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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. Residues in the acetyl CoA binding site of pyruvate carboxylase involved in allosteric regulation.

    PubMed

    Choosangtong, Kamonman; Sirithanakorn, Chaiyos; Adina-Zada, Abdul; Wallace, John C; Jitrapakdee, Sarawut; Attwood, Paul V

    2015-07-22

    We have examined the roles of Asp1018, Glu1027, Arg469 and Asp471 in the allosteric domain of Rhizobium etli pyruvate carboxylase. Arg469 and Asp471 interact directly with the allosteric activator acetyl coenzyme A (acetyl CoA) and the R469S and R469K mutants showed increased enzymic activity in the presence and absence of acetyl CoA, whilst the D471A mutant exhibited no acetyl CoA-activation. E1027A, E1027R and D1018A mutants had increased activity in the absence of acetyl CoA, but not in its presence. These results suggest that most of these residues impose restrictions on the structure and/or dynamics of the enzyme to affect activity. PMID:26149215

  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

    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.

  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

    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.

  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. Acetyl CoA Carboxylase 2 Is Dispensable for CD8+ T Cell Responses

    PubMed Central

    Lee, Jang Eun; Walsh, Matthew C.; Hoehn, Kyle L.; James, David E.; Wherry, E. John; Choi, Yongwon

    2015-01-01

    Differentiation of T cells is closely associated with dynamic changes in nutrient and energy metabolism. However, the extent to which specific metabolic pathways and molecular components are determinative of CD8+ T cell fate remains unclear. It has been previously established in various tissues that acetyl CoA carboxylase 2 (ACC2) regulates fatty acid oxidation (FAO) by inhibiting carnitine palmitoyltransferase 1 (CPT1), a rate-limiting enzyme of FAO in mitochondria. Here, we explore the cell-intrinsic role of ACC2 in T cell immunity in response to infections. We report here that ACC2 deficiency results in a marginal increase of cellular FAO in CD8+ T cells, but does not appear to influence antigen-specific effector and memory CD8+ T cell responses during infection with listeria or lymphocytic choriomeningitis virus. These results suggest that ACC2 is dispensable for CD8+ T cell responses. PMID:26367121

  10. Antitumor/Antifungal Celecoxib Derivative AR-12 is a Non-Nucleoside Inhibitor of the ANL-Family Adenylating Enzyme Acetyl CoA Synthetase

    PubMed Central

    2016-01-01

    AR-12/OSU-03012 is an antitumor celecoxib-derivative that has progressed to Phase I clinical trial as an anticancer agent and has activity against a number of infectious agents including fungi, bacteria and viruses. However, the mechanism of these activities has remained unclear. Based on a chemical-genetic profiling approach in yeast, we have found that AR-12 is an ATP-competitive, time-dependent inhibitor of yeast acetyl coenzyme A synthetase. AR-12-treated fungal cells show phenotypes consistent with the genetic reduction of acetyl CoA synthetase activity, including induction of autophagy, decreased histone acetylation, and loss of cellular integrity. In addition, AR-12 is a weak inhibitor of human acetyl CoA synthetase ACCS2. Acetyl CoA synthetase activity is essential in many fungi and parasites. In contrast, acetyl CoA is primarily synthesized by an alternate enzyme, ATP-citrate lyase, in mammalian cells. Taken together, our results indicate that AR-12 is a non-nucleoside acetyl CoA synthetase inhibitor and that acetyl CoA synthetase may be a feasible antifungal drug target. PMID:27088128

  11. Enhanced activity of acetyl CoA synthetase adsorbed on smart microgel: an implication for precursor biosynthesis.

    PubMed

    Dubey, Nidhi Chandrama; Tripathi, Bijay Prakash; Müller, Martin; Stamm, Manfred; Ionov, Leonid

    2015-01-28

    Acetyl coenzyme A (acetyl CoA) is an essential precursor molecule for synthesis of metabolites such as the polyketide-based drugs (tetracycline, mitharamycin, Zocor, etc.) fats, lipids, and cholesterol. Acetyl CoA synthetase (Acs) is one of the enzymes that catalyzes acetyl CoA synthesis, and this enzyme is essentially employed for continuous supply of the acetyl CoA for the production of these metabolites. To achieve reusable and a more robust entity of the enzyme, we carried out the immobilization of Acs on poly(N-isopropylacrylamide)-poly(ethylenimine) (PNIPAm-PEI) microgels via adsorption. Cationic PNIPAm-PEI microgel was synthesized by one-step graft copolymerization of NIPAm and N,N-methylene bis-acrylamide (MBA) from PEI. Adsorption studies of Acs on microgel indicated high binding of enzymes, with a maximum binding capacity of 286 μg/mg of microgel for Acs was achieved. The immobilized enzymes showed improved biocatalytic efficiency over free enzymes, beside this, the reaction parameters and circular dichroism (CD) spectroscopy studies indicated no significant changes in the enzyme structure after immobilization. This thoroughly characterized enzyme bioconjugate was further immobilized on an ultrathin membrane to assess the same reaction in flow through condition. Bioconjugate was covalently immobilized on a thin layer of preformed microgel support upon polyethylene terephthalate (PET) track etched membrane. The prepared membrane was used in a dead end filtration device to monitor the bioconversion efficiency and operational stability of cross-linked bioconjugate. The membrane reactor showed consistent operational stability and maintained >70% of initial activity after 7 consecutive operation cycles. PMID:25561344

  12. Correlation of ATP Citrate Lyase and Acetyl CoA Levels with Trichothecene Production in Fusarium graminearum

    PubMed Central

    Sakamoto, Naoko; Tsuyuki, Rie; Yoshinari, Tomoya; Usuma, Jermnak; Furukawa, Tomohiro; Nagasawa, Hiromichi; Sakuda, Shohei

    2013-01-01

    Thecorrelation of ATP citrate lyase (ACL) and acetyl CoA levels with trichothecene production in Fusarium graminearum was investigated using an inhibitor (precocene II) and an enhancer (cobalt chloride) of trichothecene production by changing carbon sources in liquid medium. When precocene II (30 µM) was added to inhibit trichothecene production in a trichothecene high-production medium containing sucrose, ACL expression was reduced and ACL mRNA level as well as acetyl CoA amount in the fungal cells were reduced to the levels observed in a trichothecene trace-production medium containing glucose or fructose. The ACL mRNA level was greatly increased by addition of cobalt chloride in the trichothecene high-production medium, but not in the trichothecene trace-production medium. Levels were reduced to those level in the trichothecene trace-production medium by addition of precocene II (300 µM) together with cobalt chloride. These results suggest that ACL expression is activated in the presence of sucrose and that acetyl CoA produced by the increased ALC level may be used for trichothecene production in the fungus. These findings also suggest that sucrose is important for the action of cobalt chloride in activating trichothecene production and that precocene II may affect a step down-stream of the target of cobalt chloride. PMID:24284828

  13. 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

  14. 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

  15. 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

  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. 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

  19. 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

  20. 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 Å and the C-O bond to the attacking water nucleophile=2.30 Å. The MacroD1 TS provides mechanistic understanding of the OAADPr esterase chemistry. PMID:25051211

  1. 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 C–O ester bond = 1.60 Å and the C–O bond to the attacking water nucleophile = 2.30 Å. The MacroD1 TS provides mechanistic understanding of the OAADPr esterase chemistry. PMID:25051211

  2. 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

  3. Analysis of sucrose acetates in a crude 6-O-acetyl sucrose product by on-line hydrolysis-high-performance liquid chromatography with pulsed amperometric detection.

    PubMed

    Yan, Wenwu; Wang, Nani; Zhang, Peimin; Zhang, Jiajie; Wu, Shuchao; Zhu, Yan

    2016-06-01

    A standard-free and sensitive method was developed for analysis of sucrose acetates in a crude 6-O-acetyl sucrose (S-6-a) product by on-line hydrolysis-high-performance liquid chromatography with pulsed amperometric detection (PAD). Sucrose, three regio-isomers of acetyl sucrose and five regio-isomers of diacetyl sucrose were separated on a C18 column using 3% (v/v) acetonitrile in water as eluent within 25min. After purification with LC followed by semi-preparative HPLC, their chemical structures were identified by 1D, 2D NMR and LC-MS. Moreover, quantification of those regio-isomers was achieved by on-line alkaline hydrolysis to liberate sucrose using a post-column delivery system, and then detected by PAD for indirect estimation of the sucrose acetate content. Under optimal conditions, the linear ranges were from 0.03 to 150μmolL(-1) for sucrose corresponding to sucrose acetates with coefficient of determination as 0.9997 and detection limit as 0.01μmolL(-1) (S/N=3). Good repeatability was obtained (RSD<3%, n=6). Furthermore, this method has been successfully applied to the analysis of sucrose and sucrose acetates in a crude S-6-a product during synthesis, purification and structure elucidation studies. The recoveries were from 94.89% to 102.31% for sucrose and sucrose acetates. PMID:27139218

  4. 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.

  5. Synthesis and Kinetics of Hydrolysis of 3,5-Dimethyl-N-acetyl-p-benzoquinone Imine: An Undergraduate Laboratory

    NASA Astrophysics Data System (ADS)

    Buccigross, Jeanne M.; Metz, Christa; Elliot, Lori; Becker, Pamela; Earley, Angela S.; Hayes, Jerry W.; Novak, Michael; Underwood, Gayl A.

    1996-04-01

    The synthesis of the title compound by a three-step procedure is described. The hydrolysis kinetics, which involve two consecutive psuedo-first-order processes, are also described. The synthesis and kinetics experiments described here are proposed for incorporation into undergraduate laboratory courses under a variety of formats. The compound described here is related to a toxic metabolite of the common analgesics acetaminophen and phenacetin.

  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. In vitro stability and metabolism of O2', O3', O5'-tri-acetyl-N6-(3-hydroxylaniline) adenosine in rat, dog and human plasma: chemical hydrolysis and role of plasma esterases.

    PubMed

    Liu, Yin; He, Jiuming; Abliz, Zeper; Zhu, Haibo

    2011-07-01

    O2', O3', O5'-tri-acetyl-N(6)-(3-hydroxylaniline)adenosine (WS070117), a new structure-type lipid regulator, is being developed in pre-clinical study. In order to monitor drug kinetics it is essential to understand pre-analytical factors that may affect drug assay. In vitro stability and metabolism were investigated using high-performance liquid chromatography (HPLC) method in this study. The hydrolysis products were identified by HPLC-mass spectrometry (MS)/MS method. The esterases involved in WS070117 hydrolysis was assigned via inhibition rate assay. It was found that WS070117 was chemically unstable in alkaline solutions compared to acidic and near neutral solutions. Enzymatic hydrolysis was even more rapid. Hydrolytic rate constants differ between species, being 4.24, 5.96 × 10(-3) and 6.85 × 10(-2) min(-1) in rat, dog and human plasma at 37°C, respectively. The hydrolysis was catalyzed by plasma esterase because NaF (sodium fluoride: a general esterase inhibitor) inhibited WS070117 hydrolysis and metabolite production. Hydrolysis was fast in rat plasma and was catalysed by carboxylesterase and butyrylcholinesterase. In dog plasma, carboxylesterase, butyrylcholinesterase and paraoxonase were mainly responsible. Butyrylcholinesterase was the major esterase involved in WS070117 hydrolysis in human plasma. The WS070117 hydrolysis in plasma proceeded by gradual loss of acetyl groups. The knowledge of in vitro drug stability and metabolic pathways identified in this study will be essential for future pre-clinical and clinical pharmacokinetics studies. PMID:21486191

  8. 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.

  9. 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

  10. Acetate/acetyl-CoA metabolism associated with cancer fatty acid synthesis: overview and application.

    PubMed

    Yoshii, Yukie; Furukawa, Takako; Saga, Tsuneo; Fujibayashi, Yasuhisa

    2015-01-28

    Understanding cancer-specific metabolism is important for identifying novel targets for cancer diagnosis and therapy. Induced acetate/acetyl CoA metabolism is a notable feature that is related to fatty acid synthesis supporting tumor growth. In this review, we focused on the recent findings related to cancer acetate/acetyl CoA metabolism. We also introduce [1-¹¹C]acetate positron emission tomography (PET), which is a useful tool to visualize up-regulation of acetate/acetyl CoA metabolism in cancer, and discuss the utility of [1-¹¹C]acetate PET in cancer diagnosis and its application to personalized medicine. PMID:24569091

  11. A novel neutral xylanase with high SDS resistance from Volvariella volvacea: characterization and its synergistic hydrolysis of wheat bran with acetyl xylan esterase.

    PubMed

    Zheng, Fei; Huang, Jingxuan; Yin, Yuhao; Ding, Shaojun

    2013-10-01

    A neutral xylanase (XynII) from Volvariella volvacea was identified and characterized. Unlike other modular xylanases, it consists of only a single GH10 catalytic domain with a unique C-terminal sequence (W-R-W-F) and a phenylalanine and proline-rich motif (T-P-F-P-P-F) at N-terminus, indicating that it is a novel GH10 xylanase. XynII exhibited optimal activity at pH 7 and 60 °C and stability over a broad range of pH 4.0-10.0. XynII displayed extreme highly SDS resistance retaining 101.98, 92.99, and 69.84 % activity at the presence of 300 mM SDS on birchwood, soluble oat spelt, and beechwood xylan, respectively. It remained largely intact after 24 h of incubation with proteinase K at a protease to protein ratio of 1:50 at 37 °C. The kinetic constants K(m) value towards beechwood xylan was 0.548 mg ml⁻¹, and the k(cat)/K(m) ratio, reflecting the catalytic efficiency of the enzyme, was 126.42 ml mg⁻¹ s⁻¹ at 60 °C. XynII was a true endo-acting xylanase lacking cellulase activity. It has weak activity towards xylotriose but efficiently hydrolyzed xylans and xylooligosaccharides larger than xylotriose mainly to xylobiose. Synergistic action with acetyl xylan esterase (AXEI) from V. volvacea was observed for de-starched wheat bran. The highest degree of synergy (DS 1.42) was obtained in sequential reactions with AXEI digestion preceding XynII. The high SDS resistance and intrinsic stability suggested XynII may have potential applications in various industrial processes especially for the detergent and textile industries and animal feed industries. PMID:23903903

  12. 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 relaxation–response 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 μM PaCoA and acetyl CoA, respectively); the apparent pA2 value for PaCoA was 6.44. 3′-dephospho-palmitoyl-CoA (10 μM) 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

  13. Inhibition of Acetyl-CoA Carboxylase Activity by Haloxyfop and Tralkoxydim.

    PubMed

    Secor, J; Cséke, C

    1988-01-01

    Acetyl-coenzyme A (CoA) carboxylase from maize (Zea mays L.) is inhibited by nanomolar concentrations of both haloxyfop, an aryloxyphenoxypropionate, and tralkoxydim, a cyclohexanedione herbicide. These results suggest that acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid biosynthesis, may be the target of these herbicides, contrary to an earlier report suggesting that aryloxyphenoxypropionate herbicides do not inhibit acetyl-CoA carboxylase. PMID:16665846

  14. Inhibition of Acetyl-CoA Carboxylase Activity by Haloxyfop and Tralkoxydim

    PubMed Central

    Secor, Jacob; Cséke, Csaba

    1988-01-01

    Acetyl-coenzyme A (CoA) carboxylase from maize (Zea mays L.) is inhibited by nanomolar concentrations of both haloxyfop, an aryloxyphenoxypropionate, and tralkoxydim, a cyclohexanedione herbicide. These results suggest that acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid biosynthesis, may be the target of these herbicides, contrary to an earlier report suggesting that aryloxyphenoxypropionate herbicides do not inhibit acetyl-CoA carboxylase. PMID:16665846

  15. 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

  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. 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

  18. 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

  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. Inhibition of neutral lipase from castor bean lipid bodies by coenzyme A (CoA) and Oleoyl-CoA

    SciTech Connect

    Not Available

    1989-03-01

    The neutral lipase (EC 3.1.1.3) in lipid body membranes isolated from the endosperm of 4 day old castor (Ricinus communis L.) seedlings catalyzes the hydrolysis of ({sup 14}C)trioleoylglycerol, releasing ({sup 14}C)oleic acid for up to 4 hours. However, the addition of Mg-ATP and coenzyme A (CoA), which are present in the cytoplasm of plant cells, caused a progressive inhibition of the neutral lipase such that after 15 minutes, release of ({sup 14}C)oleic acid was almost undetectable. A fatty acyl CoA synthetase was found in the lipid body membrane which converts ({sup 14}C)oleic acid produced from the lipase reaction to ({sup 14}C)oleoyl-CoA under these conditions. The concentration of free oleoyl-CoA in the reaction mixture when the lipase was inhibited by 50% was calculated to be about 21 micromolar. It was found that a mixture of exogenously added oleoyl-CoA and CoA was most effective in causing lipase inhibition. Little inhibition of lipase was detected in the presence of CoA alone. It is possible that this effect is important in vivo in coordinating lipase activity with fatty acid oxidation.

  2. 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

  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. 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....

  5. 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

  6. 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).

  7. Molecular cloning, characterization, and elicitation of acetyl-CoA carboxylase from alfalfa.

    PubMed Central

    Shorrosh, B S; Dixon, R A; Ohlrogge, J B

    1994-01-01

    Acetyl-CoA carboxylase [ACCase; acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] catalyzes the ATP-dependent carboxylation of acetyl CoA to produce malonyl CoA. In plants, malonyl CoA is needed for plastid localized fatty acid biosynthesis and for a variety of pathways in the cytoplasm including flavonoid biosynthesis. We have determined the full nucleotide sequence of an ACCase from alfalfa, which appears to represent a cytoplasmic isozyme. Partial cDNAs were isolated from a cDNA library of suspension culture cells that had been elicited for isoflavonoid phytoalexin synthesis. The full-length sequence was obtained by primer extension and amplification of the cDNA with synthetic primers. The sequence codes for a protein of 2257 amino acids with a calculated M(r) of 252,039. The biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase domains, respectively, show approximately 72%, 50%, and 65% sequence similarity to those of animal, diatom, and yeast ACCase sequences. ACCase enzyme activity and transcripts are induced severalfold upon addition of yeast or fungal elicitors to alfalfa cell cultures. Images PMID:7910406

  8. Molecular cloning, characterization, and elicitation of acetyl-CoA carboxylase from alfalfa.

    PubMed

    Shorrosh, B S; Dixon, R A; Ohlrogge, J B

    1994-05-10

    Acetyl-CoA carboxylase [ACCase; acetyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.2] catalyzes the ATP-dependent carboxylation of acetyl CoA to produce malonyl CoA. In plants, malonyl CoA is needed for plastid localized fatty acid biosynthesis and for a variety of pathways in the cytoplasm including flavonoid biosynthesis. We have determined the full nucleotide sequence of an ACCase from alfalfa, which appears to represent a cytoplasmic isozyme. Partial cDNAs were isolated from a cDNA library of suspension culture cells that had been elicited for isoflavonoid phytoalexin synthesis. The full-length sequence was obtained by primer extension and amplification of the cDNA with synthetic primers. The sequence codes for a protein of 2257 amino acids with a calculated M(r) of 252,039. The biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase domains, respectively, show approximately 72%, 50%, and 65% sequence similarity to those of animal, diatom, and yeast ACCase sequences. ACCase enzyme activity and transcripts are induced severalfold upon addition of yeast or fungal elicitors to alfalfa cell cultures. PMID:7910406

  9. 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.

  10. 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.

  11. 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 α- and β-subunits of carboxyltransferase (α- and β-CT). At least three different cDNAs were isolated from germinating soybean seeds that encode BC, two that encode BCCP, and four that encode α-CT. Whereas BC, BCCP, and α-CT are products of nuclear genes, the DNA that encodes soybean β-CT is located in chloroplasts. Translation products from cDNAs for BC, BCCP, and α-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 α- and β-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

  12. Doping Induced Itinerant Ferromagnetism in CoAs

    NASA Astrophysics Data System (ADS)

    Chen, Chih-Wei; Morosan, Emilia

    2013-03-01

    The magnetism in α-CoAs is dominated by strong spin fluctuations. In this study, we explore the effects of Phosphorus doping in α-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.

  13. 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

  14. Hypophagic effects of propionate increase with elevated hepatic acetyl coenzyme A concentration for cows in the early postpartum period.

