated higher hydrolysis rate and extent than the enzymatic hydrolysis. The granular appearance and chromatograph profile demonstrated that ?-amylase and hydrochloric acid exhibited different patterns of hydrolysis. The rise in the ratio of absorbance 1047/1022cm(-1), the melting temperature range (Tc-To), and the melting enthalpy (?H) were observed during the combined hydrolysis. These results suggest that ?-amylase simultaneously cleaves the amorphous and crystalline regions, whereas the amorphous regions of starch granules are preferentially hydrolyzed during the acid hydrolysis. Furthermore, the combined hydrolysis increased rapidly digestible starch (RDS) while decreased slowly digestible starch (SDS) and resistant starch (RS), indicating that the hydrolysis mode affected the digestion property of native waxy rice starch. PMID:23357798

  7. Genetic Evidence that the Acetylation of the Smc3p Subunit of Cohesin Modulates Its ATP-Bound State to Promote Cohesion Establishment in Saccharomyces cerevisiae

    PubMed Central

    Heidinger-Pauli, Jill M.; Onn, Itay; Koshland, Douglas

    2010-01-01

    Sister chromatid cohesion refers to the process by which sister chromatids are tethered together until the metaphase-to-anaphase transition. The evolutionarily conserved cohesin complex mediates sister chromatid cohesion. Cohesin not only ensures proper chromosome segregation, but also promotes high-fidelity DNA repair and transcriptional regulation. Two subunits of cohesin (Smc1p, Smc3p) are members of the structural maintenance of chromosomes (SMC) family. The SMC family is recognized by their large coiled-coil arms and conserved ATP-binding cassette-like ATPase domain. While both Smc1p and Smc3p ATP binding and hydrolysis are essential for cohesin function in vivo, little is known about how this core enzymatic activity is regulated to facilitate sister chromatid cohesion. Here we use SMC mutant proteins to block specific steps in cohesin's ATPase cycle in Saccharomyces cerevisiae. We show that blocking Smc3p-mediated ATP binding or Smc3p ATP hydrolysis traps unique functional states in cohesion. Finally, we provide evidence that Smc3p acetylation, which has an essential role in cohesion establishment, modulates the Smc3p ATP-bound state. PMID:20498298

  8. Involvement of protein acetylation in glucose-induced transcription of a stress-responsive promoter.

    PubMed

    Lima, Bruno P; Antelmann, Haike; Gronau, Katrin; Chi, Bui Khanh; Becher, Dörte; Brinsmade, Shaun R; Wolfe, Alan J

    2011-09-01

    In eukaryotes, lysine acetylation is a well-established post-translational modification that has been implicated in virtually all aspects of eukaryotic physiology. Although homologues of the enzymes that catalyse protein acetylation are widely conserved and distributed among bacterial species, not much is known about the impact of protein acetylation on bacterial physiology. Here, we present evidence that the Gcn5-like acetyltransferase YfiQ and the sirtuin deacetylase CobB play crucial roles in the transcription regulation of the periplasmic stress-responsive promoter cpxP when cells of Escherichia coli grow in the presence of glucose, an environment that induces protein acetylation. Under this growth condition, several acetylation sites were detected on three of the RNA polymerase subunits: β, β' and α. We focused on acetylations of the carboxy-terminal domain (CTD) of α because of its relative small size and its limited acetylation. We determined that K298 of α is acetylated in a glucose and YfiQ-dependent manner and that K298 is specifically required for glucose-induced cpxP transcription. Because the αCTD aids in promoter recognition by RNA polymerase, we propose its acetylation may influence bacterial physiology through effects on gene expression. PMID:21696463

  9. Enzymatic 2'-N-acetylation of arbekacin and antibiotic activity of its product.

    PubMed

    Hotta, K; Zhu, C B; Ogata, T; Sunada, A; Ishikawa, J; Mizuno, S; Ikeda, Y; Kondo, S

    1996-05-01

    Aminoglycoside antibiotics (AGs) with a free 2'-amino group were subjected to enzymatic N-acetylation using a cell free extract that contained an aminoglycoside 2'-N-acetyltransferase, AAC (2'), derived from a kasugamycin-producing strain of Streptomyces kasugaensis. TLC and antibiotic assay of the incubated reaction mixtures revealed that a modified compound retaining substantial antibiotic activity was formed from arbekacin (ABK), while modification of the other AGs resulted in the marked decrease in antibiotic activity. Structure determination following isolation from a large scale reaction mixture showed the modified ABK to be 2'-N-acetyl ABK. In addition, 2',6'-di-N-acetyl ABK was formed as a minor product. The same conversion also occurred with dibekacin (DKB) resulting in the formation of 2'-N-acetyl DKB and 2',6'-di-N-acetyl DKB. MIC determination showed antibacterial activity (1.56 approximately 3.13 micrograms/ml) of 2'-N-acetyl ABK against a variety of organisms. By contrast, 2'-N-acetyl DKB showed no substantial antibiotic activity. We believe 2'-N-acetyl ABK has the highest and broadest antibacterial activity, compared with known N-acetylated AGs. PMID:8682723

  10. Acetylation of cyclin-dependent kinase 5 is mediated by GCN5

    SciTech Connect

    Lee, Juhyung; Yun, Nuri; Kim, Chiho; Song, Min-Young; Park, Kang-Sik; Oh, Young J.