    PubMed

    Stocks, S E; Allen, M S

    2012-06-01

    Thirty multiparous lactating dairy cows were used in a randomized block design experiment to evaluate factors related to the degree of hypophagia from intraruminal infusion of propionate. Cows between 3 and 40 d postpartum at the start of the experiment were blocked by calving date and randomly assigned to treatment. Treatments were 1.0 mol/L propionic acid or 1.0 mol/L acetic acid adjusted to pH 6 with sodium hydroxide and infused at 0.5 mol of volatile fatty acid/h from 6h before feeding until 12h after feeding. Propionate infusion decreased dry matter intake by 20.0%, total metabolizable energy intake by 22.5%, and plasma β-hydroxybutyrate concentration by 54.3% compared with acetate infusion. Effects of treatment on dry matter intake were related to concentration of acetyl coenzyme A (CoA) in the liver; hypophagic effects of propionate compared with acetate increased as liver acetyl CoA concentration increased. Hypophagic effects of propionate are greater for cows with elevated concentrations of acetyl CoA in the liver. PMID:22612960

  15. Cloning, sequencing, and expression of the pantothenate kinase (coaA) gene of Escherichia coli.

    PubMed Central

    Song, W J; Jackowski, S

    1992-01-01

    Pantothenate kinase catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis. The structural gene (coaA) located at 90 min of the Escherichia coli chromosome was cloned and sequenced. The coaA gene was transcribed in the opposite direction to the flanking genes birA and thrU and produced a single 1.1-kb transcript. Translation of the coaA gene produced two protein products (36.4 and 35.4 kDa) that differed by eight amino acids at the amino terminus. The poor homology of the coaA promoter region to consensus E. coli promoter sequences and the low frequency of optimal codon usage (0.565) were consistent with the low abundance of pantothenate kinase. Strains containing multiple copies of the coaA gene possessed 76-fold-higher specific activity of pantothenate kinase; however, there was only a 2.7-fold increase in the steady-state level of CoA. These data corroborate the conclusion that regulation of pantothenate kinase activity by feedback inhibition is the critical factor controlling the intracellular CoA concentration. Images PMID:1328157

  16. Site-specific acetylation of ISWI by GCN5

    PubMed Central

    Ferreira, Roger; Eberharter, Anton; Bonaldi, Tiziana; Chioda, Mariacristina; Imhof, Axel; Becker, Peter B

    2007-01-01

    Background The tight organisation of eukaryotic genomes as chromatin hinders the interaction of many DNA-binding regulators. The local accessibility of DNA is regulated by many chromatin modifying enzymes, among them the nucleosome remodelling factors. These enzymes couple the hydrolysis of ATP to disruption of histone-DNA interactions, which may lead to partial or complete disassembly of nucleosomes or their sliding on DNA. The diversity of nucleosome remodelling factors is reflected by a multitude of ATPase complexes with distinct subunit composition. Results We found further diversification of remodelling factors by posttranslational modification. The histone acetyltransferase GCN5 can acetylate the Drosophila remodelling ATPase ISWI at a single, conserved lysine, K753, in vivo and in vitro. The target sequence is strikingly similar to the N-terminus of histone H3, where the corresponding lysine, H3K14, can also be acetylated by GCN5. The acetylated form of ISWI represents a minor species presumably associated with the nucleosome remodelling factor NURF. Conclusion Acetylation of histone H3 and ISWI by GCN5 is explained by the sequence similarity between the histone and ISWI around the acetylation site. The common motif RKT/SxGx(Kac)xPR/K differs from the previously suggested GCN5/PCAF recognition motif GKxxP. This raises the possibility of co-regulation of a nucleosome remodelling factor and its nucleosome substrate through acetylation of related epitopes and suggests a direct crosstalk between two distinct nucleosome modification principles. PMID:17760996

  17. 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.

  18. 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

  19. Automated IPB in support of wargaming and COA analysis and comparison

    NASA Astrophysics Data System (ADS)

    Mitchell, Charles H.; Williams, James K.

    2001-09-01

    In the area of Modeling and Simulations for Wargaming and Exercise Support, Intelligence Preparation of the Battlespace (IPB) is a flexible process that assists commanders and their staffs in planning and executing campaigns and missions. The Automated Assistance with IPB (A2IPB) tool, being developed for the USAF, enables the leveraging of information about the adversary's capabilities, potential centers of gravity, and possible courses of action (COAs) across all dimensions of the battlespace. Operational staffs heavily depend on IPB products prepared during the analysis of the adversary situation and the evaluation of the battlepace's effects in order to forumlate initial friendly force dispositions and schemes of maneuver. Wargaming is a conscious attempt to visualize the flow of a military operation, given friendly strengths and dispositions, adversary assets, and possible COAs, and a specific battlespace environment. It attempts to foresee the action, reaction, and counteraction dynamics between a pair of friendly and adversary COAs. A2IPB has the potential to allow the commander and his staff to conduct a wargame from within the software. Friendly COAs will be graphically portrayed against the adversary's most likely COA and then against the most dangerous COA. By having all relevent infomration loaded into the same database, conduct of the wargame is facilitated, data modification is easier, and the ability to plan back a particular set of COAs multiple times is provided. Using A2IPB facilities the commander's decision upon a COA believed to be the most advantegeous to the operation. Using the results of wargaming associated with the COA, the staff prpared OPORDs that implement the commander's decision.

  20. 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

  1. 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

  2. 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.

  3. Underlying Resistance Mechanisms in the Cynosurus echinatus Biotype to Acetyl CoA Carboxylase-Inhibiting Herbicides

    PubMed Central

    Fernández, Pablo; Alcántara-de la Cruz, Ricardo; Cruz-Hipólito, Hugo; Osuna, María D.; De Prado, Rafael

    2016-01-01

    Hedgehog dogtail (Cynosurus echinatus) is an annual grass, native to Europe, but also widely distributed in North and South America, South Africa, and Australia. Two hedgehog dogtail biotypes, one diclofop-methyl (DM)-resistant and one DM-susceptible were studied in detail for experimental dose-response resistance mechanisms. Herbicide rates that inhibited shoot growth by 50% (GR50) were determined for DM, being the resistance factor (GR50R/GR50S) of 43.81. When amitrole (Cyt. P450 inhibitor) was applied before treatment with DM, the R biotype growth was significantly inhibited (GR50 of 1019.9 g ai ha-1) compared with the GR50 (1484.6 g ai ha-1) found for the R biotype without pretreatment with amitrole. However, GR50 values for S biotype do not vary with or without amitrole pretreatment. Dose-response experiments carried out to evaluate cross-resistance, showed resistance to aryloxyphenoxypropionate (APP), cyclohexanedione (CHD) and phenylpyrazoline (PPZ) inhibiting herbicides. Both R and S biotypes had a similar 14C-DM uptake and translocation. The herbicide was poorly distributed among leaves, the rest of the shoot and roots with unappreciable acropetal and/or basipetal DM translocation at 96 h after treatment (HAT). The metabolism of 14C-DM, D-acid and D-conjugate metabolites were identified by thin-layer chromatography. The results showed that DM resistance in C. echinatus is likely due to enhanced herbicide metabolism, involving Cyt. P450 as was demonstrated by indirect assays (amitrole pretreatment). The ACCase in vitro assays showed that the target site was very sensitive to APP, CHD and PPZ herbicides in the C. echinatus S biotype, while the R biotype was insensitive to the previously mentioned herbicides. DNA sequencing studies confirmed that C. echinatus cross-resistance to ACCase inhibitors has been conferred by specific ACCase double point mutations Ile-2041-Asn and Cys-2088-Arg. PMID:27148285

  4. Isolation and characterization of temperature-sensitive pantothenate kinase (coaA) mutants of Escherichia coli.

    PubMed Central

    Vallari, D S; Rock, C O

    1987-01-01

    Escherichia coli mutants conditionally defective in the conversion of pantothenate to coenzyme A were isolated and characterized. The gene was designated coaA and localized between argEH and rpoB near min 90 of the chromosome. The coaA15(Ts) mutation caused a temperature-sensitive growth phenotype and temperature-dependent inactivation of pantothenate kinase activity assayed both in vivo and in vitro. At 30 degrees C, coaA15(Ts) extracts contained less than 20% of the wild-type pantothenate kinase activity; the kinase had near normal kinetic constants for the substrates ATP and pantothenate and was inhibited by coenzyme A to the same degree as the wild-type enzyme. These data define the coaA gene as the structural gene for pantothenate kinase. PMID:2824448

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. Exome Sequence Reveals Mutations in CoA Synthase as a Cause of Neurodegeneration with Brain Iron Accumulation

    PubMed Central

    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, Conceição; 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

  13. Progressing batch hydrolysis process

    DOEpatents

    Wright, John D.

    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.

  14. Bad Ac-etylated Tau.

    PubMed

    Hettinger, Jane C; Cirrito, John R

    2016-04-20

    By using a tau construct with two mimicked acetylation sites as identified in AD brains, Tracy et al. (2016) found that acetylated tau promotes synaptic dysfunction through disruption of postsynaptic KIBRA signaling pathways, actin dynamics, and AMPA receptor trafficking. PMID:27100190

  15. Cryogenic Optical Assembly (COA) cooldown analysis for the Cosmic Background Explorer (COBE)

    NASA Technical Reports Server (NTRS)

    Coladonato, Robert J.; Irish, Sandra M.; Mosier, Carol L.

    1990-01-01

    The Cosmic Background Explorer (COBE) spacecraft, developed by Goddard Space Flight Center (GSFC), was successfully launched on November 18, 1989 aboard a Delta expendable launch vehicle. Two of the three instruments for this mission were mounted inside a liquid helium (LHe) dewar which operates at a temperature of 2 K. These two instruments are the Diffuse Infrared Background Experiment (DIRBE) and the Far Infrared Absolute Spectrophotometer (FIRAS). They are mounted to a common Instrument Interface Structure (IIS) and the entire assembly is called the Cryogenic Optical Assembly (COA). As part of the structural verification requirement, it was necessary to show that the entire COA exhibited adequate strength and would be capable of withstanding the launch environment. This requirement presented an unique challenge for COBE because the COA is built and assembled at room temperature (300 K), cooled to 2 K, and then subjected to launch loads. However, strength testing of the entire COA at 2 K could not be done because of facility limitations. Therefore, it was decided to perform the strength verification of the COA by analysis.

  16. Structural and docking studies of Leucaena leucocephala Cinnamoyl CoA reductase.

    PubMed

    Prasad, Nirmal K; Vindal, Vaibhav; Kumar, Vikash; Kabra, Ashish; Phogat, Navneet; Kumar, Manoj

    2011-03-01

    Lignin, a major constituent of plant call wall, is a phenolic heteropolymer. It plays a major role in the development of plants and their defense mechanism against pathogens. Therefore Lignin biosynthesis is one of the critical metabolic pathways. In lignin biosynthesis, the Cinnamoyl CoA reductase is a key enzyme which catalyzes the first step in the pathway. Cinnamoyl CoA reductase provides the substrates which represent the main transitional molecules of lignin biosynthesis pathway, exhibits a high in vitro kinetic preference for feruloyl CoA. In present study, the three-dimensional model of cinnamoyl CoA reductase was constructed based on the crystal structure of Grape Dihydroflavonol 4-Reductase. Furthermore, the docking studies were performed to understand the substrate interactions to the active site of CCR. It showed that residues ARG51, ASN52, ASP54 and ASN58 were involved in substrate binding. We also suggest that residue ARG51 in CCR is the determinant residue in competitive inhibition of other substrates. This structural and docking information have prospective implications to understand the mechanism of CCR enzymatic reaction with feruloyl CoA, however the approach will be applicable in prediction of substrates and engineering 3D structures of other enzymes as well. PMID:20512516

  17. Distinct roles of carbohydrate esterase family CE16 acetyl esterases and polymer-acting acetyl xylan esterases in xylan deacetylation.

    PubMed

    Koutaniemi, S; van Gool, M P; Juvonen, M; Jokela, J; Hinz, S W; Schols, H A; Tenkanen, M

    2013-12-01

    Mass spectrometric analysis was used to compare the roles of two acetyl esterases (AE, carbohydrate esterase family CE16) and three acetyl xylan esterases (AXE, families CE1 and CE5) in deacetylation of natural substrates, neutral (linear) and 4-O-methyl glucuronic acid (MeGlcA) substituted xylooligosaccharides (XOS). AEs were similarly restricted in their action and apparently removed in most cases only one acetyl group from the non-reducing end of XOS, acting as exo-deacetylases. In contrast, AXEs completely deacetylated longer neutral XOS but had difficulties with the shorter ones. Complete deacetylation of neutral XOS was obtained after the combined action of AEs and AXEs. MeGlcA substituents partially restricted the action of both types of esterases and the remaining acidic XOS were mainly substituted with one MeGlcA and one acetyl group, supposedly on the same xylopyranosyl residue. These resisting structures were degraded to great extent only after inclusion of α-glucuronidase, which acted with the esterases in a synergistic manner. When used together with xylan backbone degrading endoxylanase and β-xylosidase, both AE and AXE enhanced the hydrolysis of complex XOS equally. PMID:24140638

  18. Molecular typing of Staphylococcus aureus based on PCR-RFLP of coa gene and RAPD analysis.

    PubMed

    Karakulska, J; Pobucewicz, A; Nawrotek, P; Muszyńska, M; Furowicz, A J; Czernomysy-Furowicz, D

    2011-01-01

    The aim of this study was molecular identification of S. aureus strains isolated from mastitic milk samples and establishing the genetic relationship between strains isolated from cows belonging to the same herd. In all 43 isolated strains the gap gene (930 bp) was amplified, which enabled their affiliation to the Staphylococcus genus to be established. PCR-RFLP with AluI endonuclease of the gap gene as well as nuc (450 bp) and coa (1130 bp) gene amplification allowed precise S. aureus species identification. One hundred percent of the genetic relationship between strains was established via RAPD-PCR and coa-typing. PMID:21721416

  19. 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

  20. 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

  1. Control of Acetyl-Coenzyme A Synthetase (AcsA) Activity by Acetylation/Deacetylation without NAD+ Involvement in Bacillus subtilis†

    PubMed Central

    Gardner, Jeffrey G.; Grundy, Frank J.; Henkin, Tina M.; Escalante-Semerena, Jorge C.

    2006-01-01

    Posttranslational modification is an efficient mechanism for controlling the activity of structural proteins, gene expression regulators, and enzymes in response to rapidly changing physiological conditions. Here we report in vitro and in vivo evidence that the acuABC operon of the gram-positive soil bacterium Bacillus subtilis encodes a protein acetyltransferase (AcuA) and a protein deacetylase (AcuC), which may control the activity of acetyl-coenzyme A (CoA) synthetase (AMP-forming, AcsA) in this bacterium. Results from in vitro experiments using purified proteins show that AcsA is a substrate for the acetyl-CoA-dependent AcuA acetyltransferase. Mass spectrometry analysis of a tryptic digest of acetylated AcsA (AcsAAc) identified residue Lys549 as the sole modification site in the protein. Unlike sirtuins, the AcuC protein did not require NAD+ as cosubstrate to deacetylate AcsAAc. The function of the putative AcuB protein remains unknown. PMID:16855235

  2. Regulation of Glycolysis and Gluconeogenesis by Acetylation of PKM and PEPCK

    PubMed Central

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

    2016-01-01

    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

  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. 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.

  6. 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...

  7. The COA360: a tool for assessing the cultural competency of healthcare organizations.

    PubMed

    LaVeist, Thomas A; Relosa, Rachel; Sawaya, Nadia

    2008-01-01

    The U.S. Census Bureau projects that by 2050, non-Hispanic whites will be in the numerical minority. This rapid diversification requires healthcare organizations to pay closer attention to cross-cultural issues if they are to meet the healthcare needs of the nation and continue to maintain a high standard of care. Although scorecards and benchmarking are widely used to gauge healthcare organizations' performance in various areas, these tools have been underused in relation to cultural preparedness or initiatives. The likely reason for this is the lack of a validated tool specifically designed to examine cultural competency. Existing validated cultural competency instruments evaluate individuals, not organizations. In this article, we discuss a study to validate the Cultural Competency Organizational Assessment--360 or the COA360, an instrument designed to appraise a healthcare organization's cultural competence. The Office of Minority Health and the Joint Commission have each developed standards for measuring the cultural competency of organizations. The COA360 is designed to assess adherence to both of these sets of standards. For this validation study, we enlisted a panel of national experts. The panel rated each dimension of the COA360, and the combination of items for each of the scale's 14 dimensions was rated above 4.13 (on 5-point scale). Our conclusion points to the validity of the COA360. As such, it is a valuable tool not only for assessing a healthcare organization's cultural readiness but also for benchmarking its progress in addressing cultural and diversity issues. PMID:18720687

  8. Coenzyme A esters of 2-aryloxyphenoxypropionate herbicides and 2-arylpropionate antiinflammatory drugs are potent and stereoselective inhibitors of rat liver acetyl-CoA carboxylase.

    PubMed

    Kemal, C; Casida, J E

    1992-01-01

    The CoA esters of diclofop, haloxyfop and fluazifop are up to 425-fold more potent than the corresponding unconjugated herbicides as inhibitors of rat liver acetyl-CoA carboxylase (EC 6.4.1.2); the most potent inhibitor is (R)-fluazifopyl-CoA2 (Ki = 0.03 microM). The binding site is stereoselective for (R)-diclofop, the herbicidally active enantiomer, and for (R)-diclofopyl-CoA. The CoA esters of the antiinflammatory drugs ibuprofen and fenoprofen also strongly inhibit this carboxylase. (S)-Ibuprofenyl-CoA (Ki = 0.7 microM), the CoA ester of the enantiomer with antiinflammatory activity, is 15-fold more potent as an inhibitor than (R)-ibuprofenyl-CoA. These results suggest that some of the biological effects of these herbicides and antiinflammatory drugs in animals may be due to the inhibition of acetyl-CoA carboxylase by their acyl-CoA derivatives. PMID:1347398

  9. 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.

  10. 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.

  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. 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

  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. 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

  15. The Antibiotic CJ-15,801 is an Antimetabolite which Hijacks and then Inhibits CoA Biosynthesis

    PubMed Central

    van der Westhuyzen, Renier; Hammons, Justin C.; Meier, Jordan L.; Dahesh, Samira; Moolman, Wessel J. A.; Pelly, Stephen C.; Nizet, Victor; Burkart, Michael D.; Strauss, Erick

    2012-01-01

    SUMMARY The natural product CJ-15,801 is an inhibitor of Staphylococcus aureus, but not other bacteria. Its close structural resemblance to pantothenic acid, the vitamin precursor of coenzyme A (CoA), and its Michael acceptor moiety suggest that it irreversibly inhibits an enzyme involved in CoA biosynthesis or utilization. However, its mode of action and the basis for its specificity have not been elucidated to date. We demonstrate that CJ-15,801 is transformed by the uniquely selective S. aureus pantothenate kinase, the first CoA biosynthetic enzyme, into a substrate for the next enzyme, phosphopantothenoylcysteine synthetase, which is inhibited through formation of a tight-binding structural mimic of its native reaction intermediate. These findings reveal CJ-15,801 as a vitamin biosynthetic pathway antimetabolite with a mechanism similar to that of the sulfonamide antibiotics, and highlight CoA biosynthesis as a viable antimicrobial drug target. PMID:22633408

  16. Variations in the Localization of Acetyl-Coenzyme A Synthetase in Aerobic Yeast Cells

    PubMed Central

    Klein, Harold P.; Jahnke, Linda

    1971-01-01

    In cells of Saccharomyces cerevisiae growing aerobically for 24 hr, acetyl-coenzyme A synthetase [acetate: CoA ligase (AMP), EC 6.2.1.1] was localized principally in the microsomal fraction. On density gradients, the enzyme in such cells behaved as a low-density particle, readily separable from the soluble proteins. After 48 hr of incubation, the cells showed a bimodal distribution of enzyme, with most of the activity now sedimenting with the mitochondrial fraction and only a smaller amount with the microsomal fraction. By using density gradients, two forms of synthetase were obtained from these cells: one band denser and the other band less dense than the intact mitochondria. In all preparations containing synthetase activity, appreciable levels of phospholipids were also detected. Images PMID:4102333

  17. Bacterial protein acetylation: new discoveries unanswered questions.

    PubMed

    Wolfe, Alan J

    2016-05-01

    Nε-acetylation is emerging as an abundant post-translational modification of bacterial proteins. Two mechanisms have been identified: one is enzymatic, dependent on an acetyltransferase and acetyl-coenzyme A; the other is non-enzymatic and depends on the reactivity of acetyl phosphate. Some, but not most, of those acetylations are reversed by deacetylases. This review will briefly describe the current status of the field and raise questions that need answering. PMID:26660885

  18. Germline Deletion of Pantothenate Kinases 1 and 2 Reveals the Key Roles for CoA in Postnatal Metabolism

    PubMed Central

    Garcia, Matthew; Leonardi, Roberta; Zhang, Yong-Mei; Rehg, Jerold E.; Jackowski, Suzanne

    2012-01-01

    Pantothenate kinase (PanK) phosphorylates pantothenic acid (vitamin B5) and controls the overall rate of coenzyme A (CoA) biosynthesis. Pank1 gene deletion in mice results in a metabolic phenotype where fatty acid oxidation and gluconeogenesis are impaired in the fasted state, leading to mild hypoglycemia. Inactivating mutations in the human PANK2 gene lead to childhood neurodegeneration, but Pank2 gene inactivation in mice does not elicit a phenotype indicative of the neuromuscular symptoms or brain iron accumulation that accompany the human disease. Pank1/Pank2 double knockout (dKO) mice were derived to determine if the mild phenotypes of the single knockout mice are due to the ability of the two isoforms to compensate for each other in CoA biosynthesis. Postnatal development was severely affected in the dKO mice. The dKO pups developed progressively severe hypoglycemia and hyperketonemia by postnatal day 10 leading to death by day 17. Hyperketonemia arose from impaired whole-body ketone utilization illustrating the requirement for CoA in energy generation from ketones. dKO pups had reduced CoA and decreased fatty acid oxidation coupled with triglyceride accumulation in liver. dKO hepatocytes could not maintain the NADH levels compared to wild-type hepatocytes. These results revealed an important link between CoA and NADH levels, which was reflected by deficiencies in hepatic oleate synthesis and gluconeogenesis. The data indicate that PanK1 and PanK2 can compensate for each other to supply tissue CoA, but PanK1 is more important to CoA levels in liver whereas PanK2 contributes more to CoA synthesis in the brain. PMID:22815849

  19. Copper supplementation restores cytochrome c oxidase assembly defect in a mitochondrial disease model of COA6 deficiency

    PubMed Central

    Ghosh, Alok; Trivedi, Prachi P.; Timbalia, Shrishiv A.; Griffin, Aaron T.; Rahn, Jennifer J.; Chan, Sherine S. L.; Gohil, Vishal M.

    2014-01-01

    Mitochondrial respiratory chain biogenesis is orchestrated by hundreds of assembly factors, many of which are yet to be discovered. Using an integrative approach based on clues from evolutionary history, protein localization and human genetics, we have identified a conserved mitochondrial protein, C1orf31/COA6, and shown its requirement for respiratory complex IV biogenesis in yeast, zebrafish and human cells. A recent next-generation sequencing study reported potential pathogenic mutations within the evolutionarily conserved Cx9CxnCx10C motif of COA6, implicating it in mitochondrial disease biology. Using yeast coa6Δ cells, we show that conserved residues in the motif, including the residue mutated in a patient with mitochondrial disease, are essential for COA6 function, thus confirming the pathogenicity of the patient mutation. Furthermore, we show that zebrafish embryos with zfcoa6 knockdown display reduced heart rate and cardiac developmental defects, recapitulating the observed pathology in the human mitochondrial disease patient who died of neonatal hypertrophic cardiomyopathy. The specific requirement of Coa6 for respiratory complex IV biogenesis, its intramitochondrial localization and the presence of the Cx9CxnCx10C motif suggested a role in mitochondrial copper metabolism. In support of this, we show that exogenous copper supplementation completely rescues respiratory and complex IV assembly defects in yeast coa6Δ cells. Taken together, our results establish an evolutionarily conserved role of Coa6 in complex IV assembly and support a causal role of the COA6 mutation in the human mitochondrial disease patient. PMID:24549041

  20. 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; Krüger, 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 Ran•GTP/Ran•GDP 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

  1. Acetyl Coenzyme A Carboxylase Activity in Developing Seedlings and Chloroplasts of Barley and Its Virescens Mutant 1

    PubMed Central

    Thomson, Lawrence W.; Zalik, Saul

    1981-01-01

    Acetyl coenzyme A (CoA) carboxylase activity of whole tissue homogenates and chloroplast preparations was analyzed as the acetyl-CoA-dependent incorporation of [14C]bicarbonate into an acid-stable product. The absolute requirement for ATP and MgCl2, the complete inhibition with avidin, and end-product analysis were consistent with the presence of acetyl-CoA carboxylase activity. Little difference was found between the mutant and normal tissue homogenates from the 1- to 3-day growth stages, during which period both showed a 3-fold increase. However, by 4 days, the activity of the mutant exceeded that of the normal. Fractionation studies showed that the enzyme was a soluble protein present in the stromal fraction of chloroplasts. The biotin content was also highest in the stroma, although it was found in the lamellar fraction as well. For both the mutant and the normal, the highest acetyl-CoA carboxylase activities were obtained in the stromal preparations from 4-day seedlings (54 and 31 nmoles per milligram protein per minute for the mutant and the normal, respectively) with a progressive decline by 6 and 8 days. The difference between the mutant and the normal was not due to the accumulation of an inhibitor in the normal. PMID:16661731

  2. Cooperation between COA6 and SCO2 in COX2 maturation during cytochrome c oxidase assembly links two mitochondrial cardiomyopathies.

    PubMed

    Pacheu-Grau, David; Bareth, Bettina; Dudek, Jan; Juris, Lisa; Vögtle, F-Nora; Wissel, Mirjam; Leary, Scot C; Dennerlein, Sven; Rehling, Peter; Deckers, Markus

    2015-06-01

    Three mitochondria-encoded subunits form the catalytic core of cytochrome c oxidase, the terminal enzyme of the respiratory chain. COX1 and COX2 contain heme and copper redox centers, which are integrated during assembly of the enzyme. Defects in this process lead to an enzyme deficiency and manifest as mitochondrial disorders in humans. Here we demonstrate that COA6 is specifically required for COX2 biogenesis. Absence of COA6 leads to fast turnover of newly synthesized COX2 and a concomitant reduction in cytochrome c oxidase levels. COA6 interacts transiently with the copper-containing catalytic domain of newly synthesized COX2. Interestingly, similar to the copper metallochaperone SCO2, loss of COA6 causes cardiomyopathy in humans. We show that COA6 and SCO2 interact and that corresponding pathogenic mutations in each protein affect complex formation. Our analyses define COA6 as a constituent of the mitochondrial copper relay system, linking defects in COX2 metallation to cardiac cytochrome c oxidase deficiency. PMID:25959673

  3. Kinetic studies on two isoforms of acetyl-CoA carboxylase from maize leaves.

    PubMed Central

    Herbert, D; Price, L J; Alban, C; Dehaye, L; Job, D; Cole, D J; Pallett, K E; Harwood, J L

    1996-01-01

    The steady-state kinetics of two multifunctional isoforms of acetyl-CoA carboxylase (ACCase) from maize leaves (a major isoform, ACCase1 and a minor isoform, ACCase2) have been investigated with respect to reaction mechanism, inhibition by two graminicides of the aryloxyphenoxypropionate class (quizalofop and fluazifop) and some cellular metabolites. Substrate interaction and product inhibition patterns indicated that ADP and P(i) products from the first partial reaction were not released before acetyl-CoA bound to the enzymes. Product inhibition patterns did not match exactly those predicted for an ordered Ter Ter or a random Ter Ter mechanism, but were close to those postulated for an ordered mechanism. ACCase2 was about 1/2000 as sensitive as ACCase1 to quizalofop but only about 1/150 as sensitive to fluazifop. Fitting inhibition data to the Hill equation indicated that binding of quizalofop or fluazifop to ACCase1 was non-cooperative, as shown by the Hill constant (n(app)) values of 0.86 and 1.16 for quizalofop and fluazifop respectively. Apparent inhibition constant values (K' from the Hill equation) for ACCase1 were 0.054 microM for quizalofop and 21.8 microM for fluazifop. On the other hand, binding of quizalofop or fluazifop to ACCase2 exhibited positive co-operativity, as shown by the (napp) values of 1.85 and 1.59 for quizalofop and fluazifop respectively. K' values for ACCase2 were 1.7 mM for quizalofop and 140 mM for fluazifop. Kinetic parameters for the co-operative binding of quizalofop to maize ACCase2 were close to those of another multifunctional ACCase of limited sensitivity to graminicide, ACC220 from pea. Inhibition of ACCase1 by quizalofop was mixed-type with respect to acetyl-CoA or ATP, but the concentration of acetyl-CoA had the greater effect on the level of inhibition. Neither ACCase1 nor ACCase2 was appreciably sensitive to CoA esters of palmitic acid (16:0) or oleic acid (18:1). Approximate IC50 values were 10 microM (ACCase2) and 50 microM (ACCase1) for both CoA esters. Citrate concentrations up to 1 mM had no effect on ACCase1 activity. Above this concentration, citrate was inhibitory. ACCase2 activity was slightly stimulated by citrate over a broad concentration range (0.25-10 mM). The significance of possible effects of acyl-CoAs or citrate in vivo is discussed. PMID:8836149

  4. 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...

  5. 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...

  6. Hydrolysis reactor for hydrogen production

    DOEpatents

    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.

  7. A Chemo-Enzymatic Road Map to the Synthesis of CoA Esters.

    PubMed

    Peter, Dominik M; Vögeli, Bastian; Cortina, Niña Socorro; Erb, Tobias J

    2016-01-01

    Coenzyme A (CoA) is a ubiquitous cofactor present in every known organism. The thioesters of CoA are core intermediates in many metabolic processes, such as the citric acid cycle, fatty acid biosynthesis and secondary metabolism, including polyketide biosynthesis. Synthesis of CoA-thioesters is vital for the study of CoA-dependent enzymes and pathways, but also as standards for metabolomics studies. In this work we systematically tested five chemo-enzymatic methods for the synthesis of the three most abundant acyl-CoA thioester classes in biology; saturated acyl-CoAs, α,β-unsaturated acyl-CoAs (i.e., enoyl-CoA derivatives), and α-carboxylated acyl-CoAs (i.e., malonyl-CoA derivatives). Additionally we report on the substrate promiscuity of three newly described acyl-CoA dehydrogenases that allow the simple conversion of acyl-CoAs into enoyl-CoAs. With these five methods, we synthesized 26 different CoA-thioesters with a yield of 40% or higher. The CoA esters produced range from short- to long-chain, include branched and α,β-unsaturated representatives as well as other functional groups. Based on our results we provide a general guideline to the optimal synthesis method of a given CoA-thioester in respect to its functional group(s) and the commercial availability of the precursor molecule. The proposed synthetic routes can be performed in small scale and do not require special chemical equipment, making them convenient also for biological laboratories. PMID:27104508

  8. 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. PMID:26937394

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

    PubMed

    Stroud, David A; Maher, Megan J; Lindau, Caroline; Vögtle, 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

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

    PubMed

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

    2015-11-01

    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

  11. 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

  12. 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

  13. 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.

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

    NASA Astrophysics Data System (ADS)

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

  15. Association between the enterotoxin production and presence of Coa, Nuc genes among Staphylococcus aureus isolated from various sources, in Shiraz

    PubMed Central

    Moghassem Hamidi, R; Hosseinzadeh, S; Shekarforoush, S. S.; Poormontaseri, M; Derakhshandeh, A

    2015-01-01

    The present study was aimed to identify the frequency of coagulase (Coa) and thermonuclease (Nuc) genes and Staphylococcal enterotoxin A (Sea) production among Staphylococcus aureus isolated from various sources in Shiraz. Moreover, the correlation between the Sea gene and coagulase and thermonuclease enzymes is also considered. A total of 100 S. aureus were isolated from various sources including 40 humans, 30 animals and 30 food samples by the routine biochemical tests. The frequency of Coa, Nuc and Sea genes was evaluated by PCR assay. Correlation among those genes was finally evaluated by statistical analysis. The PCR results showed that the prevalence of Coa, Nuc and Sea genes was 91%, 100% and 14%, respectively. The evaluation of the enterotoxin production indicated that 78.6% of the Sea gene was expressed. The presence of enterotoxin A was not necessarily correlated to the production of toxin. As a final conclusion to detect the enterotoxigenic strains, both genotypic and phenotypic methods are highly recommended. PMID:27175208

  16. 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.

  17. UNIQUE ACETYLATION OF OLIGOSACCHARIDES BY TRICHODERMA REESEI ACETYL ESTERASE IN WATER - VINYL ACETATE MIXTURE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Purified T. reesei RUT C-30 acetyl esterase catalyzes acetyl transfer to a variety of carbohydrates in water in the presence of vinyl acetate as the acetyl group donor. The degree of conversion and the number of formed acetates depended on the acceptor used. With some acceptors, such as methyl or ...

  18. 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

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

    PubMed

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

    2015-07-01

    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

  1. 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...

  2. Effects of CoA and acyl-CoA on Ca(2+)-permeability of endoplasmic-reticulum membranes from rat liver.

    PubMed Central

    Rich, G T; Comerford, J G; Graham, S; Dawson, A P

    1995-01-01

    We have studied the effects of CoA and palmitoyl-CoA on Ca2+ movements and GTP-dependent vesicle fusion in rat liver microsomes. (1) Inhibition of membrane fusion by CoA depends on esterification of CoA to long-chain acyl-CoA using endogenous non-esterified fatty acids. (2) Binding of long-chain acyl-CoA to microsomal membranes is inhibited by BSA, which also relieves inhibition of membrane fusion. (3) Under conditions where acyl-CoA binding is inhibited, CoA causes increased Ca2+ accumulation, apparently by decreasing the Ca2+ leak rate. (4) Conversely, palmitoyl-CoA, in the presence of BSA, causes Ca2+ efflux. (5) The decrease in Ca(2+)-permeability caused by CoA does not depend on the presence of ATP or GTP, and is irreversible in the short term. (6) Using 14C-labelled CoA we show that CoA derivatives can be formed from endogenous components of microsomal membranes in the absence of ATP. (7) The results are interpreted in terms of a Ca(2+)-permeability which is controlled by CoA and/or long-chain acyl-CoA esters. PMID:7702563

  3. 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

  4. 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.

  5. 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

  6. 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

  7. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Multipurpose Additives § 172.828 Acetylated monoglycerides. The food additive acetylated... of catalytic agents that are not food additives or are authorized by regulation, followed by...

  8. A Method to Determine Lysine Acetylation Stoichiometries

    PubMed Central

    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, Ljiljiana; Qian, Wei-Jun; Smith, Richard D.; Adkins, Joshua N.; Ansong, Charles

    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 sodium 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. PMID:25143833

  9. 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.; Pérez-Cerdá, 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

  10. 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...

  11. 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 (α-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

  12. 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.

  13. 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.

  14. 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...

  15. 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). ...

  16. Acetylation-mediated suppression of transcription-independent memory: bidirectional modulation of memory by acetylation.

    PubMed

    Merschbaecher, Katja; Haettig, Jakob; Mueller, Uli

    2012-01-01

    Learning induced changes in protein acetylation, mediated by histone acetyl transferases (HATs), and the antagonistic histone deacetylases (HDACs) play a critical role in memory formation. The status of histone acetylation affects the interaction between the transcription-complex and DNA and thus regulates transcription-dependent processes required for long-term memory (LTM). While the majority of studies report on the role of elevated acetylation in memory facilitation, we address the impact of both, increased and decreased acetylation on formation of appetitive olfactory memory in honeybees. We show that learning-induced changes in the acetylation of histone H3 at aminoacid-positions H3K9 and H3K18 exhibit distinct and different dynamics depending on the training strength. A strong training that induces LTM leads to an immediate increase in acetylation at H3K18 that stays elevated for hours. A weak training, not sufficient to trigger LTM, causes an initial increase in acetylation at H3K18, followed by a strong reduction in acetylation at H3K18 below the control group level. Acetylation at position H3K9 is not affected by associative conditioning, indicating specific learning-induced actions on the acetylation machinery. Elevating acetylation levels by blocking HDACs after conditioning leads to an improved memory. While memory after strong training is enhanced for at least 2 days, the enhancement after weak training is restricted to 1 day. Reducing acetylation levels by blocking HAT activity after strong training leads to a suppression of transcription-dependent LTM. The memory suppression is also observed in case of weak training, which does not require transcription processes. Thus, our findings demonstrate that acetylation-mediated processes act as bidirectional regulators of memory formation that facilitate or suppress memory independent of its transcription-requirement. PMID:23028801

  17. 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

  18. Determination of 3-hydroxy-3-methylglutaryl CoA reductase activity in plants.

    PubMed

    Campos, Narciso; Arró, Montserrat; Ferrer, Albert; Boronat, Albert

    2014-01-01

    The enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyzes the NADPH-mediated reductive deacylation of HMG-CoA to mevalonic acid, which is the first committed step of the mevalonate pathway for isoprenoid biosynthesis. In agreement with its key regulatory role in the pathway, plant HMG-CoA reductase is modulated by many diverse external stimuli and endogenous factors and can be detected to variable levels in every plant tissue. A fine determination of HMG-CoA reductase activity levels is required to understand its contribution to plant development and adaptation to changing environmental conditions. Here, we report a procedure to reliably determine HMG-CoA reductase activity in plants. The method includes the sample collection and homogenization strategies as well as the specific activity determination based on a classical radiochemical assay. PMID:24777788

  19. 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

  20. 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...

  1. Carnitine palmitoyl transferase activity in Morris Hepatoma 7777 mitochondria and its sensitivity to malonyl CoA inhibition

    SciTech Connect

    Woldegiorgis, G.; Shrago, E.

    1986-05-01

    Earlier reports in the literature have indicated no detectable Carnitine Palymitoyl Transferase (CPT) activity in homogenates prepared from Morris Hepatoma 7777. In its study CPT activity in isolated mitochondria (mito) was measured by butanol extraction of the (/sup 3/H)palmitoyl carnitine formed as outlined by Bremer et al. Contrary to the earlier work where no appreciable activity of CPT was observed the authors find significant levels of CPT (2.6 nMol/min/mg protein) in isolated mito from Morris Hepatoma 7777 (MH 7777). The level of CPT activity observed in MH 7777 mito was, however, 36% lower compared to the host liver CPT activity (4.1 nMol/min/mg protein). The enzyme in MH 7777 mito showed 83% inhibition in the presence of 10 ..mu..M malonyl CoA, in agreement with the degree of sensitivity observed with the host liver isolated mito. On freeze thawing host mito, total CPT activity increased and the sensitivity of the enzyme to malonyl CoA decreased. Frozen thawed MH 7777 mito showed a similar response to malonyl CoA but no change in the total CPT level was observed. The authors results establish for the first time the presence of a malonyl CoA sensitive CPT in MH 7777 mito, which may have slightly different properties from normal due to the membrane environment of the enzyme.

  2. 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 35°C 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

  3. 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

  4. 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

  5. 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

  6. Wheat acetyl-CoA carboxylase.

    PubMed

    Gornicki, P; Haselkorn, R

    1993-06-01

    The acetyl-CoA carboxylase present in both wheat germ and total wheat leaf protein contains ca. 220 kDa subunits. It is the major biotin-dependent carboxylase present in wheat chloroplasts. Active acetyl-CoA carboxylase purified from wheat germ is a homodimer with an apparent molecular mass of ca. 500 kDa. The enzyme from wheat germ or from wheat chloroplasts is sensitive to the herbicide haloxyfop at micromolar levels. The incorporation of 14C-acetate into fatty acids in freshly cut wheat seedling leaves provides a convenient in vivo assay for both acetyl-CoA carboxylase and haloxyfop. PMID:8101104

  7. 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

  8. 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

  9. Phthalate hydrolysis under landfill conditions.

    PubMed

    Jonsson, S; Vavilin, V A; Svensson, B H

    2006-01-01

    Experimental data from a study using a landfill simulation reactor were used to develop and calibrate a one-dimensional distributed model of co-digestion of municipal solid waste and three phthalic acid diesters with different water solubilities. The three diesters were diethyl phthalate, dibutyl phthalate, and di-2-ethylhexyl phthalate. Two types of municipal solid wastes were assumed, easily degradable and recalcitrant. The model considered inhibition of hydrolysis of the recalcitrant fraction and phthalic acid esters, and also methanogenesis at acidic pH. The results indicated that the prolonged steady-state concentrations of the diesters in the leachates could be explained by equilibrium between physicochemical desorption and sorption processes for the three diesters. When methanogenic conditions were induced in the acidogenic landfill simulation reactor, inhibition of both hydrolysis of recalcitrant MSW and of phthalic acid esters ceased. PMID:16784196

  10. Hydrolysis of an acetylthiocholine by pralidoxime iodide (2-PAM).

    PubMed

    Sakurada, Koichi; Ikegaya, Hiroshi; Ohta, Hikoto; Akutsu, Tomoko; Takatori, Takehiko

    2006-10-25

    Pralidoxime iodide (2-PAM), an antidote approved for the reactivation of inhibited acetylcholinesterase (AChE) in organophosphate poisoning, dose-dependently hydrolyzed an acetylthiocholine iodide (ASCh). The AChE (0.3 U) activity inhibited by VX analog (ENMP, 0.1 microM) increased to approximately 200% of normal levels after a dosage of 5 mM 2-PAM (control 0.132+/-0.012 U/ml, 5 mM 0.253+/-0.026 U/ml). This result indicates that 2-PAM produced a thiocholine from the ASCh by hydrolysis. High-performance liquid chromatography (HPLC) analysis was then performed to further clarify the hydrolysis of ASCh with 2-PAM. It was clear that 2-PAM was converted to acetylated 2-PAM with acetic acid produced from ASCh by hydrolysis. Next, we tried to compare this esterase-like activity of 2-PAM with that of obidoxime, which is known as a strong reactivator of inhibited AChE, and with diacetylmonoxime, known as a weak reactivator. All of these oximes showed esterase-like activity, and their strengths were consistent with those of known reactivators of inhibited AChE. These results indicate that a great deal of the data obtained previously with ASCh relating to the effects of oximes must be rechecked. It is clear that oximes easily hydrolyze ASCh. We therefore strongly caution that the method of determining AChE activity with ASCh is not suitable for examining the effects of oximes. PMID:16971069

  11. Peripheral Effects of FAAH Deficiency on Fuel and Energy Homeostasis: Role of Dysregulated Lysine Acetylation

    PubMed Central

    Vaitheesvaran, Bhavapriya; Yang, Li; Hartil, Kirsten; Glaser, Sherrye; Yazulla, Stephen; Bruce, James E.; Kurland, Irwin J.

    2012-01-01

    Background FAAH (fatty acid amide hydrolase), primarily expressed in the liver, hydrolyzes the endocannabinoids fatty acid ethanolamides (FAA). Human FAAH gene mutations are associated with increased body weight and obesity. In our present study, using targeted metabolite and lipid profiling, and new global acetylome profiling methodologies, we examined the role of the liver on fuel and energy homeostasis in whole body FAAH−/− mice. Methodology/Principal Findings FAAH−/− mice exhibit altered energy homeostasis demonstrated by decreased oxygen consumption (Indirect calorimetry). FAAH−/− mice are hyperinsulinemic and have adipose, skeletal and hepatic insulin resistance as indicated by stable isotope phenotyping (SIPHEN). Fed state skeletal muscle and liver triglyceride levels was increased 2–3 fold, while glycogen was decreased 42% and 57% respectively. Hepatic cholesterol synthesis was decreased 22% in FAAH−/− mice. Dysregulated hepatic FAAH−/− lysine acetylation was consistent with their metabolite profiling. Fasted to fed increases in hepatic FAAH−/− acetyl-CoA (85%, p<0.01) corresponded to similar increases in citrate levels (45%). Altered FAAH−/− mitochondrial malate dehydrogenase (MDH2) acetylation, which can affect the malate aspartate shuttle, was consistent with our observation of a 25% decrease in fed malate and aspartate levels. Decreased fasted but not fed dihydroxyacetone-P and glycerol-3-P levels in FAAH−/− mice was consistent with a compensating contribution from decreased acetylation of fed FAAH−/− aldolase B. Fed FAAH−/− alcohol dehydrogenase (ADH) acetylation was also decreased. Conclusions/Significance Whole body FAAH deletion contributes to a pre-diabetic phenotype by mechanisms resulting in impairment of hepatic glucose and lipid metabolism. FAAH−/− mice had altered hepatic lysine acetylation, the pattern sharing similarities with acetylation changes reported with chronic alcohol treatment. Dysregulated hepatic lysine acetylation seen with impaired FAA hydrolysis could support the liver's role in fostering the pre-diabetic state, and may reflect part of the mechanism underlying the hepatic effects of endocannabinoids in alcoholic liver disease mouse models. PMID:22442717

  12. Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site

    PubMed Central

    Sayer, Christopher; Finnigan, William; Isupov, Michail N.; Levisson, Mark; Kengen, Servé W. M.; van der Oost, John; Harmer, Nicholas J.; Littlechild, Jennifer A.