    2014-04-25

    Highlights: • Cyclin-dependent kinase 5 (CDK5) is present as an acetylated form. • CDK5 is acetylated by GCN5. • CDK5’s acetylation site is mapped at Lys33. • Its acetylation may affect CDK5’s kinase activity. - Abstract: Cyclin-dependent kinase 5 (CDK5), a member of atypical serine/threonine cyclin-dependent kinase family, plays a crucial role in pathophysiology of neurodegenerative disorders. Its kinase activity and substrate specificity are regulated by several independent pathways including binding with its activator, phosphorylation and S-nitrosylation. In the present study, we report that acetylation of CDK5 comprises an additional posttranslational modification within the cells. Among many candidates, we confirmed that its acetylation is enhanced by GCN5, a member of the GCN5-related N-acetyl-transferase family of histone acetyltransferase. Co-immunoprecipitation assay and fluorescent localization study indicated that GCN5 physically interacts with CDK5 and they are co-localized at the specific nuclear foci. Furthermore, liquid chromatography in conjunction with a mass spectrometry indicated that CDK5 is acetylated at Lys33 residue of ATP binding domain. Considering this lysine site is conserved among a wide range of species and other related cyclin-dependent kinases, therefore, we speculate that acetylation may alter the kinase activity of CDK5 via affecting efficacy of ATP coordination.

  11. Lysine Acetylation of CREBH Regulates Fasting-Induced Hepatic Lipid Metabolism.

    PubMed

    Kim, Hyunbae; Mendez, Roberto; Chen, Xuequn; Fang, Deyu; Zhang, Kezhong

    2015-12-15

    Cyclic AMP-responsive element-binding protein 3-like 3, hepatocyte specific (CREBH), is a hepatic transcription factor that functions as a key regulator of energy homeostasis. Here, we defined a regulatory CREBH posttranslational modification process, namely, lysine-specific acetylation, and its functional involvement in fasting-induced hepatic lipid metabolism. Fasting induces CREBH acetylation in mouse livers in a time-dependent manner, and this event is critical for CREBH transcriptional activity in regulating hepatic lipid homeostasis. The histone acetyltransferase PCAF-mediated acetylation and the deacetylase sirtuin-1-mediated deacetylation coexist to maintain CREBH acetylation states under fasting conditions. Site-directed mutagenesis and functional analyses revealed that the lysine (K) residue at position 294 (K294) within the bZIP domain of the CREBH protein is the site where fasting-induced acetylation/deacetylation occurs. Introduction of the acetylation-deficient (K294R) or acetylation-mimicking (K294Q) mutation inhibited or enhanced CREBH transcriptional activity, respectively. Importantly, CREBH acetylation at lysine 294 was required for the interaction and synergy between CREBH and peroxisome proliferator-activated receptor ? (PPAR?) in activating their target genes upon fasting or glucagon stimulation. Introduction of the CREBH lysine 294 mutation in the liver leads to hepatic steatosis and hyperlipidemia in animals under prolonged fasting. In summary, our study reveals a molecular mechanism by which fasting or glucagon stimulation modulates lipid homeostasis through acetylation of CREBH. PMID:26438600

  12. Acetyl-CoA and the regulation of metabolism: mechanisms and consequences.

    PubMed

    Shi, Lei; Tu, Benjamin P

    2015-04-01

    Acetyl-CoA represents a key node in metabolism due to its intersection with many metabolic pathways and transformations. Emerging evidence reveals that cells monitor the levels of acetyl-CoA as a key indicator of their metabolic state, through distinctive protein acetylation modifications dependent on this metabolite. We offer the following conceptual model for understanding the role of this sentinel metabolite in metabolic regulation. High nucleocytosolic acetyl-CoA amounts are a signature of a 'growth' or 'fed' state and promote its utilization for lipid synthesis and histone acetylation. In contrast, under 'survival' or 'fasted' states, acetyl-CoA is preferentially directed into the mitochondria to promote mitochondrial-dependent activities such as the synthesis of ATP and ketone bodies. Fluctuations in acetyl-CoA within these subcellular compartments enable the substrate-level regulation of acetylation modifications, but also necessitate the function of sirtuin deacetylases to catalyze removal of spontaneous modifications that might be unintended. Thus, understanding the sources, fates, and consequences of acetyl-CoA as a carrier of two-carbon units has started to reveal its underappreciated but profound influence on the regulation of numerous life processes. PMID:25703630

  13. Metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil

    SciTech Connect

    Triggiani, M.; D'Souza, D.M.; Chilton, F.H. )

    1991-04-15

    The biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) together with that of 1-alkyl-2-acetyl-GPC (platelet-activating factor) has been demonstrated in a variety of inflammatory cells and tissues. It has been hypothesized that the relative proportion of these phospholipids produced upon cell activation may be influenced by their rates of catabolism. We studied the catabolism of 1-acyl-2-acetyl-GPC in resting and activated human neutrophils and compared it to that of 1-alkyl-2-acetyl-GPC. Neutrophils rapidly catabolize both 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC; however, the rate of catabolism of 1-acyl-2-acetyl-GPC is approximately 2-fold higher than that of 1-alkyl-2-acetyl-GPC. In addition, most of 1-acyl-2-acetyl-GPC is catabolized through a pathway different from that of 1-alkyl-2-acetyl-GPC. The main step in the catabolism of 1-acyl-2-acetyl-GPC is the removal of the long chain at the sn-1 position; the long chain residue is subsequently incorporated either into triglycerides or into phosphatidylcholine. The 1-lyso-2-acetyl-GPC formed in this reaction is then further degraded to glycerophosphocholine, choline, or phosphocholine. 1-Acyl-2-acetyl-GPC is also catabolized, to a lesser extent, through deacetylation at the sn-2 position and reacylation with a long chain fatty acid. Stimulation of neutrophils by A23187 results in a higher rate of catabolism of 1-acyl-2-acetyl-GPC by increasing both the removal of the long chain at the sn-1 position and the deacetylation-reacylation at the sn-2 position. In a broken cell preparation, the cytosolic fraction of the neutrophil was shown to contain an enzyme activity which cleaved the sn-1 position of 1-acyl-2-acetyl-GPC and 1-acyl-2-lyso-GPC but not of 1,2-diacyl-GPC.