    2016-01-01

    A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions. PMID:27160974

  13. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... molecular distillation or by steam stripping; or (2) The direct acetylation of edible monoglycerides with acetic anhydride without the use of catalyst or molecular distillation, and with the removal by...

  14. 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

  15. 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

  16. The acetylator phenotypes of Saudi Arabian diabetics.

    PubMed Central

    Evans, D A; Paterson, S; Francisco, P; Alvarez, G

    1985-01-01

    There is a significant association between the rapid acetylator phenotype and diabetes in European populations. Diabetes is a common problem in Saudi Arabians with some clinical features differentiating it from the disorder in Europeans. A series of 100 Saudi diabetics and 100 Saudi controls has been acetylator phenotyped. The controls showed 33 rapid acetylators (R) and 67 slow acetylators (S), a result closely similar to that previously published for the Saudi population. Overall the diabetics showed 27 R and 73 S which is not significantly different from the controls. The type I diabetics, however, showed two R and 22 S which is not only significantly different from the controls and the type II diabetics, but also the reverse of the association found in European populations. PMID:4078878

  17. 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

  18. 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

  19. 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

  20. 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

  1. Proximal ADP-ribose Hydrolysis in Trypanosomatids is Catalyzed by a Macrodomain

    PubMed Central

    Haikarainen, Teemu; Lehtiö, Lari

    2016-01-01

    ADP-ribosylation is a ubiquitous protein modification utilized by both prokaryotes and eukaryotes for several cellular functions, such as DNA repair, proliferation, and cell signaling. Higher eukaryotes, such as humans, utilize various enzymes to reverse the modification and to regulate ADP-ribose dependent signaling. In contrast, some lower eukaryotes, including trypanosomatids, lack many of these enzymes and therefore have a much more simplified ADP-ribose metabolism. Here we identified and characterized ADP-ribose hydrolases from Trypanosoma brucei and Trypanosoma cruzi, which are homologous to human O-acetyl-ADP-ribose deacetylases MacroD1 and MacroD2. The enzymes are capable for hydrolysis of protein linked ADP-ribose and a product of sirtuin-mediated lysine deacetylation, O-acetyl-ADP-ribose. Crystal structures of the trypanosomatid macrodomains revealed a conserved catalytic site with distinct differences to human MacroD1 and MacroD2. PMID:27064071

  2. Proximal ADP-ribose Hydrolysis in Trypanosomatids is Catalyzed by a Macrodomain.

    PubMed

    Haikarainen, Teemu; Lehtiö, Lari

    2016-01-01

    ADP-ribosylation is a ubiquitous protein modification utilized by both prokaryotes and eukaryotes for several cellular functions, such as DNA repair, proliferation, and cell signaling. Higher eukaryotes, such as humans, utilize various enzymes to reverse the modification and to regulate ADP-ribose dependent signaling. In contrast, some lower eukaryotes, including trypanosomatids, lack many of these enzymes and therefore have a much more simplified ADP-ribose metabolism. Here we identified and characterized ADP-ribose hydrolases from Trypanosoma brucei and Trypanosoma cruzi, which are homologous to human O-acetyl-ADP-ribose deacetylases MacroD1 and MacroD2. The enzymes are capable for hydrolysis of protein linked ADP-ribose and a product of sirtuin-mediated lysine deacetylation, O-acetyl-ADP-ribose. Crystal structures of the trypanosomatid macrodomains revealed a conserved catalytic site with distinct differences to human MacroD1 and MacroD2. PMID:27064071

  3. 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...

  4. Purification and Characterization of Acetyl-Coenzyme A Carboxylase from Diclofop-Resistant and -Susceptible Lolium multiflorum.

    PubMed Central

    Evenson, K. J.; Gronwald, J. W.; Wyse, D. L.

    1994-01-01

    Acetyl-coenzyme A carboxylase (ACCase) was purified >100-fold (specific activity 3.5 units mg-1) from leaf tissue of diclofopresistant and -susceptible biotypes of Lolium multiflorum. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified fractions from both biotypes contained a single 206-kD biotinylated polypeptide. The molecular mass of the native enzyme from both biotypes was approximately 520 kD. In some cases the native dimer from both biotypes dissociated during gel filtration to form a subunit of approximately 224 kD. The inclusion of 5% (w/v) polyethylene glycol 3350 (PEG) in the elution buffer prevented this dissociation. Steady-state substrate kinetics were analyzed in both the presence and absence of 5% PEG. For ACCase from both biotypes, addition of PEG increased the velocity 22% and decreased the apparent Km values for acetyl-coenzyme A (acetyl-CoA), but increased the Km values for bicarbonate and ATP. In the presence of PEG, the Km values for bicarbonate and ATP were approximately 35% higher for the enzyme from the susceptible biotype compared with the resistant enzyme. In the absence of PEG, no differences in apparent Km values were observed for the enzymes from the two biotypes. Inhibition constants (Ki app) were determined for CoA, malonyl-CoA, and diclofop. CoA was an S-hyperbolic (slope replots)-I-hyperbolic (intercept replots) noncompetitive inhibitor with respect to acetyl-CoA, with Ki app values of 711 and 795 [mu]M for enzymes from the resistant and susceptible biotypes, respectively. Malonyl-CoA competitively inhibited both enzymes (versus acetyl-CoA) with Ki app values of 140 and 104 [mu]M for ACCase from resistant and susceptible biotypes, respectively. Diclofop was a linear noncompetitive inhibitor of ACCase from the susceptible biotype and a nonlinear, or S-hyperbolic-I-hyperbolic, noncompetitive inhibitor of ACCase from the resistant biotype. For ACCase from the susceptible biotype the slope (Kis) and intercept (Kii) inhibition constants for diclofop versus acetyl-CoA were 0.08 and 0.44 [mu]M, respectively. ACCase from the resistant biotype had a Ki app value of 6.5 [mu]M. At a subsaturating acetyl-CoA concentration of 50 [mu]M, the Hill coefficients for diclofop binding were 0.61 and 1.2 for ACCase from the resistant and susceptible biotypes, respectively. The Hill coefficients for diclofop binding and the inhibitor replots suggest that the resistant form of ACCase exhibits negative cooperativity in binding diclofop. However, the possibility that the nonlinear inhibition of ACCase activity by diclofop in the enzyme fraction isolated from the resistant biotype is due to the presence of both resistant and susceptible forms of ACCase cannot be excluded. PMID:12232234

  5. Expression and Characterization of α-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 α-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

  6. Cloning, Expression and Purification of an Acetoacetyl CoA Thiolase from Sunflower Cotyledon

    PubMed Central

    Dyer, James H.; Maina, Anthony; Gomez, Iris D.; Cadet, Melissa; Oeljeklaus, Silke; Schiedel, Anke C.

    2009-01-01

    Thiolase I and II coexist as part of the glyoxysomal β-oxidation system in sunflower (Helianthus annuus L.) cotyledons, the only system shown to have both forms. The importance of thiolases can be underscored not only by their ubiquity, but also by their involvement in a wide variety of processes in plants, animals and bacteria. Here we describe the cloning, expression and purification of acetoacetyl CoA thiolase (AACT) in enzymatically active form. Use of the extensive amount of sequence information from the databases facilitated the efficient generation of the gene-specific primers used in the RACE protocols. The recombinant AACT (1233 bp) shares 75% similarity with other plant AACTs. Comparison of specific activity of this recombinant AACT to a previously reported enzyme purified from primary sunflower cotyledon tissue was very similar (263 nkat/mg protein vs 220 nkat/mg protein, respectively). Combining the most pure fractions from the affinity column, the enzyme was purified 88-fold with a 55% yield of the enzymatically active, 47 kDa AACT. PMID:20011134

  7. Contribution of CoA Ligases to Benzenoid Biosynthesis in Petunia Flowers[W

    PubMed Central

    Klempien, Antje; Kaminaga, Yasuhisa; Qualley, Anthony; Nagegowda, Dinesh A.; Widhalm, Joshua R.; Orlova, Irina; Shasany, Ajit Kumar; Taguchi, Goro; Kish, Christine M.; Cooper, Bruce R.; D’Auria, John C.; Rhodes, David; Pichersky, Eran; Dudareva, Natalia

    2012-01-01

    Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the β-oxidative or nonoxidative pathways. The first step in the β-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the β-oxidative pathway. PMID:22649270

  8. 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

  9. 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.

  10. 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

  11. 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...

  12. 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.

  13. Quantitative Profiling of Lysine Acetylation Reveals Dynamic Crosstalk between Receptor Tyrosine Kinases and Lysine Acetylation

    PubMed Central

    Bryson, Bryan D.; White, Forest M.

    2015-01-01

    Lysine acetylation has been primarily investigated in the context of transcriptional regulation, but a role for acetylation in mediating other cellular responses has emerged. Multiple studies have described global lysine acetylation profiles for particular biological states, but none to date have investigated the temporal dynamics regulating cellular response to perturbation. Reasoning that lysine acetylation may be altered in response to growth factors, we implemented quantitative mass spectrometry-based proteomics to investigate the temporal dynamics of lysine acetylation in response to growth factor stimulation in cultured carcinoma cell lines. We found that lysine acetylation changed rapidly in response to activation of several different receptor tyrosine kinases by their respective ligands. To uncover the effects of lysine acetylation dynamics on tyrosine phosphorylation signaling networks, cells were treated with an HDAC inhibitor. This short-term pharmacological inhibition of histone deacetylase activity modulated signaling networks involving phosphorylated tyrosine and thereby altered the response to receptor tyrosine kinase activation. This result highlights the interconnectivity of lysine acetylation and tyrosine phosphorylation signaling networks and suggests that HDAC inhibition may influence cellular responses by affecting both types of post-translational modifications. PMID:25978619

  14. Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism.

    PubMed

    Rivière, Loïc; van Weelden, Susanne W H; Glass, Patricia; Vegh, Patricia; Coustou, Virginie; Biran, Marc; van Hellemond, Jaap J; Bringaud, Frédéric; Tielens, Aloysius G M; Boshart, Michael

    2004-10-29

    Acetyl:succinate CoA-transferase (ASCT) is an acetate-producing enzyme shared by hydrogenosomes, mitochondria of trypanosomatids, and anaerobically functioning mitochondria. The gene encoding ASCT in the protozoan parasite Trypanosoma brucei was identified as a new member of the CoA transferase family. Its assignment to ASCT activity was confirmed by 1) a quantitative correlation of protein expression and activity upon RNA interference-mediated repression, 2) the absence of activity in homozygous Deltaasct/Deltaasct knock out cells, 3) mitochondrial colocalization of protein and activity, 4) increased activity and acetate excretion upon transgenic overexpression, and 5) depletion of ASCT activity from lysates upon immunoprecipitation. Genetic ablation of ASCT produced a severe growth phenotype, increased glucose consumption, and excretion of beta-hydroxybutyrate and pyruvate, indicating accumulation of acetyl-CoA. Analysis of the excreted end products of (13)C-enriched and (14)C-labeled glucose metabolism showed that acetate excretion was only slightly reduced. Adaptation to ASCT deficiency, however, was an infrequent event at the population level, indicating the importance of this enzyme. These studies show that ASCT is indeed involved in acetate production, but is not essential, as apparently it is not the only enzyme that produces acetate in T. brucei. PMID:15326192

  15. High consistency enzymatic hydrolysis of hardwood substrates.

    PubMed

    Zhang, Xiao; Qin, Wenjuan; Paice, Michael G; Saddler, John N

    2009-12-01

    The feasibility of using a laboratory peg mixer to carry out high consistency enzymatic hydrolysis of lignocellulosic substrates was investigated. Two hardwood substrates, unbleached hardwood pulp (UBHW) and organosolv pretreated poplar (OPP), were used in this study. Hydrolysis of UBHW and OPP at 20% substrate consistency led to a high glucose concentration in the final hydrolysate. For example, a 48 h enzymatic hydrolysis of OPP resulted in a hydrolysate with 158 g/L of glucose. This is the highest glucose concentration ever obtained from enzymatic hydrolysis of lignocellulosic substrates. Fermentation of UBHW and OPP hydrolysates with high glucose content led to high ethanol concentrations, 50.4 and 63.1 g/L, respectively after fermentation. Our results demonstrate that using common pulping equipment to carry out high consistency hydrolysis can overcome the rheological problems and greatly increase the sugar and ethanol concentrations after the hydrolysis and fermentation. PMID:19643602

  16. Site of intestinal dipeptide hydrolysis.

    PubMed

    Wiseman, G

    1977-12-01

    1. Sacs of everted small intestine of the hamster have been used to study the site of final hydrolysis of twelve dipeptides. 2. The results suggest that L-alanyl-glycine, glycyl-glycine, L-valyl-L-valine, L-alanyl-L-valine, L-valyl-L-alanine and L-prolyl-glycine are hydrolysed beyond the locus of the active transport mechanism for D-glucose, perhaps even within the cell. These may be designated class 1 (deep) dipeptides. 3. In contrast, superficial (perhaps even surface) hydrolysis seems to occur with L-alanyl-L-alanine, L-leucly-L-leucine, glycyl-L-alanine, L-alanyl-L-leucine, L-leucyl-L-alanine and glycyl-L-proline. These may be designated class 2 (superficial) dipeptides. 4. All the dipeptides were able to partially inhibit D-glucose active transport, the findings supporting the view that more than one mechanism may exist for the active absorption of the sugar. PMID:604456

  17. 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 Nε-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 ε-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 Nε-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 Nε-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

  18. Acid hydrolysis of cellulose to yield glucose

    DOEpatents

    Tsao, George T.; Ladisch, Michael R.; Bose, Arindam

    1979-01-01

    A process to yield glucose from cellulose through acid hydrolysis. Cellulose is recovered from cellulosic materials, preferably by pretreating the cellulosic materials by dissolving the cellulosic materials in Cadoxen or a chelating metal caustic swelling solvent and then precipitating the cellulose therefrom. Hydrolysis is accomplished using an acid, preferably dilute sulfuric acid, and the glucose is yielded substantially without side products. Lignin may be removed either before or after hydrolysis.

  19. 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

  20. 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

  1. 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

  2. Histone Acetylation Regulates Intracellular pH

    PubMed Central

    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.

    2014-01-01

    SUMMARY 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 (pHi). As pHi 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 pHi. Conversely, global histone acetylation increases as pHi rises, such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pHi, particularly compromising pHi 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 pHi with important implications for mechanism of action and therapeutic use of HDAC inhibitors. PMID:23201122

  3. Acetylation Is Indispensable for p53 Activation

    PubMed Central

    Tang, Yi; Zhao, Wenhui; Chen, Yue; Zhao, Yingming; Gu, Wei

    2010-01-01

    SUMMARY The activation of the tumor suppressor p53 facilitates the cellular response to genotoxic stress; however, the p53 response can only be executed if its interaction with its inhibitor Mdm2 is abolished. There have been conflicting reports on the question of whether p53 posttranslational modifications, such as phosphorylation or acetylation, are essential or only play a subtle, fine-tuning role in the p53 response. Thus, it remains unclear whether p53 modification is absolutely required for its activation. We have now identified all major acetylation sites of p53. Although unacetylated p53 retains its ability to induce the p53-Mdm2 feedback loop, loss of acetylation completely abolishes p53-dependent growth arrest and apoptosis. Notably, acetylation of p53 abrogates Mdm2-mediated repression by blocking the recruitment of Mdm2 to p53-responsive promoters, which leads to p53 activation independent of its phosphorylation status. Our study identifies p53 acetylation as an indispensable event that destabilizes the p53-Mdm2 interaction and enables the p53-mediated stress response. PMID:18485870

  4. 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)

  5. 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

  6. 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

  7. 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

  8. 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

  9. Identification of 9α-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, 9α-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 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid to the CoA ester of 9α-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

  10. 4-coumarate: CoA ligase partitions metabolites for eugenol biosynthesis.

    PubMed

    Rastogi, Shubhra; Kumar, Ritesh; Chanotiya, Chandan S; Shanker, Karuna; Gupta, Madan M; Nagegowda, Dinesh A; Shasany, Ajit K

    2013-08-01

    Biosynthesis of eugenol shares its initial steps with that of lignin, involving conversion of hydroxycinnamic acids to their corresponding coenzyme A (CoA) esters by 4-coumarate:CoA ligases (4CLs). In this investigation, a 4CL (OS4CL) was identified from glandular trichome-rich tissue of Ocimum sanctum with high sequence similarity to an isoform (OB4CL_ctg4) from Ocimum basilicum. The levels of OS4CL and OB4CL_ctg4-like transcripts were highest in O. sanctum trichome, followed by leaf, stem and root. The eugenol content in leaf essential oil was positively correlated with the expression of OS4CL in the leaf at different developmental stages. Recombinant OS4CL showed the highest activity with p-coumaric acid, followed by ferulic, caffeic and trans-cinnamic acids. Transient RNA interference (RNAi) suppression of OS4CL in O. sanctum leaves caused a reduction in leaf eugenol content and trichome transcript level, with a considerable increase in endogenous p-coumaric, ferulic, trans-cinnamic and caffeic acids. A significant reduction in the expression levels was observed for OB4CL_ctg4-related transcripts in suppressed trichome compared with transcripts similar to the other four isoforms (OB4CL_ctg1, 2, 3 and 5). Sinapic acid and lignin content were also unaffected in RNAi suppressed leaf samples. Transient expression of OS4CL-green fluorescent protein fusion protein in Arabidopsis protoplasts was associated with the cytosol. These results indicate metabolite channeling of intermediates towards eugenol by a specific 4CL and is the first report demonstrating the involvement of 4CL in creation of virtual compartments through substrate utilization and committing metabolites for eugenol biosynthesis at an early stage of the pathway. PMID:23677922

  11. 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

  12. 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

  13. 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

  14. Isotope fractionation during peptide bond hydrolysis

    SciTech Connect

    Bada, J.L.; Schimmelmann, A. ); Schoeninger, M.J. )

    1989-12-01

    Isotopic fractionation of nitrogen and carbon is considered during peptide bond hydrolysis. Theoretical considerations suggest that hydrolysis will enrich residual, unhydrolyzed protein in {sup 15}N while {sup 13}C should be relatively unaffected. Preliminary experimental results support this conclusion, although further studies are required to quantify the magnitude of this effect as a function of protein degradation, in particular under natural environmental conditions.

  15. 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.

  16. 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)

  17. 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...

  18. 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 E1α, E1β, 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

  19. New improved synthesis of 2-deoxy-2-(/sup 18/F)fluoro-d-glucose from /sup 18/F-labeled acetyl hypofluorite

    SciTech Connect

    Shiue, C.Y.; Salvadori, P.A.; Wolf, A.P.; Fowler, J.S.; MacGregor, R.R.

    1982-10-01

    A new improved synthesis of 2-deoxy-2-(/sup 18/F)fluoro-D-glucose (2-/sup 18/FDG, 4) from /sup 18/F-labeled acetyl hypofluorite (1) is described. Reaction of 3,4,6-tri-O-acetyl-D-glucal (2) with 1, generated in situ from (/sup 18/F)F/sub 2/ and ammonium acetate in acetic acid solution at room temperature, gave 2-deoxy-2-(/sup 18/F)fluoro-1,3,4,6-tetra-O-acetyl-..cap alpha..-D-glucopyranose (3). Hydrolysis of compound 3 with 2N HCl gave 2-deoxy-2-(/sup 18/F)fluoro-D-glucose (4) in radiochemical yields of approx.20% in a synthesis time of 70 min from EOB.

  20. 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.

  1. A slow dance for microtubule acetylation.

    PubMed

    Kull, F Jon; Sloboda, Roger D

    2014-06-01

    Microtubules contribute to diverse cellular processes through balancing dynamic, short-lived and stable, long-lived populations. One way in which long-lived microtubules are marked is by posttranslational acetylation of α-tubulin by tubulin acetyltransferase (TAT). Szyk et al. now provide insight into TAT's mechanism of action and its unique time-stamping ability. PMID:24906144

  2. 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

  3. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    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.

  4. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... molecular distillation or by steam stripping; or (2) 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...

  5. 21 CFR 172.828 - Acetylated monoglycerides.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... molecular distillation or by steam stripping; or (2) 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...

  6. Property enhancement of optically transparent bionanofiber composites by acetylation

    NASA Astrophysics Data System (ADS)

    Nogi, Masaya; Abe, Kentaro; Handa, Keishin; Nakatsubo, Fumiaki; Ifuku, Shinsuke; Yano, Hiroyuki

    2006-12-01

    The authors studied acetylation of bacterial cellulose (BC) nanofibers to widen the applications of BC nanocomposites in optoelectronic devices. The slight acetylation of BC nanofibers significantly reduces the hygroscopicity of BC nanocomposites, while maintaining their high optical transparency and thermal stability. Furthermore, the degradation in optical transparency at elevated temperature (200°C) was significantly reduced by acetylation treatment. Therefore, the acetylation of bionanofibers has an extraordinary potential as treatment for property enhancement of bionanofiber composites.

  7. 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...

  8. 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...

  9. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false N-Acetyl-L-methionine. 172.372 Section 172.372... CONSUMPTION Special Dietary and Nutritional Additives § 172.372 N-Acetyl-L-methionine. The food additive N...) N-Acetyl-L-methionine (Chemical Abstracts Service Registry No. 65-82-7) is the derivative of...

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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

  16. Bromodomains shake the hegemony of pan-acetyl antibodies

    PubMed Central

    Champleboux, Morgane; Govin, Jérôme

    2015-01-01

    Acetylation signaling pathways are involved in numerous cellular processes and are used as therapeutic targets in several disease contexts. However, acetylated proteins only represent a minor fraction of the full proteome, and the identification and quantification of acetylated sites remain a technological challenge. Currently, pan-acetyl antibodies are used to increase the abundance of acetylated peptides through affinity purification before MS analysis. These antibodies are powerful reagents, but they are hampered by a lack of specificity, affinity, and batch-to-batch reproducibility. In this issue, Bryson et al. (Proteomics 2015 15, 1470–1475) present an interesting alternative to these antibodies, in the form of bromodomains. These domains specifically recognize acetylated lysines, and were successfully used in this study to enrich for acetylated peptides before MS analysis. Future development of this pioneering approach could help overcome this limiting step in the characterization of acetylproteomes. PMID:25825234

  17. Bromodomains shake the hegemony of pan-acetyl antibodies.

    PubMed

    Champleboux, Morgane; Govin, Jérôme

    2015-05-01

    Acetylation signaling pathways are involved in numerous cellular processes and are used as therapeutic targets in several disease contexts. However, acetylated proteins only represent a minor fraction of the full proteome, and the identification and quantification of acetylated sites remain a technological challenge. Currently, pan-acetyl antibodies are used to increase the abundance of acetylated peptides through affinity purification before MS analysis. These antibodies are powerful reagents, but they are hampered by a lack of specificity, affinity, and batch-to-batch reproducibility. In this issue, Bryson et al. (Proteomics 2015 15, 1470-1475) present an interesting alternative to these antibodies, in the form of bromodomains. These domains specifically recognize acetylated lysines, and were successfully used in this study to enrich for acetylated peptides before MS analysis. Future development of this pioneering approach could help overcome this limiting step in the characterization of acetylproteomes. PMID:25825234

  18. Dynamic Protein Acetylation in Plant–Pathogen Interactions

    PubMed Central

    Song, Gaoyuan; Walley, Justin W.

    2016-01-01

    Pathogen infection triggers complex molecular perturbations within host cells that results in either resistance or susceptibility. Protein acetylation is an emerging biochemical modification that appears to play central roles during host–pathogen interactions. To date, research in this area has focused on two main themes linking protein acetylation to plant immune signaling. Firstly, it has been established that proper gene expression during defense responses requires modulation of histone acetylation within target gene promoter regions. Second, some pathogens can deliver effector molecules that encode acetyltransferases directly within the host cell to modify acetylation of specific host proteins. Collectively these findings suggest that the acetylation level for a range of host proteins may be modulated to alter the outcome of pathogen infection. This review will focus on summarizing our current understanding of the roles of protein acetylation in plant defense and highlight the utility of proteomics approaches to uncover the complete repertoire of acetylation changes triggered by pathogen infection. PMID:27066055

  19. 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

  20. 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.

  1. Hydrolysis and alcoholysis of diorganylphosphinous iodides

    SciTech Connect

    Gomelya, N.D.; Feschenko, N.G.

    1988-01-20

    It was shown that unlike diorganylphosphinous chlorides, diorganylphosphinous iodides in hydrolysis and alcoholysis reactions react with the hydrogen iodide formed. There then occurs the reduction and oxidation of the diorganylphosphinous iodides, for which those reactions had a general character.

  2. Effect of surfactants on cellulose hydrolysis

    SciTech Connect

    Helle, S.S.; Duff, S.J.B. . Pulp and Paper Centre and Dept. of Chemical Engineering); Cooper, D.G. . Chemical Engineering Dept.)

    1993-08-20

    The effect of surfactants on the heterogeneous enzymatic hydrolysis of Sigmacell 100 cellulose and of steam-exploded wood was studied. Certain biosurfactants (sophorolipid, rhamnolipid, bacitracin) and Tween 80 increased the rate of hydrolysis of Sigmacell 100, as measured by the amount of reducing sugar produced, by as much as seven times. The hydrolysis of steam-exploded wood was increased by 67% in the presence of sophorolipid. At the same time, sophorolipid was found to decrease the amount of enzyme absorbed onto the cellulose at equilibrium. Sophorolipid had the greatest effect on cellulose hydrolysis when it was present from the beginning of the experiment and when the enzyme/cellulose ratio was low.

  3. 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

  4. 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.

  5. 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

  6. Measurement of bile acid CoA esters by high-performance liquid chromatography-electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS).

    PubMed

    Gan-Schreier, H; Okun, J G; Kohlmueller, D; Langhans, C-D; Peters, V; Ten Brink, H J; Verhoeven, N M; Jakobs, C; Voelkl, A; Hoffmann, G F

    2005-07-01

    The novel and rapid assay presented here combines high-performance liquid chromatography and electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) to directly measure and quantify the CoA esters of 3alpha,7alpha,12alpha-trihydroxy- and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic acid (THCA and DHCA). The latter are converted inside peroxisomes to the primary bile acids, cholic and chenodeoxycholic acids, respectively. Prior to MS/MS, esters were separated by reversed-phase HPLC on a C(18) column using an isocratic mobile phase (acetonitrile/water/2-propanol) and subsequently detected by multiple reaction monitoring. For quantification, the CoA ester of deuterium-labelled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid (d(4)-CA) was used as internal standard. To complete an assay took less than 8 min. To verify the validity of the assay, the effect of peroxisomal proteins on the efficacy of extraction of the CoA esters was tested. To this end, variable amounts of the CoA esters were spiked with a fixed amount of either intact peroxisomes or peroxisomal matrix proteins and then extracted using a solid-phase extraction system. The CoA esters could be reproducibly recovered in the range of 0.1-4 micromol l(-1) (linear correlation coefficient R(2) > 0.99), with a detection limit of 0.1 micromol l(-1). In summary, electrospray ionization tandem mass spectrometry combined with HPLC as described here proved to be a rapid and versatile technique for the determination of bile acid CoA esters in a mixture with peroxisomal proteins. This suggests this technique to become a valuable tool in studies dealing with the multi-step biosynthesis of bile acids and its disturbances in disorders like the Zellweger syndrome. PMID:15892178

  7. The neurobiology of acetyl-L-carnitine.

    PubMed

    Traina, Giovanna

    2016-01-01

    A large body of evidence points to the positive effects of dietary supplementation of acetyl-L-carnitine (ALC). Its use has shown health benefits in neuroinflammation, which is a common denominator in a host of neurodegenerative diseases. ALC is the principal acetyl ester of L-Carnitine (LC), and it plays an essential role in intermediary metabolism, acting as a donor of acetyl groups and facilitating the transfer of fatty acids from cytosol to mitochondria during beta-oxidation. Dietary supplementation of ALC exerts neuroprotective, neurotrophic, antidepressive and analgesic effects in painful neuropathies. ALC also has antioxidant and anti-apoptotic activity. Moreover, ALC exhibits positive effects on mitochondrial metabolism, and shows promise in the treatment of aging and neurodegenerative pathologies by slowing the progression of mental deterioration. In addition, ALC plays neuromodulatory effects on both synaptic morphology and synaptic transmission. These effects are likely due to affects of ALC through modulation of gene expression on several targets in the central nervous system. Here, we review the current state of knowledge on effects of ALC in the nervous system. PMID:27100509

  8. [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

  9. Poly-acetylated chromatin signatures are preferred epitopes for site-specific histone H4 acetyl antibodies

    PubMed Central

    Rothbart, Scott B.; Lin, Shu; Britton, Laura-Mae; Krajewski, Krzysztof; Keogh, Michael-C; Garcia, Benjamin A.; Strahl, Brian D.

    2012-01-01

    Antibodies specific for histone post-translational modifications (PTMs) have been central to our understanding of chromatin biology. Here, we describe an unexpected and novel property of histone H4 site-specific acetyl antibodies in that they prefer poly-acetylated histone substrates. By all current criteria, these antibodies have passed specificity standards. However, we find these site-specific histone antibodies preferentially recognize chromatin signatures containing two or more adjacent acetylated lysines. Significantly, we find that the poly-acetylated epitopes these antibodies prefer are evolutionarily conserved and are present at levels that compete for these antibodies over the intended individual acetylation sites. This alarming property of acetyl-specific antibodies has far-reaching implications for data interpretation and may present a challenge for the future study of acetylated histone and non-histone proteins. PMID:22761995

  10. Poly-acetylated chromatin signatures are preferred epitopes for site-specific histone H4 acetyl antibodies.

    PubMed

    Rothbart, Scott B; Lin, Shu; Britton, Laura-Mae; Krajewski, Krzysztof; Keogh, Michael-C; Garcia, Benjamin A; Strahl, Brian D

    2012-01-01

    Antibodies specific for histone post-translational modifications (PTMs) have been central to our understanding of chromatin biology. Here, we describe an unexpected and novel property of histone H4 site-specific acetyl antibodies in that they prefer poly-acetylated histone substrates. By all current criteria, these antibodies have passed specificity standards. However, we find these site-specific histone antibodies preferentially recognize chromatin signatures containing two or more adjacent acetylated lysines. Significantly, we find that the poly-acetylated epitopes these antibodies prefer are evolutionarily conserved and are present at levels that compete for these antibodies over the intended individual acetylation sites. This alarming property of acetyl-specific antibodies has far-reaching implications for data interpretation and may present a challenge for the future study of acetylated histone and non-histone proteins. PMID:22761995

  11. New insights into structure-function relationships of oxalyl CoA decarboxylase from Escherichia coli.

    PubMed

    Werther, Tobias; Zimmer, Agnes; Wille, Georg; Golbik, Ralph; Weiss, Manfred S; König, Stephan

    2010-06-01

    The gene yfdU from Escherichia coli encodes a putative oxalyl coenzyme A decarboxylase, a thiamine diphosphate-dependent enzyme that is potentially involved in the degradation of oxalate. The enzyme has been purified to homogeneity. The kinetic constants for conversion of the substrate oxalyl coenzyme A by the enzyme in the absence and presence of the inhibitor coenzyme A, as well as in the absence and presence of the activator adenosine 5'-diphosphate, were determined using a novel continuous optical assay. The effects of these ligands on the solution and crystal structure of the enzyme were studied using small-angle X-ray scattering and X-ray crystal diffraction. Analyses of the obtained crystal structures of the enzyme in complex with the cofactor thiamine diphosphate, the activator adenosine 5'-diphosphate and the inhibitor acetyl coenzyme A, as well as the corresponding solution scattering patterns, allow comparison of the oligomer structures of the enzyme complexes under various experimental conditions, and provide insights into the architecture of substrate and effector binding sites. PMID:20553497

  12. 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.

  13. 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

  14. 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

  15. 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

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

    PubMed Central

    Délye, Christophe; Zhang, Xiao-Qi; Michel, Séverine; Matéjicek, 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

  17. 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.

  18. Acetylation and characterization of spruce (Picea abies) galactoglucomannans.

    PubMed

    Xu, Chunlin; Leppänen, Ann-Sofie; Eklund, Patrik; Holmlund, Peter; Sjöholm, Rainer; Sundberg, Kenneth; Willför, Stefan

    2010-04-19

    Acetylated galactoglucomannans (GGMs) are the main hemicellulose type in most softwood species and can be utilized as, for example, bioactive polymers, hydrocolloids, papermaking chemicals, or coating polymers. Acetylation of spruce GGM using acetic anhydride with pyridine as catalyst under different conditions was conducted to obtain different degrees of acetylation on a laboratory scale, whereas, as a classic method, it can be potentially transferred to the industrial scale. The effects of the amount of catalyst and acetic anhydride, reaction time, temperature and pretreatment by acetic acid were investigated. A fully acetylated product was obtained by refluxing GGM for two hours. The structures of the acetylated GGMs were determined by SEC-MALLS/RI, (1)H and (13)C NMR and FTIR spectroscopy. NMR studies also indicated migration of acetyl groups from O-2 or O-3 to O-6 after a heating treatment in a water bath. The thermal stability of the products was investigated by DSC-TGA. PMID:20144827

  19. 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

  20. 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.

  1. 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

  2. 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

  3. Survey of the human acetylator polymorphism in spontaneous disorders.

    PubMed Central

    Evans, D A

    1984-01-01

    There is ample evidence that the human acetylator phenotypes are associated with drug induced phenomena. It is principally the slow acetylators who exhibit toxic adverse effects because of their relative inability to detoxify the original drug compounds. In rare instances, however, it is the rapid acetylators who are at a disadvantage. In the matter of association of spontaneous disease with either acetylator phenotype, there are two groups of disorders to consider. First, disorders in which carcinogenic amines are known to be an aetiological factor. This is because these amines are substrates for the polymorphic N-acetyltransferase activity and hence there is a possible rational basis for searching for an association. Secondly, other disorders where searches for associations are based more on hunches. In the first group there is a definite statistical association between cancer of the bladder and the slow acetylator phenotype. In prevalence studies the slow phenotype is 39% more associated with bladder cancer than is the rapid phenotype. On the basis of the evidence now available it is not possible to say whether this association is because slow acetylators develop the disease more frequently or whether they survive longer. In the second group the relevant studies show (1) a greatly increased prevalence of slow acetylators in Gilbert's disease; (2) a confirmed association between the rapid acetylator phenotype and diabetes; (3) a possible association between the rapid acetylator phenotype and breast cancer; (4) a possible association between the slow acetylator phenotype and leprosy in Chinese patients; (5) an earlier age of onset of thyrotoxicosis (Graves' disease) in slow acetylators than in rapid acetylators; (6) no evidence of an association between either phenotype and spontaneous systemic lupus erythematosus. PMID:6387123

  4. 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

  5. Hydrolysis of epithelial junctional proteins by Porphyromonas gingivalis gingipains.

    PubMed

    Katz, Jannet; Yang, Qiu-Bo; Zhang, Ping; Potempa, Jan; Travis, James; Michalek, Suzanne M; Balkovetz, Daniel F

    2002-05-01

    Porphyromonas gingivalis has been implicated as an etiologic agent of adult periodontitis. We have previously shown that P. gingivalis can degrade the epithelial cell-cell junction complexes, thus suggesting that this bacterium can invade the underlying connective tissues via a paracellular pathway. However, the precise mechanism(s) involved in this process has not been elucidated. The purpose of this study was to determine if the arginine- and lysine-specific gingipains of P. gingivalis (i.e., HRgpA and RgpB, and Kgp, respectively) were responsible for the degradation of E-cadherin, the cell-cell adhesion protein in the adherens junctions. In addition, we compared the degradative abilities of the whole gingipains HRgpA and Kgp to those of their catalytic domains alone. In these studies, immunoprecipitated E-cadherin as well as monolayers of polarized Madin-Darby canine kidney (MDCK) epithelial cell cultures were incubated with the gingipains and hydrolysis of E-cadherin was assessed by Western blot analysis. Incubation of P. gingivalis cells with immunoprecipitated E-cadherin resulted in degradation, whereas prior exposure of P. gingivalis cells to leupeptin and especially acetyl-Leu-Val-Lys-aldehyde (which are arginine- and lysine-specific inhibitors, respectively) reduced this activity. Furthermore, incubation of E-cadherin immunoprecipitates with the different gingipains resulted in an effective and similar hydrolysis of the protein. However, when monolayers of MDCK cells were exposed to the gingipains, Kgp was most effective in hydrolyzing the E-cadherin molecules in the adherens junction. Kgp was more effective than its catalytic domain in degrading E-cadherin at 500 nM but not at a lower concentration (250 nM). These results suggest that the hemagglutinin domain of Kgp plays a role in degradation and that there is a critical threshold concentration for this activity. Taken together, these results provide evidence that the gingipains, especially Kgp, are involved in the degradation of the adherens junction of epithelial cells, which may be important in the invasion of periodontal connective tissue by P. gingivalis. PMID:11953390

  6. Determination of Acetylation of the Gli Transcription Factors.

    PubMed

    Coni, Sonia; Di Magno, Laura; Canettieri, Gianluca

    2015-01-01

    The Gli transcription factors (Gli1, Gli2, and Gli3) are the final effectors of the Hedgehog (Hh) signaling and play a key role in development and cancer. The activity of the Gli proteins is finely regulated by covalent modifications, such as phosphorylation, ubiquitination, and acetylation. Both Gli1 and Gli2 are acetylated at a conserved lysine, and this modification causes the inhibition of their transcriptional activity. Thus, the acetylation status of these proteins represents a useful marker to monitor Hh activation in pathophysiological conditions. Herein we describe the techniques utilized to detect in vitro and intracellular acetylation of the Gli transcription factors. PMID:26179046

  7. 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

  8. 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)-κB 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-κB (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-κB 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

  9. 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

  10. 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

  11. 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...

  12. 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

  13. Camptothecin-catalyzed phospholipid hydrolysis in liposomes.

    PubMed

    Saetern, Ann Mari; Skar, Merete; Braaten, Asmund; Brandl, Martin

    2005-01-01

    Hydrolysis of phospholipid (PL) within camptothecin (CPT)-containing liposomes was studied systematically, after elevated lyso-phosphatidylcholine (LPC)-concentrations in pH 5, CPT-containing liposomes (22.1+/-0.9 mol%) relative to control-liposomes (7.3+/-0.5 mol%) occasionally had been observed after four months storage in fridge. Liposomes were prepared by dispersing freeze-dried PL/CPT mixtures in 25 mM phosphate buffered saline (PBS) of varying pH (5.0-7.8) and CPT concentrations (0, 3 and 6 mM). PL-hydrolysis was monitored by HPTLC, quantifying LPC. In an accelerated stability study (60 degrees C), a catalytic effect of CPT on PL-hydrolysis was observed after 40 h, but not up to 30 h of incubation. The pH profile of the hydrolysis indicated a stability optimum at pH 6.0 for the liposomes independent of CPT. The equilibrium point between the more active lactone- and the carboxylate-form of CPT was found to be pH 6.8. As a compromise, pH 6.0 was chosen, assuring >85% CPT to be present in the lactone form. At this pH, both control- and CPT-liposomes showed only minor hydrolysis after autoclaving (121 degrees C, 15 min). Storage at room temperature and in fridge (2 months), as well as accelerated ageing (70 degrees C, 25 h), gave a significant elevation of LPC content in CPT-liposomes relative to control-liposomes. This study demonstrates a catalytic effect of CPT on PL-hydrolysis, the onset of which seems to require a certain threshold level of hydrolytic degradation. PMID:15607259

  14. Importance of product inhibition in the kinetics of the acylase hydrolysis reaction by differential stopped flow microcalorimetry.

    PubMed

    Stödeman, Magnus; Schwarz, Frederick P

    2002-09-15

    The hydrolysis of N-acetyl-L-methionine, N-acetylglycine, N-acetyl-L-phenylalanine, and N-acetyl-L-alanine at 298.35K by porcine kidney acylase I (EC 3.5.1.14) was monitored by the heat released upon mixing of the substrate and enzyme in a differential stopped flow microcalorimeter. Values for the Michaelis constant (K(m)) and the catalytic constant (k(cat)) were determined from the progress of the reaction curve employing the integrated form of the Michaelis-Menten equation for each reaction mixture. When neglecting acetate product inhibition of the acylase, values for k(cat) were up to a factor of 2.3 larger than those values determined from reciprocal initial velocity-initial substrate concentration plots for at least four different reaction mixtures. In addition, values for K(m) were observed to increase linearly with an increase in the initial substrate concentration. When an acetate product inhibition constant of 600+/-31M(-1), determined by isothermal titration calorimetry, was used in the progress curve analysis, values for K(m) and k(cat) were in closer agreement with their values determined from the reciprocal initial velocity versus initial substrate concentration plots. The reaction enthalpies, Delta(r)H(cal), which were determined from the integrated heat pulse per amount of substrate in the reaction mixture, ranged from -4.69+/-0.09kJmol(-1) for N-acetyl-L-phenylalanine to -1.87+/-0.23kJmol(-1) for N-acetyl-L-methionine. PMID:12419341

  15. N-acetylaspartate catabolism determines cytosolic acetyl-CoA levels and histone acetylation in brown adipocytes.

    PubMed

    Prokesch, A; Pelzmann, H J; Pessentheiner, A R; Huber, K; Madreiter-Sokolowski, C T; Drougard, A; Schittmayer, M; Kolb, D; Magnes, C; Trausinger, G; Graier, W F; Birner-Gruenberger, R; Pospisilik, J A; Bogner-Strauss, J G

    2016-01-01

    Histone acetylation depends on the abundance of nucleo-cytoplasmic acetyl-CoA. Here, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. N-acetylaspartate (NAA) is a highly abundant brain metabolite catabolized by aspartoacylase yielding aspartate and acetate. The latter can be further used for acetyl-CoA production. Prior to this work, the presence of NAA has not been described in adipocytes. Here, we show that accumulation of NAA decreases the brown adipocyte phenotype. We increased intracellular NAA concentrations in brown adipocytes via media supplementation or knock-down of aspartoacylase and measured reduced lipolysis, thermogenic gene expression, and oxygen consumption. Combinations of approaches to increase intracellular NAA levels showed additive effects on lipolysis and gene repression, nearly abolishing the expression of Ucp1, Cidea, Prdm16, and Ppara. Transcriptome analyses of aspartoacylase knock-down cells indicate deficiencies in acetyl-CoA and lipid metabolism. Concordantly, cytoplasmic acetyl-CoA levels and global histone H3 acetylation were decreased. Further, activating histone marks (H3K27ac and H3K9ac) in promoters/enhancers of brown marker genes showed reduced acetylation status. Taken together, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. Thereby, we mechanistically connect the NAA pathway to the epigenomic regulation of gene expression, modulating the phenotype of brown adipocytes. PMID:27045997

  16. N-acetylaspartate catabolism determines cytosolic acetyl-CoA levels and histone acetylation in brown adipocytes

    PubMed Central

    Prokesch, A.; Pelzmann, H. J.; Pessentheiner, A. R.; Huber, K.; Madreiter-Sokolowski, C. T.; Drougard, A.; Schittmayer, M.; Kolb, D.; Magnes, C.; Trausinger, G.; Graier, W. F.; Birner-Gruenberger, R.; Pospisilik, J. A.; Bogner-Strauss, J. G.