  14. Aspirin-mediated acetylation induces structural alteration and aggregation of bovine pancreatic insulin.

    PubMed

    Yousefi, Reza; Taheri, Behnaz; Alavi, Parnian; Shahsavani, Mohammad Bagher; Asadi, Zahra; Ghahramani, Maryam; Niazi, Ali; Alavianmehr, Mohammad Mehdi; Moosavi-Movahedi, Ali Akbar

    2016-02-01

    The simple aggregation of insulin under various chemical and physical stresses is still an important challenge for both pharmaceutical production and clinical formulation. In the storage form, this protein is subjected to various chemical modifications which alter its physicochemical and aggregation properties. Aspirin (acetylsalicylic acid) which is the most widely used medicine worldwide has been indicated to acetylate a large number of proteins both in vitro and in vivo. In this study, as insulin treated with aspirin at 37C, a significant level of acetylation was observed by flourescamine and o-phthalaldehyde assay. Also, different spectroscopic techniques, gel electrophoresis, and microscopic assessment were applied to compare the structural variation and aggregation/fibrillation propensity among acetylated and non-acetylated insulin samples. The results of spectroscopic assessments elucidate that acetylation induces insulin unfolding which is accompanied with the exposure of protein hydrophobic patches, a transition from alpha-helix to beta-sheet and increased propensity of the protein for aggregation. The kinetic studies propose that acetylation increases aggregation rate of insulin under both thermal and chemical stresses. Also, gel electrophoresis and dynamic light scattering experiments suggest that acetylation induces insulin oligomerization. Additionally, the results of Thioflavin T fluorescence study, Congo red absorption assessment, and microscopic analysis suggest that acetylation with aspirin enhances the process of insulin fibrillation. Overall, the increased susceptibility of acetylated insulin for aggregation may reflect the fact that this type of modification has significant structural destabilizing effect which finally makes the protein more vulnerable for pathogenic aggregation/fibrillation. PMID:25994118

  15. Aurora B is regulated by acetylation/deacetylation during mitosis in prostate cancer cells

    PubMed Central

    Fadri-Moskwik, Maria; Weiderhold, Kimberly N.; Deeraksa, Arpaporn; Chuang, Carol; Pan, Jing; Lin, Sue-Hwa; Yu-Lee, Li-Yuan

    2012-01-01

    Protein acetylation has been implicated in playing an important role during mitotic progression. Aurora B kinase is known to play a critical role in mitosis. However, whether Aurora B is regulated by acetylation is not known. Using IP with an anti-acetyl lysine antibody, we identified Aurora B as an acetylated protein in PC3 prostate cancer cells. Knockdown of HDAC3 or inhibiting HDAC3 deacetylase activity led to a significant increase (P<0.01 and P<0.05, respectively) in Aurora B acetylation as compared to siLuc or vehicle-treated controls. Increased Aurora B acetylation is correlated with a 30% reduction in Aurora B kinase activity in vitro and resulted in significant defects in Aurora B-dependent mitotic processes, including kinetochore-microtubule attachment and chromosome congression. Furthermore, Aurora B transiently interacts with HDAC3 at the kinetochore-microtubule interface of congressing chromosomes during prometaphase. This window of interaction corresponded with a transient but significant reduction (P=0.02) in Aurora B acetylation during early mitosis. Together, these results indicate that Aurora B is more active in its deacetylated state and further suggest a new mechanism by which dynamic acetylation/deacetylation acts as a rheostat to fine-tune Aurora B activity during mitotic progression.Fadri-Moskwik, M., Weiderhold, K. N., Deeraksa, A., Chuang, C., Pan, J., Lin, S.-H., Yu-Lee, L.-Y. Aurora B is regulated by acetylation/deacetylation during mitosis in prostate cancer cells. PMID:22751009

  16. The Mechanisms of Plant Cell Wall Deconstruction during Enzymatic Hydrolysis

    PubMed Central

    Thygesen, Lisbeth G.; Thybring, Emil E.; Johansen, Katja S.; Felby, Claus

    2014-01-01

    Mechanical agitation during enzymatic hydrolysis of insoluble plant biomass at high dry matter contents is indispensable for the initial liquefaction step in biorefining. It is known that particle size reduction is an important part of liquefaction, but the mechanisms involved are poorly understood. Here we put forward a simple model based on mechanical principles capable of capturing the result of the interaction between mechanical forces and cell wall weakening via hydrolysis of glucosidic bonds. This study illustrates that basic material science insights are relevant also within biochemistry, particularly when it comes to up-scaling of processes based on insoluble feed stocks. PMID:25232741

  17. Enzymatic hydrolysis of ammonia-treated sugar beet pulp.

    PubMed

    Foster, B L; Dale, B E; Doran-Peterson, J B

    2001-01-01

    Sugar beet pulp is a carbohydrate-rich coproduct generated by the table sugar industry. Beet pulp has shown promise as a feedstock for ethanol production using enzymes to hydrolyze polymeric carbohydrates and engineered bacteria to ferment sugars to ethanol. In this study, sugar beet pulp underwent an ammonia pressurization depressurization (APD) pretreatment in which the pulp was exploded by the sudden evaporation of ammonia in a reactor vessel. APD was found to substantially increase hydrolysis efficiency of the cellulose component, but when hemicellulose- and pectin-degrading enzymes were added, treated pulp hydrolysis was no better than the untreated control. PMID:11963856

  18. Benzene/nitrous oxide flammability in the precipitate hydrolysis process

    SciTech Connect

    Jacobs, R A

    1989-09-18

    The HAN (hydroxylamine nitrate) process for destruction of nitrite in precipitate hydrolysis produces nitrous oxide (N2O) gas as one of the products. N2O can form flammable mixtures with benzene which is also present due to radiolysis and hydrolysis of tetraphenylborate. Extensive flame modeling and explosion testing was undertaken to define the minimum oxidant for combustion of N2O/benzene using both nitrogen and carbon dioxide as diluents. The attached memorandum interprets and documents the results of the studies.