    2016-01-01

    Histone acetylation depends on the abundance of nucleo-cytoplasmic acetyl-CoA. Here, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. N-acetylaspartate (NAA) is a highly abundant brain metabolite catabolized by aspartoacylase yielding aspartate and acetate. The latter can be further used for acetyl-CoA production. Prior to this work, the presence of NAA has not been described in adipocytes. Here, we show that accumulation of NAA decreases the brown adipocyte phenotype. We increased intracellular NAA concentrations in brown adipocytes via media supplementation or knock-down of aspartoacylase and measured reduced lipolysis, thermogenic gene expression, and oxygen consumption. Combinations of approaches to increase intracellular NAA levels showed additive effects on lipolysis and gene repression, nearly abolishing the expression of Ucp1, Cidea, Prdm16, and Ppara. Transcriptome analyses of aspartoacylase knock-down cells indicate deficiencies in acetyl-CoA and lipid metabolism. Concordantly, cytoplasmic acetyl-CoA levels and global histone H3 acetylation were decreased. Further, activating histone marks (H3K27ac and H3K9ac) in promoters/enhancers of brown marker genes showed reduced acetylation status. Taken together, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. Thereby, we mechanistically connect the NAA pathway to the epigenomic regulation of gene expression, modulating the phenotype of brown adipocytes. PMID:27045997

  17. Temperature dependence of the kinetics of the acylase hydrolysis reaction by differential stopped flow microcalorimetry.

    PubMed

    Stödeman, Magnus; Schwarz, Frederick P

    2003-10-01

    The rates of the hydrolysis of N-acetylglycine, N-acetyl-L-methionine, and N-acetyl-L-phenylalanine by porcine acylase I in 0.1M phosphate buffer, which is inhibited by acetate product formation, were monitored calorimetrically at temperatures between 15.2 and 45.3 degrees C by a differential stopped-flow microcalorimeter. Since the acylase is thermally stable and the pH of the phosphate buffer changes by less than 0.01 pH units over this temperature range, any temperature effect on the hydrolysis reaction can be attributed to the effect of temperature on the kinetics of the reaction. Analysis of the integrated heat released by the reaction as a function of time with regard to the integrated Michaelis-Menten equation yields apparent values for k(cat) and apparent values for Km that depend on the product inhibition constant. The apparent values for Km also exhibited a dependence on the initial substrate concentration because of the acetate product inhibition at each temperature. By assuming that the inhibition constant is independent of temperature over this temperature range and from extrapolation of Km to its value at zero substrate concentration, intrinsic values of Km and k(cat) were determined over the temperature range from 15.2 to 45.3 degrees C. The intrinsic values of Km exhibited very little variation over this temperature range while the intrinsic values of k(cat) exhibited an increase over the same temperature range. The heats of reaction also exhibited an increase with temperature over this range with an average heat capacity change of -94 Jmol(-1)K(-1) for the three substrates. PMID:12963049

  18. Enhanced enzymatic hydrolysis of cellulose in microgels.

    PubMed

    Chang, Aiping; Wu, Qingshi; Xu, Wenting; Xie, Jianda; Wu, Weitai

    2015-07-01

    A cellulose-based microgel, where an individual microgel contains approximately one cellulose chain on average, is synthesized via free radical polymerization of a difunctional small-molecule N,N'-methylenebisacrylamide in cellulose solution. This microgelation leads to a low-ordered cellulose, favoring enzymatic hydrolysis of cellulose to generate glucose. PMID:26035077

  19. 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...

  20. 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.

  1. 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...

  2. 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 ...

  3. Structure of a Ternary Naa50p (NAT5/SAN) N-terminal Acetyltransferase Complex Reveals the Molecular Basis for Substrate-specific Acetylation*

    PubMed Central

    Liszczak, Glen; Arnesen, Thomas; Marmorstein, Ronen

    2011-01-01

    The co-translational modification of N-terminal acetylation is ubiquitous among eukaryotes and has been reported to have a wide range of biological effects. The human N-terminal acetyltransferase (NAT) Naa50p (NAT5/SAN) acetylates the α-amino group of proteins containing an N-terminal methionine residue and is essential for proper sister chromatid cohesion and chromosome condensation. The elevated activity of NATs has also been correlated with cancer, making these enzymes attractive therapeutic targets. We report the x-ray crystal structure of Naa50p bound to a native substrate peptide fragment and CoA. We found that the peptide backbone of the substrate is anchored to the protein through a series of backbone hydrogen bonds with the first methionine residue specified through multiple van der Waals contacts, together creating an α-amino methionine-specific pocket. We also employed structure-based mutagenesis; the results support the importance of the α-amino methionine-specific pocket of Naa50p and are consistent with the proposal that conserved histidine and tyrosine residues play important catalytic roles. Superposition of the ternary Naa50p complex with the peptide-bound Gcn5 histone acetyltransferase revealed that the two enzymes share a Gcn5-related N-acetyltransferase fold but differ in their respective substrate-binding grooves such that Naa50p can accommodate only an α-amino substrate and not a side chain lysine substrate that is acetylated by lysine acetyltransferase enzymes such as Gcn5. The structure of the ternary Naa50p complex also provides the first molecular scaffold for the design of NAT-specific small molecule inhibitors with possible therapeutic applications. PMID:21900231

  4. Histone H4 lysine 16 acetylation breaks the genome's silence

    PubMed Central

    Shia, Wei-Jong; Pattenden, Samantha G; Workman, Jerry L

    2006-01-01

    Acetylation at histone H4 lysine 16 is involved in many cellular processes in organisms as diverse as yeast and humans. A recent biochemical study pinpoints this particular acetylation mark as a switch for changing chromatin from a repressive to a transcriptionally active state. PMID:16689998

  5. Emerging Functions for N-Terminal Protein Acetylation in Plants.

    PubMed

    Gibbs, Daniel J

    2015-10-01

    N-terminal (Nt-) acetylation is a widespread but poorly understood co-translational protein modification. Two reports now shed light onto the proteome-wide dynamics and protein-specific consequences of Nt-acetylation in relation to plant development, stress-response, and protein stability, identifying this modification as a key regulator of diverse aspects of plant growth and behaviour. PMID:26319188

  6. 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

  7. HIV-1 tat transcriptional activity is regulated by acetylation.

    PubMed

    Kiernan, R E; Vanhulle, C; Schiltz, L; Adam, E; Xiao, H; Maudoux, F; Calomme, C; Burny, A; Nakatani, Y; Jeang, K T; Benkirane, M; Van Lint, C

    1999-11-01

    The human immunodeficiency virus (HIV) trans- activator protein, Tat, stimulates transcription from the viral long-terminal repeats (LTR) through an RNA hairpin element, trans-activation responsive region (TAR). We and others have shown that trans-activator protein (Tat)-associated histone acetyltransferases (TAHs), p300 and p300/CBP-associating factor (PCAF), assist functionally in the activation of chromosomally integrated HIV-1 LTR. Here, we show that p300 and PCAF also directly acetylate Tat. We defined two sites of acetylation located in different functional domains of Tat. p300 acetylated Lys50 in the TAR RNA binding domain, while PCAF acetylated Lys28 in the activation domain of Tat. In support of a functional role for acetylation in vivo, histone deacetylase inhibitor (trichostatin A) synergized with Tat in transcriptional activation of the HIV-1 LTR. Synergism was TAR-dependent and required the intact presence of both Lys28 and Lys50. Mechanistically, acetylation at Lys28 by PCAF enhanced Tat binding to the Tat-associated kinase, CDK9/P-TEFb, while acetylation by p300 at Lys50 of Tat promoted the dissociation of Tat from TAR RNA that occurs during early transcription elongation. These data suggest that acetylation of Tat regulates two discrete and functionally critical steps in transcription, binding to an RNAP II CTD-kinase and release of Tat from TAR RNA. PMID:10545121

  8. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... Special Dietary and Nutritional Additives § 172.372 N-Acetyl-L-methionine. The food additive N-acetyl-L... safe use of the additive, the label and labeling of the additive and any premix thereof shall bear, in... use solely under medical supervision to meet nutritional requirements in specific medical...

  9. 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

  10. 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

  11. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.372 N-Acetyl-L-methionine. The food additive N-acetyl-L-methionine may be safely added to...) When the food additive is added as a nutrient to special dietary foods that are intended for use...

  12. 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

  13. 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

  14. 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-β-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: β-D-mannopyranose; G: β-D-glucopyranose; a: O-acetyl group. PMID:25498655

  15. 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.

  16. 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

  17. 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

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

    PubMed Central

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

    2013-01-01

    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

  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. HYDROLYSIS RATE CONSTANTS FOR ENHANCING PROPERTY-REACTIVITY RELATIONSHIPS

    EPA Science Inventory

    Rate constants for hydrolysis in water of ten classes of organic compounds are examined with the objective of establishing new, or expanding existing, property reactivity correlations. These relationships then can be used to predict the environmental hydrolysis of chemicals that ...

  1. 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.

  2. 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

  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. Technical bases for precipitate hydrolysis process operating parameters

    SciTech Connect

    Bannochie, C.J.

    1992-10-05

    This report provides the experimental data and rationale in support of the operating parameters for precipitate hydrolysis specified in WSRC-RP-92737. The report is divided into two sections, the first dealing with lab-scale precipitate hydrolysis experimentation while the second part addresses large-scale runs conducted to demonstrate the revised operating parameters in the Precipitate Hydrolysis Experimental Facility (PHEF).

  6. 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

  7. 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

  8. 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

  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. 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.

  11. 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.

  12. 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).

  13. Impact of single-dose nandrolone decanoate on gonadotropins, blood lipids and HMG CoA reductase in healthy men.

    PubMed

    Gårevik, N; Börjesson, A; Choong, E; Ekström, L; Lehtihet, M

    2016-06-01

    The aim was to study the effect and time profile of a single dose of nandrolone decanoate (ND) on gonadotropins, blood lipids and HMG CoA reductase [3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR)] in healthy men. Eleven healthy male participants aged 29-46 years were given a single dose of 150 mg ND as an intramuscular dose of Deca Durabol®, Organon. Blood samples for sex hormones, lipids and HMGCR mRNA analysis were collected prior to ND administration day 0, 4 and 14. A significant suppression of luteinising hormone (LH) and follicle-stimulating hormone (FSH) was seen after 4 days. Total testosterone and bioavailable testosterone level decreased significantly throughout the observed study period. A small but significant decrease in sexual hormone-binding globulin (SHBG) was seen after 4 days but not after 14 days. Total serum (S)-cholesterol and plasma (P)-apolipoprotein B (ApoB) increased significantly after 14 days. In 80% of the individuals, the HMGCR mRNA level was increased 4 days after the ND administration. Our results show that a single dose of 150 mg ND increases (1) HMGCR mRNA expression, (2) total S-cholesterol and (3) P-ApoB level. The long-term consequences on cardiovascular risk that may appear in users remain to be elucidated. PMID:26370185

  14. Long-Chain Acyl CoA Synthetase 4A regulates Smad activity and dorsoventral patterning in the zebrafish embryo

    PubMed Central

    Miyares, Rosa Linda; Stein, Cornelia; Renisch, Björn; Anderson, Jennifer Lynn; Hammerschmidt, Matthias; Farber, Steven Arthur

    2013-01-01

    Summary Long-chain polyunsaturated fatty acids (LC-PUFA) and their metabolites are critical players in cell biology and embryonic development. Here we show that long-chain acyl CoA synthetase 4a (Acsl4a), an LC-PUFA activating enzyme, is essential for proper patterning of the zebrafish dorsoventral axis. Loss of Acsl4a results in dorsalized embryos due to attenuated Bmp signaling. We demonstrate that Acsl4a modulates the activity of Smad transcription factors, the downstream mediators of Bmp signaling. Acsl4a promotes the inhibition of p38 MAPK and the Akt-mediated inhibition of glycogen synthase kinase 3 (GSK3), critical inhibitors of Smad activity. Consequently, introduction of a constitutively active Akt can rescue the dorsalized phenotype of Acsl4a deficient embryos. Our results reveal a critical role for Acsl4a in modulating Bmp-Smad activity and provide a potential avenue for LC-PUFAs to influence a variety of developmental processes. PMID:24332754

  15. The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.

    PubMed

    Schumacher, Marc M; Elsabrouty, Rania; Seemann, Joachim; Jo, Youngah; DeBose-Boyd, Russell A

    2015-01-01

    Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD. PMID:25742604

  16. 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.

  17. 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

  18. 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

  19. 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 β- 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 β-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

  20. 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

  1. Protein N-terminal acetylation: NAT 2007–2008 Symposia

    PubMed Central

    Arnesen, Thomas

    2009-01-01

    Protein N-terminal acetylation is a very common modification, but has during the past decades received relatively little attention. In order to put this neglected field back on the scientific map, we have in May 2007 and September 2008 arranged two international NAT symposia in Bergen, Norway. This supplement contains selected proceedings from these symposia reflecting the current status of the field, including an overview of protein N-terminal acetylation in yeast and humans, a novel nomenclature system for the N-terminal acetyltransferases (NATs) and methods for studying protein N-terminal acetylation in vitro and in vivo. PMID:19660094

  2. Molecular, cellular, and physiological significance of N-terminal acetylation.

    PubMed

    Aksnes, Henriette; Hole, Kristine; Arnesen, Thomas

    2015-01-01

    Protein N-terminal acetylation is catalyzed by N-terminal acetyltransferases and represents one of the most common protein modifications in eukaryotes. An increasing number of studies report on the importance of N-terminal acetylation for protein degradation, complex formation, subcellular targeting, and protein folding. N-terminal acetyltransferases are recognized to play important roles in a diversity of cellular processes like apoptosis, cell proliferation, sister chromatid cohesion, and chromatin silencing and are even linked to the development of rare genetic disorders and cancer. This article summarizes our current knowledge on the implications of N-terminal acetylation at the protein, cellular, and physiological levels. PMID:25805127

  3. 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

  4. CRP Is an Activator of Yersinia pestis Biofilm Formation that Operates via a Mechanism Involving gmhA and waaAE-coaD.

    PubMed

    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

  5. 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

  6. Functional Mimicry of the Acetylated C-Terminal Tail of p53 by a SUMO-1 Acetylated Domain, SAD

    PubMed Central

    CHEEMA, AMRITA; KNIGHTS, CHAD D.; RAO, MAHADEV; CATANIA, JASON; PEREZ, RICARDO; SIMONS, BRIGITTE; DAKSHANAMURTHY, SIVANESAN; KOLUKULA, VAMSI K.; TILLI, MADDALENA; FURTH, PRISCILLA A.; ALBANESE, CHRISTOPHER; AVANTAGGIATI, MARIA LAURA

    2013-01-01

    The ubiquitin-like molecule, SUMO-1, a small protein essential for a variety of biological processes, is covalently conjugated to many intracellular proteins, especially to regulatory components of the transcriptional machinery, such as histones and transcription factors. Sumoylation provides either a stimulatory or an inhibitory signal for proliferation and for transcription, but the molecular mechanisms by which SUMO-1 achieves such versatility of effects are incompletely defined. The tumor suppressor and transcription regulator p53 is a relevant SUMO-1 target. Particularly, the C-terminal tail of p53 undergoes both sumoylation and acetylation. While the effects of sumoylation are still controversial, acetylation modifies p53 interaction with chromatin embedded promoters, and enforces p53 apoptotic activity. In this study, we show that the N-terminal region of SUMO-1 might functionally mimic this activity of the p53 C-terminal tail. We found that this SUMO-1 domain possesses similarity with the C-terminal acetylable p53 tail as well as with acetylable domains of other transcription factors. SUMO-1 is, indeed, acetylated when conjugated to its substrates and to p53. In the acetylable form SUMO-1 tunes the p53 response by modifying p53 transcriptional program, by promoting binding onto selected promoters and by favoring apoptosis. By contrast, when non-acetylable, SUMO-1 enforces cell-cycle arrest and p53 binding to a different sets of genes. These data demonstrate for the first time that SUMO-1, a post-translational modification is, in turn, modified by acetylation. Further, they imply that the pleiotropy of effects by which SUMO-1 influences various cellular outcomes and the activity of p53 depends upon its acetylation state. PMID:20458745

  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. Towards a Functional Understanding of Protein N-Terminal Acetylation

    PubMed Central

    Arnesen, Thomas

    2011-01-01

    Protein N-terminal acetylation is a major modification of eukaryotic proteins. Its functional implications include regulation of protein–protein interactions and targeting to membranes, as demonstrated by studies of a handful of proteins. Fifty years after its discovery, a potential general function of the N-terminal acetyl group carried by thousands of unique proteins remains enigmatic. However, recent functional data suggest roles for N-terminal acetylation as a degradation signal and as a determining factor for preventing protein targeting to the secretory pathway, thus highlighting N-terminal acetylation as a major determinant for the life and death of proteins. These contributions represent new and intriguing hypotheses that will guide the research in the years to come. PMID:21655309

  9. 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

  10. Acetylation of banana fibre to improve oil absorbency.

    PubMed

    Teli, M D; Valia, Sanket P

    2013-01-30

    Oil spill leaves detrimental effects on the environment, living organisms and economy. In the present work, an attempt is made to provide an efficient, easily deployable method of cleaning up oil spills and recovering of the oil. The work reports the use of banana fibres which were acetylated for oil spill recovery. The product so formed was characterized by FT-IR, TG, SEM and its degree of acetylation was also evaluated. The extent of acetylation was measured by weight percent gain. The oil sorption capacity of the acetylated fibre was higher than that of the commercial synthetic oil sorbents such as polypropylene fibres as well as un-modified fibre. Therefore, these oil sorption-active materials which are also biodegradable can be used to substitute non-biodegradable synthetic materials in oil spill cleanup. PMID:23218302

  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. Can changes in histone acetylation contribute to memory formation?

    PubMed

    Lopez-Atalaya, Jose P; Barco, Angel

    2014-12-01

    Neuronal histone acetylation has been postulated to be a mnemonic substrate and a target for memory enhancers and neuropsychiatric drugs. Here we critically evaluate this view and examine the apparent conflict between the proposed instructive role for histone acetylation in memory-related transcription and the insights derived from genomic and genetic studies in other systems. We next discuss the suitability of activity-dependent neuronal histone acetylation as a mnemonic substrate and debate alternative interpretations of current evidence. We believe that further progress in our understanding of the role of histone acetylation and other epigenetic modifications in neuronal plasticity, memory, and neuropsychiatric disorders requires a clear discrimination between cause and effect so that novel epigenetics-related processes can be distinguished from classical transcriptional mechanisms. PMID:25269450

  13. 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

  14. Design and parametric evaluation of an enzymatic hydrolysis process (separate hydrolysis and fermentation)

    SciTech Connect

    Wright, J.D.; Power, A.J.; Douglas, L.J.

    1986-08-01

    A separate fungal enzyme hydrolysis and fermentation process for converting lignocellulose to ethanol was evaluated. Although the current state-of-the-art process is expensive, research now under way has the potential to reduce projected selling prices by a factor of three. While the cost of pretreatment can have a major effect on the performance of hydrolysis and enzymen production. Increases in the beta-glucosidase component of the enzyme mixture have led to greatly improved performance, and continued improvement is possible. Other important improvements include reductions in enzyme loadings, reduced agitation requirements in hydrolysis, higher productivity in enzyme manufacture, fermentation of xylose to ethanol, and better utilization of the lignin fraction. 24 refs., 8 figs., 2 tabs.

  15. 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

  16. 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

  17. 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

  18. Nitric Oxide and Histone Acetylation –Shaping Craniofacial Development

    PubMed Central

    Berghella, L.; Puri, PL

    2014-01-01

    Summary Previous work linked nitric oxide (NO) signaling to histone deacetelyases (HDACs) in the control of tissue homeostasis, and suggested that deregulation of this signaling contributes to human diseases. In the previous issue, Liao et al. (2014) show that coordinated NO signaling and histone acetylation are required for propercranial neural crest development and craniofacial morphogenesis and suggest that alterations of NO/acetylation network can contribute to the pathogenesis of craniofacial malformations. PMID:24856136

  19. 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

  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. 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

  2. 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

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

    PubMed

    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 signalling 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

  5. 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

  6. 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.

  7. 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.

  8. 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).

  9. Urea hydrolysis and calcium carbonate reaction fronts

    NASA Astrophysics Data System (ADS)

    Fox, D. T.; Redden, G. D.; Henriksen, J.; Fujita, Y.; Guo, L.; Huang, H.

    2010-12-01

    The mobility of toxic or radioactive metal contaminants in subsurface environments can be reduced by the formation of mineral precipitates that form co-precipitates with the contaminants or that isolate them from the mobile fluid phase. An engineering challenge is to control the spatial distribution of precipitation reactions with respect to: 1) the location of a contaminant, and 2) where reactants are introduced into the subsurface. One strategy being explored for immobilizing contaminants, such as Sr-90, involves stimulating mineral precipitation by forming carbonate ions and hydroxide via the in situ, microbially mediated hydrolysis of urea. A series of column experiments have been conducted to explore how the construction or design of such an in situ reactant production strategy can affect the temporal and spatial distribution of calcium carbonate precipitation, and how the distribution is coupled to changes in permeability. The columns were constructed with silica gel as the porous media. An interval midway through the column contained an adsorbed urease enzyme in order to simulate a biologically active zone. A series of influent solutions were injected to characterize hydraulic properties of the column (e.g., bromide tracer), profiles of chemical conditions and reaction products as the enzyme catalyzes urea hydrolysis (e.g., pH, ammonia, urea), and changes that occur due to CaCO3 precipitation with the introduction of a calcium+urea solutions. In one experiment, hydraulic conductivity was reduced as precipitate accumulated in a layer within the column that had a higher fraction of fine grained silica gel. Subsequent reduction of permeability and flow (for a constant head condition) resulted in displacement of the hydrolysis and precipitation reaction profiles upstream. In another experiment, which lacked the physical heterogeneity (fine grained layer), the precipitation reaction did not result in loss of permeability or flow velocity and the reaction profile, characterized by the pH profile and hydrolysis reaction species, was extended downstream of the enzyme zone. Downstream extension of the reaction profile was due partially to the partial mobility of the enzyme in the column. The experiments are helping to illustrate the complexity of transient reaction fronts as well as the needs and challenges for advanced modeling approaches. A modeling platform developed at the Idaho National Laboratory, which is capable of simulating tightly coupled physical-chemical processes (the Reactive Transport simulator), is being applied to pre-experimental simulations and post-experimental interpretation of results.