  19. Microwave-assisted hydrolysis of polysaccharides over polyoxometalate clusters.

    PubMed

    Tsubaki, Shuntaro; Oono, Kiriyo; Ueda, Tadaharu; Onda, Ayumu; Yanagisawa, Kazumichi; Mitani, Tomohiko; Azuma, Jun-ichi

    2013-09-01

    Polyoxometalate (POM) clusters were utilized as recyclable acid catalysts and microwave-absorbing agents for the microwave-assisted hydrolysis of corn starch and crystalline cellulose. Phosphotungstic (PW) and silicotungstic (SiW) acids showed high hydrolyzing activity, while phosphomolybdic acid (PMo) showed lower glucose stability. The PW catalyst could be recycled by ether extraction at least 4 times without changing its catalytic activity. The addition of PW could reduce the energy demand required for running the hydrolysis by 17-23%. The dielectric property of the aqueous PW solution was important for increasing the microwave-absorption capability of the reaction system and reducing the energy consumption. PMID:23859983

  20. Hydrolysis of sugarcane bagasse by mycelial biomass of Penicillium funiculosum

    SciTech Connect

    Rao, M.; Deshpande, V.; Seeta, R.; Srinivasan, M.C.; Mishra, C.

    1985-07-01

    Cellulose bioconversion has great promise for producing unlimited quantities of fermentable feedstocks and liquid fuels. Extensive studies on the production of extracellular cellulase and on the saccharification of various cellulosic substrates using cellulases have been reported. Use of mycelial biomass having cell bound cellulase for saccharification of cellulose was studied in Aspergillus terreus by Miller and Srinivasan. Extracellular cellulase production by P. funiculosum and its application for cellulose hydrolysis have been reported earlier by the authors. The present communication reports the hydrolysis of lignocellulose using mycelial biomass of P. funiculosum cultivated on cellulose and its reuse potential. 10 references.

  1. Comparative analysis of pharmacological treatments with N-acetyl-dl-leucine (Tanganil) and its two isomers (N-acetyl-L-leucine and N-acetyl-D-leucine) on vestibular compensation: Behavioral investigation in the cat.

    PubMed

    Tighilet, Brahim; Leonard, Jacques; Bernard-Demanze, Laurence; Lacour, Michel

    2015-12-15

    Head roll tilt, postural imbalance and spontaneous nystagmus are the main static vestibular deficits observed after an acute unilateral vestibular loss (UVL). In the UVL cat model, these deficits are fully compensated over 6 weeks as the result of central vestibular compensation. N-Acetyl-dl-leucine is a drug prescribed in clinical practice for the symptomatic treatment of acute UVL patients. The present study investigated the effects of N-acetyl-dl-leucine on the behavioral recovery after unilateral vestibular neurectomy (UVN) in the cat, and compared the effects of each of its two isomers N-acetyl-L-leucine and N-acetyl-D-leucine. Efficacy of these three drug treatments has been evaluated with respect to a placebo group (UVN+saline water) on the global sensorimotor activity (observation grids), the posture control (support surface measurement), the locomotor balance (maximum performance at the rotating beam test), and the spontaneous vestibular nystagmus (recorded in the light). Whatever the parameters tested, the behavioral recovery was strongly and significantly accelerated under pharmacological treatments with N-acetyl-dl-leucine and N-acetyl-L-leucine. In contrast, the N-acetyl-D-leucine isomer had no effect at all on the behavioral recovery, and animals of this group showed the same recovery profile as those receiving a placebo. It is concluded that the N-acetyl-L-leucine isomer is the active part of the racemate component since it induces a significant acceleration of the vestibular compensation process similar (and even better) to that observed under treatment with the racemate component only. PMID:26607469

  2. The Metabolic Fate of Deoxynivalenol and Its Acetylated Derivatives in a Wheat Suspension Culture: Identification and Detection of DON-15-O-Glucoside, 15-Acetyl-DON-3-O-Glucoside and 15-Acetyl-DON-3-Sulfate.

    PubMed

    Schmeitzl, Clemens; Warth, Benedikt; Fruhmann, Philipp; Michlmayr, Herbert; Malachová, Alexandra; Berthiller, Franz; Schuhmacher, Rainer; Krska, Rudolf; Adam, Gerhard

    2015-08-01

    Deoxynivalenol (DON) is a protein synthesis inhibitor produced by the Fusarium species, which frequently contaminates grains used for human or animal consumption. We treated a wheat suspension culture with DON or one of its acetylated derivatives, 3-acetyl-DON (3-ADON), 15-acetyl-DON (15-ADON) and 3,15-diacetyl-DON (3,15-diADON), and monitored the metabolization over a course of 96 h. Supernatant and cell extract samples were analyzed using a tailored LC-MS/MS method for the quantification of DON metabolites. We report the formation of tentatively identified DON-15-O-β-D-glucoside (D15G) and of 15-acetyl-DON-3-sulfate (15-ADON3S) as novel deoxynivalenol metabolites in wheat. Furthermore, we found that the recently identified 15-acetyl-DON-3-O-β-D-glucoside (15-ADON3G) is the major metabolite produced after 15-ADON challenge. 3-ADON treatment led to a higher intracellular content of toxic metabolites after six hours compared to all other treatments. 3-ADON was exclusively metabolized into DON before phase II reactions occurred. In contrast, we found that 15-ADON was directly converted into 15-ADON3G and 15-ADON3S in addition to metabolization into deoxynivalenol-3-O-β-D-glucoside (D3G). This study highlights significant differences in the metabolization of DON and its acetylated derivatives. PMID:26274975

  3. The Metabolic Fate of Deoxynivalenol and Its Acetylated Derivatives in a Wheat Suspension Culture: Identification and Detection of DON-15-O-Glucoside, 15-Acetyl-DON-3-O-Glucoside and 15-Acetyl-DON-3-Sulfate

    PubMed Central

    Schmeitzl, Clemens; Warth, Benedikt; Fruhmann, Philipp; Michlmayr, Herbert; Malachová, Alexandra; Berthiller, Franz; Schuhmacher, Rainer; Krska, Rudolf; Adam, Gerhard

    2015-01-01

    Deoxynivalenol (DON) is a protein synthesis inhibitor produced by the Fusarium species, which frequently contaminates grains used for human or animal consumption. We treated a wheat suspension culture with DON or one of its acetylated derivatives, 3-acetyl-DON (3-ADON), 15-acetyl-DON (15-ADON) and 3,15-diacetyl-DON (3,15-diADON), and monitored the metabolization over a course of 96 h. Supernatant and cell extract samples were analyzed using a tailored LC-MS/MS method for the quantification of DON metabolites. We report the formation of tentatively identified DON-15-O-β-D-glucoside (D15G) and of 15-acetyl-DON-3-sulfate (15-ADON3S) as novel deoxynivalenol metabolites in wheat. Furthermore, we found that the recently identified 15-acetyl-DON-3-O-β-D-glucoside (15-ADON3G) is the major metabolite produced after 15-ADON challenge. 3-ADON treatment led to a higher intracellular content of toxic metabolites after six hours compared to all other treatments. 3-ADON was exclusively metabolized into DON before phase II reactions occurred. In contrast, we found that 15-ADON was directly converted into 15-ADON3G and 15-ADON3S in addition to metabolization into deoxynivalenol-3-O-β-D-glucoside (D3G). This study highlights significant differences in the metabolization of DON and its acetylated derivatives. PMID:26274975

  4. Cyclic AMP regulation of protein lysine acetylation in Mycobacterium tuberculosis.

    PubMed

    Lee, Ho Jun; Lang, P Therese; Fortune, Sarah M; Sassetti, Christopher M; Alber, Tom

    2012-08-01

    Protein lysine acetylation networks can regulate central processes such as carbon metabolism and gene expression in bacteria. In Escherichia coli, cyclic AMP (cAMP) regulates protein lysine acetyltransferase (PAT) activity at the transcriptional level, but in Mycobacterium tuberculosis, fusion of a cyclic nucleotide-binding domain to a Gcn5-like PAT domain enables direct cAMP control of protein acetylation. Here we describe the allosteric activation mechanism of M. tuberculosis PAT. The crystal structures of the autoinhibited and cAMP-activated PAT reveal that cAMP binds to a cryptic site in the regulatory domain that is over 32 Å from the catalytic site. An extensive conformational rearrangement relieves this autoinhibition by means of a substrate-mimicking lid that covers the protein-substrate binding surface. A steric double latch couples the domains by harnessing a classic, cAMP-mediated conformational switch. The structures suggest general features that enable the evolution of long-range communication between linked domains. PMID:22773105

  5. O-Acetylation of Peptidoglycan in Gram-negative Bacteria

    PubMed Central

    Moynihan, Patrick J.; Clarke, Anthony J.

    2010-01-01

    The ape2 gene encoding a hypothetical O-acetylpeptidoglycan esterase was amplified from genomic DNA of Neisseria gonorrhoeae FA1090 and cloned to encode either the full-length protein or a truncated version lacking its hypothetical signal sequence. Expression trials revealed that production of the full-length version possessing either an N-terminal or C-terminal His6 tag was toxic to Escherichia coli transformants and that the host rapidly degraded the small amount of protein that was produced. An N-terminally truncated protein could be produced in sufficient yields for purification only if it possessed an N-terminal His6 tag. This form of the protein was isolated and purified to apparent homogeneity, and its enzymatic properties were characterized. Whereas the protein could bind to insoluble peptidoglycan, it did not function as an esterase. Phenotypic characterization of E. coli transformants producing various forms of the protein revealed that it functions instead to O-acetylate peptidoglycan within the periplasm, and it was thus renamed peptidoglycan O-acetyltransferase B. This activity was found to be dependent upon a second protein, which functions to translocate acetate from the cytoplasm to the periplasm, demonstrating that the O-acetylation of peptidoglycan in N. gonorrhoeae, and other Gram-negative bacteria, requires a two component system. PMID:20178982

  6. Acetylated Trivalent Mannobioses: Chemical Modification, Structural Elucidation, and Biological Evaluation.

    PubMed

    Rahkila, Jani; Panchadhayee, Rajib; Ardá, Ana; Jiménez-Barbero, Jesús; Savolainen, Johannes; Leino, Reko

    2016-03-17

    People suffering from allergies can be treated with repeated injections of increasing amounts of a specific allergen. This type of specific immunotherapy is currently the only way to treat the underlying pathological immune response associated with an allergy. The approach can afford long-lasting protection, but the process takes 3-5 years, can produce allergic reactions, and in severe cases treatment is often aborted due to anaphylaxis. However, treatment can be optimized with the use of specific adjuvants that modify the immune response, its duration, and that increase the production of the correct type of antibodies. In the pursuit of such adjuvants, two new trivalent acetylated β-(1→2)-linked mannobioses based on a previously discovered lead molecule were prepared. The new molecules, along with the previously developed lead, were investigated by rigorous NMR and molecular modeling experiments in order to elucidate their behavior and preferred conformations in solution. Furthermore, the molecules were subjected to a biological investigation in which their immunostimulatory properties were evaluated by assessing their effect on the production of TH 2-type cytokine interleukin-4 (IL-4) and Treg pro-inflammatory cytokine tumor necrosis factor (TNF). Treatment of peripheral mononuclear blood cell cultures from patients suffering from birch allergy with birch allergen Bet v induced a strong IL-4 response, whereas the same treatment together with the trivalent acetylated mannobioses caused significant suppression of the induced IL-4. PMID:26898175