  10. 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).

  11. 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

  12. 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.

  13. 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 ...

  14. 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

  15. Ultrasound enhanced enzymatic hydrolysis of Parthenium hysterophorus: A mechanistic investigation.

    PubMed

    Singh, Shuchi; Agarwal, Mayank; Bhatt, Aditya; Goyal, Arun; Moholkar, Vijayanand S

    2015-09-01

    This study has attempted to establish the mechanism of the ultrasound-induced enhancement of enzymatic hydrolysis of pretreated and delignified biomass of Parthenium hysterophorus. A dual approach of statistical optimization of hydrolysis followed by application of sonication at optimum conditions has been adopted. The kinetics of hydrolysis shows a marked 6× increase with sonication, while net sugar yield shows marginal rise of ∼ 20%. The statistical experimental design reveals the hydrolysis process to be enzyme limited. Profile of sugar yield in ultrasound-assisted enzymatic hydrolysis has been analyzed using HCH-1 model coupled with Genetic Algorithm optimization. The trends in the kinetic and physiological parameters of HCH-1 model reveal that sonication enhances enzyme/substrate affinity and reaction velocity of hydrolysis. The product inhibition of enzyme in all forms (free, adsorbed, complexed) also reduces with ultrasound. These effects are attributed to intense micro-convection induced by ultrasound and cavitation in the liquid medium. PMID:26094188

  16. 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.

  17. Purification and characterization of an extracellular muramidase of Clostridium acetobutylicum ATCC 824 that acts on non-N-acetylated peptidoglycan.

    PubMed Central

    Croux, C; Canard, B; Goma, G; Soucaille, P

    1992-01-01

    An extracellular enzyme showing lytic activity on non-N-acetylated peptidoglycan has been isolated from Clostridium acetobutylicum ATCC 824. The lytic enzyme was purified to homogeneity by anion-exchange chromatography and gel filtration, with a recovery of 24%. The enzyme was monomeric and had an estimated molecular weight of 41,000 and an isoelectric point of 3.8. It has been characterized as a muramidase whose 23-amino-acid N terminus displayed 39% homology with the N,O-diacetyl muramidase of the fungus Chalaropsis sp. The muramidase hydrolyzed purified cell walls at an optimum pH of 3, with a maximum velocity of 9.1 mumol of reducing sugars released min-1 mg of muramidase-1 and a concentration of cell walls giving a half-maximum rate of 0.01 mg ml-1. Its activity was inhibited by glucosamine, N-acetylglucosamine, Hg2+, Fe3+, and Ag+ but not by choline. The muramidase-peptidoglycan complex rapidly dissociated before total hydrolysis of the chain and randomly reassociated on another peptidoglycan chain. The affinity of the muramidase was affected by the protein content and the acetylation of the cell wall. Images PMID:1599233

  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. Acetate Activation in Methanosaeta thermophila: Characterization of the Key Enzymes Pyrophosphatase and Acetyl-CoA Synthetase

    PubMed Central

    Berger, Stefanie; Welte, Cornelia; Deppenmeier, Uwe

    2012-01-01

    The thermophilic methanogen Methanosaeta thermophila uses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction in Methanosarcina sp. is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction in Mt. thermophila seems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate (PPi) and was only moderately inhibited by PPi. The breakdown of PPi was performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part of PPi (KM = 0.27 ± 0.05 mM) that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides or PPi. However, it cannot be excluded that other PPi-dependent enzymes take advantage of the remaining PPi and contribute to the energy balance of the cell. PMID:22927778

  20. 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).

  1. Technical bases for precipitate hydrolysis process operating parameters

    SciTech Connect

    Bannochie, C.J.; Lambert, D.P.

    1992-11-09

    This report provides the experimental data and rationale in support of the operating parameters for tetraphenylborate precipitate hydrolysis specified in WSRC-RP-92-737. The report is divided into two sections, the first dealing with lab-scale precipitate hydrolysis experimentation while the second part addresses large-scale runs conducted to demonstrate the revised operating parameters in the Precipitate Hydrolysis Experimental Facility (PHEF). The program was in conjunction with reducing the nitrite ion level in DWPF feed.

  2. 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).

  3. 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

  4. 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

  5. Identification of cellular factors binding to acetylated HIV-1 integrase.

    PubMed

    Allouch, Awatef; Cereseto, Anna

    2011-11-01

    The viral protein integrase (IN) catalyzes the integration of the HIV-1 cDNA into the host cellular genome. We have recently demonstrated that IN is acetylated by a cellular histone acetyltransferase, p300, which modifies three lysines located in the C-terminus of the viral factor (Cereseto et al. in EMBO J 24:3070-3081, 2005). This modification enhances IN catalytic activity, as demonstrated by in vitro assays. Consistently, mutations introduced in the targeted lysines greatly decrease the efficiency of HIV-1 integration. Acetylation was proven to regulate protein functions by modulating protein-protein interactions. HIV-1 to efficiently complete its replication steps, including the integration reaction, requires interacting with numerous cellular factors. Therefore, we sought to investigate whether acetylation might modulate the interaction between IN and the cellular factors. To this aim we performed a yeast two-hybrid screening that differs from the screenings so far performed (Rain et al. in Methods 47:291-297, 2009; Studamire and Goff in Retrovirology 5:48, 2008) for using as bait IN constitutively acetylated. From this analysis we have identified thirteen cellular factors involved in transcription, chromatin remodeling, nuclear transport, RNA binding, protein synthesis regulation and microtubule organization. To validate these interactions, binding assays were performed showing that acetylation increases the affinity of IN with specific factors. Nevertheless, few two-hybrid hits bind with the same affinity the acetylated and the unmodified IN. These results further underlie the relevance of IN post-translational modification by acetylation in HIV-1 replication cycle. PMID:20016921

  6. Feedback regulation of plastidic acetyl-CoA carboxylase by 18:1-acyl carrier protein in Brassica napus

    PubMed Central

    Andre, Carl; Haslam, Richard P.; Shanklin, John

    2012-01-01

    Plant seed oil represents a major renewable source of reduced carbon, but little is known about the biochemical regulation of its synthesis. The goal of this research was to identify potential feedback regulation of fatty acid biosynthesis in Brassica napus embryo-derived cell cultures and to characterize both the feedback signals and enzymatic targets of the inhibition. Fatty acids delivered via Tween esters rapidly reduced the rate of fatty acid synthesis in a dose-dependent and reversible manner, demonstrating the existence of feedback inhibition in an oil-accumulating tissue. Tween feeding did not affect fatty acid elongation in the cytosol or the incorporation of radiolabeled malonate into nascent fatty acids, which together pinpoint plastidic acetyl-CoA carboxylase (ACCase) as the enzymatic target of feedback inhibition. To identify the signal responsible for feedback, a variety of Tween esters were tested for their effects on the rate of fatty acid synthesis. Maximum inhibition was achieved upon feeding oleic acid (18:1) Tween esters that resulted in the intracellular accumulation of 18:1 free fatty acid, 18:1-CoA, and 18:1-acyl-carrier protein (ACP). Direct, saturable inhibition of ACCase enzyme activity was observed in culture extracts and in extracts of developing canola seeds supplemented with 18:1-ACP at physiological concentrations. A mechanism for feedback inhibition is proposed in which reduced demand for de novo fatty acids results in the accumulation of 18:1-ACP, which directly inhibits plastidic ACCase, leading to reduced fatty acid synthesis. Defining this mechanism presents an opportunity for mitigating feedback inhibition of fatty acid synthesis in crop plants to increase oil yield. PMID:22665812

  7. VimA-Dependent Modulation of Acetyl Coenzyme A Levels and Lipid A Biosynthesis Can Alter Virulence in Porphyromonas gingivalis

    PubMed Central

    Aruni, A. Wilson; Lee, J.; Osbourne, D.; Dou, Y.; Roy, F.; Muthiah, A.; Boskovic, D. S.

    2012-01-01

    The Porphyromonas gingivalis VimA protein has multifunctional properties that can modulate several of its major virulence factors. To further characterize VimA, P. gingivalis FLL406 carrying an additional vimA gene and a vimA-defective mutant in a different P. gingivalis genetic background were evaluated. The vimA-defective mutant (FLL451) in the P. gingivalis ATCC 33277 genetic background showed a phenotype similar to that of the vimA-defective mutant (FLL92) in the P. gingivalis W83 genetic background. In contrast to the wild type, gingipain activity was increased in P. gingivalis FLL406, a vimA chimeric strain. P. gingivalis FLL451 had a five times higher biofilm-forming capacity than the parent strain. HeLa cells incubated with P. gingivalis FLL92 showed a decrease in invasion, in contrast to P. gingivalis FLL451 and FLL406, which showed increases of 30 and 40%, respectively. VimA mediated coenzyme A (CoA) transfer to isoleucine and reduced branched-chain amino acid metabolism. The lipid A content and associated proteins were altered in the vimA-defective mutants. The VimA chimera interacted with several proteins which were found to have an LXXTG motif, similar to the sorting motif of Gram-positive organisms. All the proteins had an N-terminal signal sequence with a putative sorting signal of L(P/T/S)X(T/N/D)G and two unique signatures of EXGXTX and HISXXGXG, in addition to a polar tail. Taken together, these observations further confirm the multifunctional role of VimA in modulating virulence possibly through its involvement in acetyl-CoA transfer and lipid A synthesis and possibly by protein sorting. PMID:22144476

  8. 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

  9. 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.

  10. Cyclohexanecarboxyl-coenzyme A (CoA) and cyclohex-1-ene-1-carboxyl-CoA dehydrogenases, two enzymes involved in the fermentation of benzoate and crotonate in Syntrophus aciditrophicus.

    PubMed

    Kung, Johannes W; Seifert, Jana; von Bergen, Martin; Boll, Matthias

    2013-07-01

    The strictly anaerobic Syntrophus aciditrophicus is a fermenting deltaproteobacterium that is able to degrade benzoate or crotonate in the presence and in the absence of a hydrogen-consuming partner. During growth in pure culture, both substrates are dismutated to acetate and cyclohexane carboxylate. In this work, the unknown enzymes involved in the late steps of cyclohexane carboxylate formation were studied. Using enzyme assays monitoring the oxidative direction, a cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA)-forming cyclohexanecarboxyl-CoA (ChCoA) dehydrogenase was purified and characterized from S. aciditrophicus and after heterologous expression of its gene in Escherichia coli. In addition, a cyclohexa-1,5-diene-1-carboxyl-CoA (Ch1,5CoA)-forming Ch1CoA dehydrogenase was characterized after purification of the heterologously expressed gene. Both enzymes had a native molecular mass of 150 kDa and were composed of a single, 40- to 45-kDa subunit; both contained flavin adenine dinucleotide (FAD) as a cofactor. While the ChCoA dehydrogenase was competitively inhibited by Ch1CoA in the oxidative direction, Ch1CoA dehydrogenase further converted the product Ch1,5CoA to benzoyl-CoA. The results obtained suggest that Ch1,5CoA is a common intermediate in benzoate and crotonate fermentation that serves as an electron-accepting substrate for the two consecutively operating acyl-CoA dehydrogenases characterized in this work. In the case of benzoate fermentation, Ch1,5CoA is formed by a class II benzoyl-CoA reductase; in the case of crotonate fermentation, Ch1,5CoA is formed by reversing the reactions of the benzoyl-CoA degradation pathway that are also employed during the oxidative (degradative) branch of benzoate fermentation. PMID:23667239

  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. 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(α2-3)Gal(β1-4)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(α2-3)Gal(β1-4)Glc (3'-sialyllactose) contained Neu4,5Ac2 (4-O-acetyl-sialic acid). These include the trisaccharide 4-O-acetyl 3'-sialyllactose, the pentasaccharide Neu4,5Ac2(α2-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (4-O-acetyl-3'-sialyllacto-N-tetraose d) and the hexasaccharide Neu4,5Ac2(α2-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)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

  13. 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

  14. Histones of Chlamydomonas reinhardtii. Synthesis, acetylation, and methylation.

    PubMed Central

    Waterborg, J H; Robertson, A J; Tatar, D L; Borza, C M; Davie, J R

    1995-01-01

    Histones of the green alga Chlamydomonas reinhardtii were prepared by a new method and fractionated by reversed-phase high-performance liquid chromatography. Acid-urea-Triton gel analysis and tritiated acetate labeling demonstrated high levels of steady-state acetylation for the single histone H3 protein, in contrast to low levels on histones H4 and H2B. Twenty percent of histone H3 is subject to dynamic acetylation with, on average, three acetylated lysine residues per protein molecule. Histone synthesis in light-dark-synchronized cultures was biphasic with pattern differences between two histone H1 variants, between two H2A variants, and between H2B and ubiquitinated H2B. Automated protein sequence analysis of histone H3 demonstrated a site-specific pattern of steady-state acetylation between 7 and 17% at five of the six amino-terminal lysines and of monomethylation between 5 and 81% at five of the eight amino-terminal lysines in a pattern that may limit dynamic acetylation. An algal histone H3 sequence was confirmed by protein sequencing with a single threonine as residue 28 instead of the serine28-alanine29 sequence, present in all other known plant and animal H3 histones. PMID:7480339

  15. 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

  16. 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

  17. Rodent models of the human acetylation polymorphism: comparisons of recombinant acetyltransferases.

    PubMed

    Hein, D W; Doll, M A; Fretland, A J; Gray, K; Deitz, A C; Feng, Y; Jiang, W; Rustan, T D; Satran, S L; Wilkie, T R

    1997-05-12

    The acetylation polymorphism is associated with differential susceptibility to drug toxicity and cancers related to aromatic and heterocyclic amine exposures. N-Acetylation is catalyzed by two cytosolic N-acetyltransferases (NAT1 and NAT2) which detoxify many carcinogenic aromatic amines. NAT1 and NAT2 also activate (via O-acetylation) the N-hydroxy metabolites of aromatic and heterocyclic amine carcinogens to electrophilic intermediates which form DNA adducts and initiate cancer. The classical N-acetylation polymorphism is regulated at the NAT2 locus, which segregates individuals into rapid, intermediate, and slow acetylator phenotypes. Some human epidemiological studies associate slow acetylator and rapid acetylator phenotypes with increased susceptibility to urinary bladder and colorectal cancers, respectively. The acetylation polymorphism has been characterized in three rodent species (mouse, Syrian hamster, and rat) to test associations between NAT2 acetylator phenotype and susceptibility to aromatic and heterocyclic amine-induced cancers in various tumor target organs. NAT1 and NAT2 from rapid and slow acetylator mouse, Syrian hamster, and rat each have been cloned and sequenced. Recombinant NAT1 and NAT2 enzymes enzymes encoded by these genes have been characterized with respect to their catalytic activities for both activation (O-acetylation) and deactivation (N-acetylation) of aromatic and heterocyclic amine carcinogens. The acetylation polymorphisms in mouse, Syrian hamster, and rat are herein reviewed and compared as models of the human acetylation polymorphism. PMID:9202744

  18. 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.

  19. 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 180°C, 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.

  20. 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

  1. Coordinate Regulation of the Nuclear and Plastidic Genes Coding for the Subunits of the Heteromeric Acetyl-Coenzyme A Carboxylase1

    PubMed Central

    Ke, Jinshan; Wen, Tuan-Nan; Nikolau, Basil J.; Wurtele, Eve Syrkin

    2000-01-01

    Plastidic acetyl-coenzyme A (CoA) carboxylase (ACCase) catalyzes the first committed reaction of de novo fatty acid biosynthesis. This heteromeric enzyme is composed of one plastid-coded subunit (β-carboxyltransferase) and three nuclear-coded subunits (biotin carboxy-carrier, biotin carboxylase, and α-carboxyltransferase). We report the primary structure of the Arabidopsis α-carboxyltransferase and β-carboxyltransferase subunits deduced from nucleotide sequences of the respective genes and/or cDNA. Co-immunoprecipitation experiments confirm that the α-carboxyltransferase and β-carboxyltransferase subunits are physically associated. The plant α-carboxyltransferases have gained a C-terminal domain relative to eubacteria, possibly via the evolutionary acquisition of a single exon. This C-terminal domain is divergent among plants and may have a structural function rather than being essential for catalysis. The four ACCase subunit mRNAs accumulate to the highest levels in tissues and cells that are actively synthesizing fatty acids, which are used either for membrane biogenesis in rapidly growing tissues or for oil accumulation in developing embryos. Development coordinately affects changes in the accumulation of the ACCase subunit mRNAs so that these four mRNAs maintain a constant molar stoichiometric ratio. These data indicate that the long-term, developmentally regulated expression of the heteromeric ACCase is in part controlled by a mechanism(s) that coordinately affects the steady-state concentrations of each subunit mRNA. PMID:10759501

  2. 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

  3. 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

  4. Recombinant human liver medium-chain acyl-CoA dehydrogenase: purification, characterization, and the mechanism of interactions with functionally diverse C8-CoA molecules.

    PubMed

    Peterson, K L; Sergienko, E E; Wu, Y; Kumar, N R; Strauss, A W; Oleson, A E; Muhonen, W W; Shabb, J B; Srivastava, D K

    1995-11-14

    We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-CoA dehydrogenase (HMCAD). This procedure routinely yield 100-150 mg of homogeneous preparation of the enzyme from 80 L of the Escherichia coli host cells. A comparative investigation of kinetic properties of the human liver and pig kidney enzymes revealed that, except for a few minor differences, both of these enzymes are nearly identical. We undertook detailed kinetic and thermodynamic investigations for the interaction of HMCAD-FAD with three C8-CoA molecules (viz., octanoyl-CoA, 2-octenoyl-CoA, and 2-octynoyl-CoA), which differ with respect to the extent of unsaturation of the alpha-beta carbon center; octanoyl-CoA and 2-octenoyl-CoA serve as the substrate and product of the enzyme, respectively, whereas 2-octynoyl-CoA is known to inactivate the enzyme. Our experimental results demonstrate that all three C8-CoA molecules first interact with HMCAD-FAD to form corresponding Michaelis complexes, followed by two subsequent isomerization reactions. The latter accompany either subtle changes in the electronic structures of the individual components (in case of 2-octenoyl-CoA and 2-octynoyl-CoA ligands), or a near-complete reduction of the enzyme-bound flavin (in case of octanoyl-CoA). The rate and equilibrium constants intrinsic to the above microscopic steps exhibit marked similarity with different C8-CoA molecules. However, the electronic structural changes accompanying the 2-octynoyl-CoA-dependent inactivation of enzyme is 3-4 orders of magnitude slower than the above isomerization reactions. Hence, the octanoyl-CoA-dependent reductive half-reaction and the 2-octynoyl-CoA-dependent covalent modification of the enzyme occur during entirely different microscopic steps. Arguments are presented that the origin of the above difference lies in the protein conformation-dependent orientation of Glu-376 in the vicinity of the C8-CoA binding site. PMID:7578106

  5. 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

  6. 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.

  7. 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

  8. 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.

  9. 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

  10. Acetylator phenotype in patients with hydralazine-induced lupoid syndrome.

    PubMed

    Strandberg, I; Boman, G; Hassler, L; Sjöqvist, F

    1976-01-01

    The acteylator phenotype has been determined (isoniazid half-life) in 31 patients, 25 of them women, who had exhibited a lupus erythematosus-like syndrome during treatment with hydralazine. Twenty-nine patients were slow acetylators, one was rapid (probably spontaneous SLE) and one uncertain. Only two patients had been given more than 200 mg of hydralazine daily. The mean duration of therapy was 32 months at the onset of symtoms. These were not serious but rather long-standing. Our study confirms that patients who risk developing hydralazine lupus are slow acetylators, especially females, treated with more than 100 mg daily. Rapid acetylators seem to develop this side-effect rarely, if at all. PMID:983808

  11. Glycolysis-mediated changes in acetyl-CoA and histone acetylation control the early differentiation of embryonic stem cells.

    PubMed

    Moussaieff, Arieh; Rouleau, Matthieu; Kitsberg, Daniel; Cohen, Merav; Levy, Gahl; Barasch, Dinorah; Nemirovski, Alina; Shen-Orr, Shai; Laevsky, Ilana; Amit, Michal; Bomze, David; Elena-Herrmann, Bénédicte; Scherf, Tali; Nissim-Rafinia, Malka; Kempa, Stefan; Itskovitz-Eldor, Joseph; Meshorer, Eran; Aberdam, Daniel; Nahmias, Yaakov

    2015-03-01

    Loss of pluripotency is a gradual event whose initiating factors are largely unknown. Here we report the earliest metabolic changes induced during the first hours of differentiation. High-resolution NMR identified 44 metabolites and a distinct metabolic transition occurring during early differentiation. Metabolic and transcriptional analyses showed that pluripotent cells produced acetyl-CoA through glycolysis and rapidly lost this function during differentiation. Importantly, modulation of glycolysis blocked histone deacetylation and differentiation in human and mouse embryonic stem cells. Acetate, a precursor of acetyl-CoA, delayed differentiation and blocked early histone deacetylation in a dose-dependent manner. Inhibitors upstream of acetyl-CoA caused differentiation of pluripotent cells, while those downstream delayed differentiation. Our results show a metabolic switch causing a loss of histone acetylation and pluripotent state during the first hours of differentiation. Our data highlight the important role metabolism plays in pluripotency and suggest that a glycolytic switch controlling histone acetylation can release stem cells from pluripotency. PMID:25738455

  12. Enhanced functional properties of tannic acid after thermal hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thermal hydrolysis processing of fresh tannic acid was carried out in a closed reactor at four different temperatures (65, 100, 150 and 200°C). Pressures reached in the system were 1.3 and 4.8 MPa at 150 and 200°C, respectively. Hydrolysis products (gallic acid and pyrogallol) were separated and qua...