  7. Autotrophic acetyl coenzyme A biosynthesis in Methanococcus maripaludis.

    PubMed Central

    Shieh, J; Whitman, W B

    1988-01-01

    To detect autotrophic CO2 assimilation in cell extracts of Methanococcus maripaludis, lactate dehydrogenase and NADH were added to convert pyruvate formed from autotrophically synthesized acetyl coenzyme A to lactate. The lactate produced was determined spectrophotometrically. When CO2 fixation was pulled in the direction of lactate synthesis, CO2 reduction to methane was inhibited. Bromoethanesulfonate (BES), a potent inhibitor of methanogenesis, enhanced lactate synthesis, and methyl coenzyme M inhibited it in the absence of BES. Lactate synthesis was dependent on CO2 and H2, but H2 + CO2-independent synthesis was also observed. In cell extracts, the rate of lactate synthesis was about 1.2 nmol min-1 mg of protein-1. When BES was added, the rate of lactate synthesis increased to 2.3 nmol min-1 mg of protein-1. Because acetyl coenzyme A did not stimulate lactate synthesis, pyruvate synthase may have been the limiting activity in these assays. Radiolabel from 14CO2 was incorporated into lactate. The percentages of radiolabel in the C-1, C-2, and C-3 positions of lactate were 73, 33, and 11%, respectively. Both carbon monoxide and formaldehyde stimulated lactate synthesis. 14CH2O was specifically incorporated into the C-3 of lactate, and 14CO was incorporated into the C-1 and C-2 positions. Low concentrations of cyanide also inhibited autotrophic growth, CO dehydrogenase activity, and autotrophic lactate synthesis. These observations are in agreement with the acetogenic pathway of autotrophic CO2 assimilation. PMID:3133359

  8. Two Arabidopsis Proteins Synthesize Acetylated Xylan in Vitro

    PubMed Central

    Urbanowicz, Breeanna R.; Pea, Maria J.; Moniz, Heather A.; Moremen, Kelley W.; York, William S.

    2014-01-01

    SUMMARY Xylan is the third most abundant glycopolymer on earth after cellulose and chitin. As a major component of wood, grain and forage, this natural biopolymer has far-reaching impacts on human life. This highly acetylated cell wall polysaccharide is a vital component of the plant cell wall, which functions as a molecular scaffold, providing plants with mechanical strength and flexibility. Mutations that impair synthesis of the xylan backbone give rise to plants that fail to grow normally due to collapsed xylem cells in the vascular system. Phenotypic analysis of these mutants has implicated many proteins in xylan biosynthesis. However, the enzymes directly responsible for elongation and acetylation of the xylan backbone have not been unambiguously identified. Here we provide direct biochemical evidence that two Arabidopsis thaliana proteins, IRREGULAR XYLEM 10-L (IRX10-L) and ESKIMO1/ TRICOME BIREFRINGENCE 29 (ESK1/TBL29), catalyze these respective processes in vitro. By identifying the elusive xylan synthase and establishing ESK1/TBL29 as the archetypal plant polysaccharide O-acetyltransferase, we have resolved two long-standing questions in plant cell wall biochemistry. These findings shed light on integral steps in the molecular pathways utilized by plants to synthesize a major component of the world's biomass and expand our toolkit for producing glycopolymers with valuable properties. PMID:25141999

  9. Cationised O-acetyl galactoglucomannans: synthesis and characterisation.

    PubMed

    Kisonen, Victor; Xu, Chunlin; Eklund, Patrik; Lindqvist, Hanna; Sundberg, Anna; Pranovich, Andrey; Sinkkonen, Jari; Vilaplana, Francisco; Willför, Stefan

    2014-01-01

    Water-soluble O-acetyl-galactoglucomannans (GGMs) can be obtained from Norway spruce by hot-water-extraction of the wood or as a side product by ultrafiltration of mechanical pulping waters. Cationic and amphiphilic polysaccharides and their derivatives are of interest for a number of applications and thus quaternary nitrogen moieties with cationic charge were grafted onto GGMs in the heterogeneous reaction to render a cationic polyelectrolyte. The degree of substitution was measured by elemental analysis of nitrogen, by quantitative (13)C NMR and interestingly also by polyelectrolyte titration and the results were congruent. NMR, matrix-assisted laser desorption/ionisation mass spectroscopy (MALDI-TOF-MS), and FT-IR analysis were used to characterise the product. THF or DMSO with water enhanced the reaction efficiency and decreased M(w) reduction in comparison to plain water as a reaction media. Cationised GGM was also successfully acetylated. The cationic derivatives of hemicelluloses can potentially be utilised as polyelectrolyte layers in packaging and pharmaceutical applications. PMID:24274567

  10. Regulation of autophagy by cytosolic acetyl-coenzyme A.

    PubMed

    Mario, Guillermo; Pietrocola, Federico; Eisenberg, Tobias; Kong, Yongli; Malik, Shoaib Ahmad; Andryushkova, Aleksandra; Schroeder, Sabrina; Pendl, Tobias; Harger, Alexandra; Niso-Santano, Mireia; Zamzami, Naoufal; Scoazec, Marie; Durand, Silvre; Enot, David P; Fernndez, lvaro F; Martins, Isabelle; Kepp, Oliver; Senovilla, Laura; Bauvy, Chantal; Morselli, Eugenia; Vacchelli, Erika; Bennetzen, Martin; Magnes, Christoph; Sinner, Frank; Pieber, Thomas; Lpez-Otn, Carlos; Maiuri, Maria Chiara; Codogno, Patrice; Andersen, Jens S; Hill, Joseph A; Madeo, Frank; Kroemer, Guido

    2014-03-01

    Acetyl-coenzyme A (AcCoA) is a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism. Here we show that nutrient starvation causes rapid depletion of AcCoA. AcCoA depletion entailed the commensurate reduction in the overall acetylation of cytoplasmic proteins, as well as the induction of autophagy, a homeostatic process of self-digestion. Multiple distinct manipulations designed to increase or reduce cytosolic AcCoA led to the suppression or induction of autophagy, respectively, both in cultured human cells and in mice. Moreover, maintenance of high AcCoA levels inhibited maladaptive autophagy in a model of cardiac pressure overload. Depletion of AcCoA reduced the activity of the acetyltransferase EP300, and EP300 was required for the suppression of autophagy by high AcCoA levels. Altogether, our results indicate that cytosolic AcCoA functions as a central metabolic regulator of autophagy, thus delineating AcCoA-centered pharmacological strategies that allow for the therapeutic manipulation of autophagy. PMID:24560926

  11. Protein acetylation sites mediated by Schistosoma mansoni GCN5

    SciTech Connect

    Moraes Maciel, Renata de; Furtado Madeiro da Costa, Rodrigo; Meirelles Bastosde Oliveira, Francisco; Rumjanek, Franklin David; Fantappie, Marcelo Rosado

    2008-05-23

    The transcriptional co-activator GCN5, a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcription activation. As in other eukaryotes, the DNA from the parasite Schistosome mansoni is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Using a series of synthetic peptides we determined that Lys-14 of histone H3 was acetylated by the recombinant SmGCN5-HAT domain. SmGCN5 was also able to acetylate schistosome non-histone proteins, such as the nuclear receptors SmRXR1 and SmNR1, and the co-activator SmNCoA-62. Electron microscopy revealed the presence of SmGCN5 protein in the nuclei of vitelline cells. Within the nucleus, SmGCN5 was found to be located in interchromatin granule clusters (IGCs), which are transcriptionally active structures. The data suggest that SmGCN5 is involved in transcription activation.

  12. Acetylation of Gly1 and Lys2 promotes aggregation of human γD-crystallin.

    PubMed

    DiMauro, Michael A; Nandi, Sandip K; Raghavan, Cibin T; Kar, Rajiv Kumar; Wang, Benlian; Bhunia, Anirban; Nagaraj, Ram H; Biswas, Ashis

    2014-11-25

    The human lens contains three major protein families: α-, β-, and γ-crystallin. Among the several variants of γ-crystallin in the human lens, γD-crystallin is a major form. γD-Crystallin is primarily present in the nuclear region of the lens and contains a single lysine residue at the second position (K2). In this study, we investigated the acetylation of K2 in γD-crystallin in aging and cataractous human lenses. Our results indicated that K2 is acetylated at an early age and that the amount of K2-acetylated γD-crystallin increased with age. Mass spectrometric analysis revealed that in addition to K2, glycine 1 (G1) was acetylated in γD-crystallin from human lenses and in γD-crystallin acetylated in vitro. The chaperone ability of α-crystallin for acetylated γD-crystallin was lower than that for the nonacetylated protein. The tertiary structure and the microenvironment of the cysteine residues were significantly altered by acetylation. The acetylated protein exhibited higher surface hydrophobicity, was unstable against thermal and chemical denaturation, and exhibited a higher propensity to aggregate at 80 °C in comparison to the nonacetylated protein. Acetylation enhanced the GdnHCl-induced unfolding and slowed the subsequent refolding of γD-crystallin. Theoretical analysis indicated that the acetylation of K2 and G1 reduced the structural stability of the protein and brought the distal cysteine residues (C18 and C78) into close proximity. Collectively, these results indicate that the acetylation of G1 and K2 residues in γD-crystallin likely induced a molten globule-like structure, predisposing it to aggregation, which may account for the high content of aggregated proteins in the nucleus of aged and cataractous human lenses. PMID:25393041

  13. Acetylation of Gly1 and Lys2 Promotes Aggregation of Human γD-Crystallin

    PubMed Central

    2015-01-01

    The human lens contains three major protein families: α-, β-, and γ-crystallin. Among the several variants of γ-crystallin in the human lens, γD-crystallin is a major form. γD-Crystallin is primarily present in the nuclear region of the lens and contains a single lysine residue at the second position (K2). In this study, we investigated the acetylation of K2 in γD-crystallin in aging and cataractous human lenses. Our results indicated that K2 is acetylated at an early age and that the amount of K2-acetylated γD-crystallin increased with age. Mass spectrometric analysis revealed that in addition to K2, glycine 1 (G1) was acetylated in γD-crystallin from human lenses and in γD-crystallin acetylated in vitro. The chaperone ability of α-crystallin for acetylated γD-crystallin was lower than that for the nonacetylated protein. The tertiary structure and the microenvironment of the cysteine residues were significantly altered by acetylation. The acetylated protein exhibited higher surface hydrophobicity, was unstable against thermal and chemical denaturation, and exhibited a higher propensity to aggregate at 80 °C in comparison to the nonacetylated protein. Acetylation enhanced the GdnHCl-induced unfolding and slowed the subsequent refolding of γD-crystallin. Theoretical analysis indicated that the acetylation of K2 and G1 reduced the structural stability of the protein and brought the distal cysteine residues (C18 and C78) into close proximity. Collectively, these results indicate that the acetylation of G1 and K2 residues in γD-crystallin likely induced a molten globule-like structure, predisposing it to aggregation, which may account for the high content of aggregated proteins in the nucleus of aged and cataractous human lenses. PMID:25393041

  14. Identification of the Acetylation and Ubiquitin-Modified Proteome during the Progression of Skeletal Muscle Atrophy

    PubMed Central

    Ryder, Daniel J.; Judge, Sarah M.; Beharry, Adam W.; Farnsworth, Charles L.; Silva, Jeffrey C.; Judge, Andrew R.

    2015-01-01

    Skeletal muscle atrophy is a consequence of several physiological and pathophysiological conditions including muscle disuse, aging and diseases such as cancer and heart failure. In each of these conditions, the predominant mechanism contributing to the loss of skeletal muscle mass is increased protein turnover. Two important mechanisms which regulate protein stability and degradation are lysine acetylation and ubiquitination, respectively. However our understanding of the skeletal muscle proteins regulated through acetylation and ubiquitination during muscle atrophy is limited. Therefore, the purpose of the current study was to conduct an unbiased assessment of the acetylation and ubiquitin-modified proteome in skeletal muscle during a physiological condition of muscle atrophy. To induce progressive, physiologically relevant, muscle atrophy, rats were cast immobilized for 0, 2, 4 or 6 days and muscles harvested. Acetylated and ubiquitinated peptides were identified via a peptide IP proteomic approach using an anti-acetyl lysine antibody or a ubiquitin remnant motif antibody followed by mass spectrometry. In control skeletal muscle we identified and mapped the acetylation of 1,326 lysine residues to 425 different proteins and the ubiquitination of 4,948 lysine residues to 1,131 different proteins. Of these proteins 43, 47 and 50 proteins were differentially acetylated and 183, 227 and 172 were differentially ubiquitinated following 2, 4 and 6 days of disuse, respectively. Bioinformatics analysis identified contractile proteins as being enriched among proteins decreased in acetylation and increased in ubiquitination, whereas histone proteins were enriched among proteins increased in acetylation and decreased in ubiquitination. These findings provide the first proteome-wide identification of skeletal muscle proteins exhibiting changes in lysine acetylation and ubiquitination during any atrophy condition, and provide a basis for future mechanistic studies into how the acetylation and ubiquitination status of these identified proteins regulates the muscle atrophy phenotype. PMID:26302492

  15. Mutations of Arabidopsis TBL32 and TBL33 Affect Xylan Acetylation and Secondary Wall Deposition

    PubMed Central

    Yuan, Youxi; Teng, Quincy; Zhong, Ruiqin; Haghighat, Marziyeh; Richardson, Elizabeth A.; Ye, Zheng-Hua

    2016-01-01

    Xylan is a major acetylated polymer in plant lignocellulosic biomass and it can be mono- and di-acetylated at O-2 and O-3 as well as mono-acetylated at O-3 of xylosyl residues that is substituted with glucuronic acid (GlcA) at O-2. Based on the finding that ESK1, an Arabidopsis thaliana DUF231 protein, specifically mediates xylan 2-O- and 3-O-monoacetylation, we previously proposed that different acetyltransferase activities are required for regiospecific acetyl substitutions of xylan. Here, we demonstrate the functional roles of TBL32 and TBL33, two ESK1 close homologs, in acetyl substitutions of xylan. Simultaneous mutations of TBL32 and TBL33 resulted in a significant reduction in xylan acetyl content and endoxylanase digestion of the mutant xylan released GlcA-substituted xylooligomers without acetyl groups. Structural analysis of xylan revealed that the tbl32 tbl33 mutant had a nearly complete loss of 3-O-acetylated, 2-O-GlcA-substituted xylosyl residues. A reduction in 3-O-monoacetylated and 2,3-di-O-acetylated xylosyl residues was also observed. Simultaneous mutations of TBL32, TBL33 and ESK1 resulted in a severe reduction in xylan acetyl level down to 15% of that of the wild type, and concomitantly, severely collapsed vessels and stunted plant growth. In particular, the S2 layer of secondary walls in xylem vessels of tbl33 esk1 and tbl32 tbl33 esk1 exhibited an altered structure, indicating abnormal assembly of secondary wall polymers. These results demonstrate that TBL32 and TBL33 play an important role in xylan acetylation and normal deposition of secondary walls. PMID:26745802

  16. Designer xylanosomes: protein nanostructures for enhanced xylan hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This work is the first report of the successful design, construction, and application of multi-functional, self-assembling biocatalysts for targeted xylan hydrolysis, termed xylanosomes. Using the architecture of cellulosomes found in some anaerobic cellulolytic microbes, four different xylanosomes...

  17. Vicinal ?,?-functionalizations of amines: cyclization versus dehydrogenative hydrolysis.

    PubMed

    Jiang, Fan; Achard, Mathieu; Bruneau, Christian

    2015-10-01

    Direct vicinal ?,?-difunctionalization of tertiary cyclic amines is achieved in the presence of ruthenium or iridium transition-metal complexes featuring phosphine-sulfonate chelates. By varying the reaction conditions, ?-alkylated lactams were obtained by a formal dehydrogenative hydrolysis in which one molecule of hydrogen is generated from water. PMID:26385286

  18. Ethanol production with dilute acid hydrolysis using partially dried lignocellulosics

    DOEpatents

    Nguyen, Quang A. (Chesterfield, MO); Keller, Fred A. (Lakewood, CO); Tucker, Melvin P. (Lakewood, CO)

    2003-12-09

    A process of converting lignocellulosic biomass to ethanol, comprising hydrolyzing lignocellulosic materials by subjecting dried lignocellulosic material in a reactor to a catalyst comprised of a dilute solution of a strong acid and a metal salt to lower the activation energy (i.e., the temperature) of cellulose hydrolysis and ultimately obtain higher sugar yields.

  19. Kinetic Modeling of Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A semimechanistic multi-reaction kinetic model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, creeping wild ryegrass (CWR; Leymus triticoides). This model incorporated one homogeneous reaction of cellobiose-to-glucose and two heterogeneous reactions of cellulose-to...

  20. Acid hydrolysis of sweet potato for ethanol production

    SciTech Connect

    Kim, K.; Hamdy, M.K.

    1985-01-01

    Studies were conducted to establish optimal conditions for the acid hydrolysis of sweet potato for maximal ethanol yield. The starch contents of two sweet potato cultivars (Georgia Red and TG-4), based on fresh weight, were 21.1 +/- 0.6% and 27.5 +/- 1.6%, respectively. The results of acid hydrolysis experiments showed the following: (1) both hydrolysis rate and hydroxymethylfurfural (HMF) concentration were a function of HCL concentration, temperature, and time; (2) the reducing sugars were rapidly formed with elevated concentrations of HCl and temperature, but also destroyed quickly; and (3) HMF concentration increased significantly with the concentration of HCl, temperature, and hydrolysis time. Maximum reducing sugar value of 84.2 DE and 0.056% HMF (based on wet weight) was achieved after heating 8% SPS for 15 min in 1N HCl at 110/sup 0/C. Degraded 8% SPS (1N HCl, 97/sup 0/C for 20 min or 110/sup 0/C for 10 min) was utilized as substrate for ethanol fermentation and 3.8% ethanol (v/v) was produced from 1400 mL fermented wort. This is equal to 41.6 g ethanol (200 proof) from 400 g of fresh sweet potato tuber (Georgia Red) or an ethanol yield potential of 431 gal of 200-proof ethanol/acre (from 500 bushel tubers/acre).