  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. 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...

  15. In-gel N-acetylation for the quantification of the degree of protein in vivo N-terminal acetylation.

    PubMed

    Van Damme, Petra; Arnesen, Thomas; Ruttens, Bart; Gevaert, Kris

    2013-01-01

    Maturation of protein N-termini occurs in all kingdoms of life, with major protein modifications being proteolytic processing (e.g., removal of initiator methionines) and N-terminal acetylation. The functional consequences of these modifications are only known for a few substrates, and techniques to study such modifications have begun to emerge only recently. We here report on a method enabling targeted, mass spectrometry based analysis of protein N-termini from polyacrylamide gel-separated proteins. In our method, stable isotope incorporation by in-gel N-acetylation of free primary amines permits calculating the extent of in vivo N-terminal acetylation, proven to reveal crucial information with reference to N-terminal protein biology. PMID:23381857

  16. Synthesis, in vitro antibacterial and antifungal activity of some n-acetylated and non-acetylated pyrazolines.

    PubMed

    Baseer, Muhammad; Ansari, Farzana Latif; Ashraf, Zaman

    2013-01-01

    Fourteen new N-acetylated and non-acetylated pyrazoline derivatives were synthesized by reacting chalcones with hydrazine in the presence of absolute ethanol however reaction was carried out in the presence of glacial acetic acid to afford N-acetylated pyrazolines. The chemical structures of the synthesized pyrazolines were confirmed by FTIR, (1)HNMR, (13)CNMR and mass spectroscopic data. The pyrazolines (1-14) were screened for antibacterial activity against ten bacterial strains using seven Gram-positive and three Gram-negative bacteria and antifungal activity against Aspergillus flavus, Aspergillus niger and Aspergillus pterus. Pyrazolines (1-14) found to exhibit good to excellent antimicrobial activities compared to the levofloxacin and fluconazole used as standard drugs. PMID:23261729

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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

  2. 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

  3. [Severe hypoglycemia and clouding of consciousness caused by deficiency of the connecting link acyl CoA dehydrogenase].

    PubMed

    Dehlholm, B; Jespersen, T W; Gregersen, N; Christensen, E

    1991-02-01

    A case of severe hypoglycaemia precipitated by fasting in a child is described. As a result of the hypoglycaemia, the patient became brain damaged. The mechanism causing the hypoglycaemia was a defect in the fatty acid beta-oxidation enzyme, the connecting link acyl-CoA dehydrogenase. During a prolonged fast, fatty acids are not converted to acetyl-CoA and ketone bodies which participate in Kreb's cycle for production of energy to a sufficient extent. This result in non-ketotic hypoglycaemia with excretion of organic acids in the urine. As a rule, the symptoms occur for the first time during the first to second years of life in connection with common infectious diseases, with vomiting followed by clouding of consciousness and possibly coma, but the condition may also present with sudden unexpected death. Treatment consists of intravenous glucose. The diagnosis is established by testing the urine for hexanoylglycin and other substances and is confirmed by culture of skin fibroblasts and measurement of beta-oxidation activity. The disease is an autosomally recessive inherited condition. In families where there have been cases of unexplained hypoglycaemia and clouding of consciousness and cases of unexplained death in infancy or "near misses", all of the family members should be offered examination for the above mentioned enzyme deficiency. PMID:2000654

  4. 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

  5. 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

  6. (Hyper)tension release by N-terminal acetylation.

    PubMed

    Aksnes, Henriette; Drazic, Adrian; Arnesen, Thomas

    2015-08-01

    A recent study links N-terminal acetylation and N-end rule degradation to blood pressure regulation. N-terminal mutants of Rgs2, a key G-protein regulator, are differentially processed by N-terminal acetyltransferases and the two branches of the N-end rule pathway. This leads to an imbalance in the signaling governing blood pressure. PMID:26027460

  7. Monitoring sterol uptake, acetylation, and export in yeast.

    PubMed

    Choudhary, Vineet; Schneiter, Roger

    2009-01-01

    Sterols are essential lipid components of eukaryotic membranes. They are synthesized in the endoplasmatic reticulum (ER) from where they are efficiently transported to the plasma membrane, which harbors ~90% of the free sterol pool of the cell. The molecular mechanisms that govern this lipid transport, however, are not well characterized and are challenging to analyze. Saccharomyces cerevisiae offers the opportunity to circumvent some of the technical limitations associated with studying this forward transport of sterols from the ER to the plasma membrane, because the organism can also take up sterols from the environment, incorporate them into the plasma membrane and transport them back to the ER, where the free sterol is converted to steryl esters. This reverse sterol transport, however, occurs only under anaerobic conditions, where the cells become sterol auxotroph, or in mutant cells that cannot synthesize heme. The reverse sterol transport pathway, however, is more amenable to experimental studies, because arrival of the sterol in the ER membrane can be monitored unambiguously by following the formation of steryl esters. Apart from sterol acylation, we have recently described a reversible sterol acetylation cycle that is operating in the lumen of the ER. Acetylation occurs on both cholesterol and pregnenolone, a steroid precursor, and serves as a signal for export of the acetylated sterols into the culture media. The time-dependent appearance of acetylated sterols in the culture supernatant thus provides a new means to monitor the forward transport of chemically modified sterols out of the ER. PMID:19784602

  8. 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

  9. Protein Acetylation Is Involved in Salmonella enterica Serovar Typhimurium Virulence.

    PubMed

    Sang, Yu; Ren, Jie; Ni, Jinjing; Tao, Jing; Lu, Jie; Yao, Yu-Feng

    2016-06-01

    Salmonella causes a range of diseases in different hosts, including enterocolitis and systemic infection. Lysine acetylation regulates many eukaryotic cellular processes, but its function in bacteria is largely unexplored. The acetyltransferase Pat and NAD(+)-dependent deacetylase CobB are involved in the reversible protein acetylation in Salmonella Typhimurium. Here, we used cell and animal models to evaluate the virulence of pat and cobB deletion mutants in S. Typhimurium and found that pat is critical for bacterial intestinal colonization and systemic infection. Next, to understand the underlying mechanism, genome-wide transcriptome was analyzed. RNA sequencing data showed that the expression of Salmonella pathogenicity island 1 (SPI-1) is partially dependent on pat In addition, we found that HilD, a key transcriptional regulator of SPI-1, is a substrate of Pat. The acetylation of HilD by Pat maintained HilD stability and was essential for the transcriptional activation of HilA. Taken together, these results suggest that a protein acetylation system regulates SPI-1 expression by controlling HilD in a posttranslational manner to mediate S. Typhimurium virulence. PMID:26810370

  10. 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

  11. 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

  12. 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...

  13. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.372 N-Acetyl-L-methionine. The food additive N... paragraph (c) of this section. (f) When the food additive is added as a nutrient to special dietary...

  14. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false N-Acetyl-L-methionine. 172.372 Section 172.372 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional...

  15. 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

  16. 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

  17. 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

  18. 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.

  19. Human acetylator polymorphism: estimate of allele frequency in Libya and details of global distribution.

    PubMed Central

    Karim, A K; Elfellah, M S; Evans, D A

    1981-01-01

    Acetylator phenotyping by means of a sulphadimidine tests revealed 65% of Libyan Arabs to be slow acetylators. Hence the frequency of the allele controlling slow acetylation (As) is estimated as q = 0.81 +/- 0.05. This estimate is similar to those previously recorded in European and adjacent Middle Eastern populations. PMID:7328611

  20. 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 β-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

  1. Effect of corosolic acid on the hydrolysis of disaccharides.

    PubMed

    Takagi, Satoshi; Miura, Toshihiro; Ishibashi, Chinami; Kawata, Takanori; Ishihara, Eriko; Gu, Yeunhwa; Ishida, Torao

    2008-06-01

    The banaba leaf (Lagerstroemia speciosa L.) has been used in traditional Oriental medicine to treat diabetes in the Philippines. It contains corosolic acid (CA), a compound which has a hypoglycemic effect. We examined the effect of CA on blood glucose levels and the hydrolysis of disaccharides in the small intestine in mice. CA (10 mg/kg body weight) improved hyperglycemia after an oral administration of sucrose, and significantly reduced the hydrolysis of sucrose in the small intestine. These results suggest that the hypoglycemic activity of CA is derived, at least in part, due to the inhibition of the hydrolysis of sucrose. PMID:18635916

  2. 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.

  3. 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

  4. 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

  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. 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

  7. Complex O-acetylation in non-typeable Haemophilus influenzae lipopolysaccharide: evidence for a novel site of O-acetylation.

    PubMed

    Yildirim, Håkan H; Li, Jianjun; Richards, James C; Hood, Derek W; Moxon, E Richard; Schweda, Elke K H

    2005-12-12

    The structure of the lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae strain 723 has been elucidated using NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS) on O-deacylated LPS and core oligosaccharide material (OS), as well as ESI-MSn on permethylated dephosphorylated OS. It was found that the LPS contains the common structural element of H. influenzae, l-alpha-D-Hepp-(1-->2)-[PEtn-->6]-l-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-l-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-Lipid A, in which the beta-D-Glcp residue (GlcI) is substituted by phosphocholine at O-6 and the distal heptose residue (HepIII) by PEtn at O-3, respectively. In a subpopulation of glycoforms O-2 of HepIII was substituted by beta-D-Galp-(1-->4)-beta-D-Glcp-(1--> or beta-D-Glcp-(1-->. Considerable heterogeneity of the LPS was due to the extent of substitution by O-acetyl groups (Ac) and ester-linked glycine of the core oligosaccharide. The location for glycine was found to be at Kdo. Prominent acetylation sites were found to be at GlcI, HepIII, and the proximal heptose (HepI) residue of the triheptosyl moiety. Moreover, GlcI was acetylated at O-3 and/or O-4 and HepI was acetylated at O-2 as evidenced by capillary electrophoresis ESI-MSn in combination with NMR analyses. This is the first study to show that an acetyl group can substitute HepI of the inner-core region of H. influenzae LPS. PMID:16199021

  8. 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

  9. 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

  10. 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-tRNA·AUG·ribosome) 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

  11. Hydrolysis of Al3+ from constrained molecular dynamics.

    PubMed

    Ikeda, Takashi; Hirata, Masaru; Kimura, Takaumi

    2006-02-21

    We investigated the hydrolysis reactions of Al(3+) in AlCl(3) aqueous solution using the constrained molecular dynamics based on the Car-Parrinello molecular-dynamics method. By employing the proton-aluminum coordination number as a reaction coordinate in the constrained molecular dynamics the deprotonation as well as dehydration processes are successfully realized. From our free-energy difference of DeltaG(0) approximately 8.0 kcal mol(-1) the hydrolysis constant pK(a1) is roughly estimated as 5.8, comparable to the literature value of 5.07. We show that the free-energy difference for the hydrolysis of Al(3+) in acidic conditions is at least 4 kcal mol(-1) higher than that in neutral condition, indicating that the hydrolysis reaction is inhibited by the presence of excess protons located around the hydrated ion, in agreement with the change of the predominant species by pH. PMID:16497053

  12. 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

  13. 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

  14. Responsive behavior of regenerated cellulose in hydrolysis under microwave radiation.

    PubMed

    Ni, Jinping; Na, Haining; She, Zhen; Wang, Jinggang; Xue, Wenwen; Zhu, Jin

    2014-09-01

    This work studied the responsive behavior of regenerated cellulose (RC) in hydrolysis under microwave radiation. Four types of RC with different crystallinity (Cr) and degree of polymerization (DP) are produced to evaluate the reactivity of RC by step-by-step hydrolysis. Results show Cr is the key factor to affect the reactivity of RCs. With hydrolysis of amorphous region and the formation of recrystallization, the Cr of RC reaches a high value and thus weakens the reactivity. As a result, the increment of cellulose conversion and sugar yield gradually reduces. Decrease of the DP of RC is helpful to increase the speed at the onset of hydrolysis and produce high sugar yield. But, there is no direct influence with the reactivity of RC to prolong the time of pretreatment. This research provides an accurate understanding to guide the RC preparation for sugar formation with relative high efficiency under mild reaction conditions. PMID:24971946

  15. 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.

  16. 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)

  17. 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

  18. 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

  19. [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

  20. CREB-binding protein regulates Ku70 acetylation in response to ionization radiation in neuroblastoma

    PubMed Central

    Subramanian, Chitra; Hada, Manila; Opipari, Anthony W.; Castle, Valerie P.; Kwok, Roland P.S.

    2012-01-01

    Ku70 was originally described as an auto-antigen, but it also functions as DNA repair protein in the nucleus and as an anti-apoptotic protein by binding to Bax in the cytoplasm, blocking Bax-mediated cell death. In neuroblastoma (NB) cells, Ku70’s binding with Bax is regulated by Ku70 acetylation such that increasing Ku70 acetylation results in Bax release, triggering cell death. While regulating cytoplasmic Ku70 acetylation is important for cell survival, the role of nuclear Ku70 acetylation in DNA repair is unclear. Here we demonstrated that Ku70 acetylation in the nucleus is regulated by the CREB-binding protein (CBP), and that Ku70 acetylation plays an important role in DNA repair in NB cells. We treated NB cells with ionization radiation and measured DNA repair activity as well as Ku70 acetylation status. Cytoplasmic and nuclear Ku70 were acetylated after ionization radiation in NB cells. Interestingly, cytoplasmic Ku70 was redistributed to the nucleus following irradiation. Depleting CBP in NB cells results in reducing Ku70 acetylation and enhancing DNA repair activity in NB cells suggesting nuclear Ku70 acetylation may have an inhibitory role in DNA repair. These results provide support for the hypothesis that enhancing Ku70 acetylation, through deacetylase inhibition, may potentiate the effect of ionization radiation in NB cells. PMID:23223795

  1. Furfural/ethanol coproduction from biomass feedstocks using acid hydrolysis

    SciTech Connect

    Barrier, J.W.; Bulls, M.M.; Broder, J.D.

    1996-12-31

    The Tennessee Valley Authority (TVA) has been involved in research and development to produce high-value chemicals from biomass for over 15 years. Use of biomass releases less carbon dioxide than use of fossil fuels, and thus represents a more environmentally friendly source of chemicals and fuels. Two biomass conversion processes have been developed as a result of TVA`s work--concentrated acid hydrolysis and dilute acid hydrolysis. Both processes use sulfuric acid as a catalyst. Early hydrolysis research focused on improving ethanol yields through hydrolysis and five-carbon sugar fermentation research. Both processes have been demonstrated at the pilot plant scale. Current work is focused on the development of integrated systems for producing ethanol and a variety of other chemicals and products from biomass. Production of furfural and ethanol from high hemicellulose feedstocks has been identified by TVA as an integrated system with technical and economic potential for commercial success. A system design has been developed to produce ethanol and furfural using dilute acid hydrolysis of sycamore. Furfural yields for the system are estimated at 180--240 pound/ton. Ethanol process yields are 25--38 gallon/ton. Capital and operating costs for a 4,500 ton/day facility are estimated to be $609 million and $183 million, respectively. The dilute acid hydrolysis process proposed by TVA will be described along with additional process economics and potential furfural markets.

  2. Site- and species-specific hydrolysis rates of heroin.

    PubMed

    Szöcs, Levente; Orgován, Gábor; Tóth, Gergő; Kraszni, Márta; Gergó, Lajos; Hosztafi, Sándor; Noszál, Béla

    2016-06-30

    The hydroxide-catalyzed non-enzymatic, simultaneous and consecutive hydrolyses of diacetylmorphine (DAM, heroin) are quantified in terms of 10 site- and species-specific rate constants in connection with also 10 site- and species-specific acid-base equilibrium constants, comprising all the 12 coexisting species in solution. This characterization involves the major and minor decomposition pathways via 6-acetylmorphine and 3-acetylmorphine, respectively, and morphine, the final product. Hydrolysis has been found to be 18-120 times faster at site 3 than at site 6, depending on the status of the amino group and the rest of the molecule. Nitrogen protonation accelerates the hydrolysis 5-6 times at site 3 and slightly less at site 6. Hydrolysis rate constants are interpreted in terms of intramolecular inductive effects and the concomitant local electron densities. Hydrolysis fraction, a new physico-chemical parameter is introduced and determined to quantify the contribution of the individual microspecies to the overall hydrolysis. Hydrolysis fractions are depicted as a function of pH. PMID:27130543

  3. 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

  4. 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

  5. DNA Catalysis of a Normally Disfavored RNA Hydrolysis Reaction

    PubMed Central

    Parker, Darren J.; Xiao, Ying; Aguilar, John M.; Silverman, Scott K.

    2013-01-01

    We recently used in vitro selection to identify many deoxyribozymes that catalyze DNA phosphodiester bond hydrolysis and create 5′-phosphate and 3′-hydroxyl termini. Alternatively, numerous deoxyribozymes have been identified for catalysis of RNA cleavage by 2′-hydroxyl transesterification, forming 2′,3′-cyclic phosphate and 5′-hydroxyl termini. In this study, we investigated the ability of DNA to catalyze RNA cleavage by hydrolysis rather than transesterification, although normally the hydrolysis reaction is substantially disfavored relative to transesterification. Via a series of in vitro selection experiments, we found that reselection of a DNA-hydrolyzing deoxyribozyme leads either to transesterification or hydrolysis, depending on exclusion or inclusion of a stringent selection pressure for hydrolysis. An entirely new selection starting from a random DNA pool, using an all-RNA substrate and imposing the same selection pressure, also leads to RNA hydrolysis. Collectively, these results establish experimentally that small DNA sequences have the catalytic ability to direct a chemical reaction down a disfavored pathway, even when a more favorable mechanism is readily available. Our view of DNA catalysis is therefore expanded beyond merely increasing the rates of reactions that would have occurred more slowly without the catalyst. PMID:23697866

  6. Nicosulfuron: alcoholysis, chemical hydrolysis, and degradation on various minerals.

    PubMed

    Sabadie, Jean

    2002-01-30

    Alcoholysis (methanol or ethanol) and hydrolysis (pH ranging from 4 to 11) of the herbicide nicosulfuron at 30 degrees C principally involves the breakdown of the urea part of the molecule. A high yield of the corresponding carbamate was obtained along with aminopyrimidine during alcoholysis. Hydrolysis led to both aminopyrimidine and pyridylsulfonamide. The latter compound may be easily cyclized (pH > or = 7). First-order kinetics describe the rates of alcoholysis and hydrolysis well. The rate constants (0.44 days(-1) for methanolysis) decreased from 0.50 to 0.002 days(-1) as pH increased from 4 to 8, then remained stable under alkaline conditions. In acidic or neutral solution, the hydrolysis path appeared prevalent (> or =70%), whereas in an alkaline medium it decreased when pH increased. The chemical degradation of nicosulfuron on various dry minerals (calcium bentonite, kaolinite, silica gel, H(+) bentonite, montmorillonite K10, and alumina) was investigated at 30 degrees C. The best conditions for the degradation are obtained on acidic minerals after herbicide deposition using the liquid method. Under these conditions an acceptable correlation with pseudo-first-order kinetics was observed, and the major degradation path is similar to that proposed for chemical hydrolysis. Conversely, alumina seemed to favor other unknown degradation processes. The hydrolysis paths of nicosulfuron and rimsulfuron appeared to be different. PMID:11804524

  7. 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

  8. 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.

  9. Structural Basis for the Design of Potent and Species-specific Inhibitors of 3-hydroxy-3-methylglutaryl CoA Synthases

    SciTech Connect

    Pojer,F.; Ferrer, J.; Richard, S.; Nagegowda, D.; Chye, M.; Bach, T.; Noel, J.

    2006-01-01

    3-Hydroxy-3-methylglutaryl CoA synthase (HMGS) catalyzes the first committed step in the mevalonate metabolic pathway for isoprenoid biosynthesis and serves as an alternative target for cholesterol-lowering and antibiotic drugs. We have determined a previously undescribed crystal structure of a eukaryotic HMGS bound covalently to a potent and specific inhibitor F-244 [(E,E)-11-[3-(hydroxymethyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4-undecadienenoic acid]. Given the accessibility of synthetic analogs of the F-244 natural product, this inhibited eukaryotic HMGS structure serves as a necessary starting point for structure-based methods that may improve the potency and species-specific selectivity of the next generation of F-244 analogs designed to target particular eukaryotic and prokaryotic HMGS.

  10. 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.

  11. Functional role of a distal (3'-phosphate) group of CoA in the recombinant human liver medium-chain acyl-CoA dehydrogenase-catalysed reaction.

    PubMed Central

    Peterson, K L; Srivastava, D K

    1997-01-01

    The X-ray crystallographic structure of medium-chain acyl-CoA dehydrogenase (MCAD)-octenoyl-CoA complex reveals that the 3'-phosphate group of CoA is confined to the exterior of the protein structure [approx. 15 A (1.5 nm) away from the enzyme active site], and is fully exposed to the outside solvent environment. To ascertain whether such a distal (3'-phosphate) fragment of CoA plays any significant role in the enzyme catalysis, we investigated the recombinant human liver MCAD (HMCAD)-catalysed reaction by using normal (phospho) and 3'-phosphate-truncated (dephospho) forms of octanoyl-CoA and butyryl-CoA substrates. The steady-state kinetic data revealed that deletion of the 3'-phosphate group from octanoyl-CoA substrate increased the turnover rate of the enzyme to about one-quarter, whereas that from butyryl-CoA substrate decreased the turnover rate of the enzyme to about one-fifth; the Km values of both these substrates were increased by 5-10-fold on deletion of the 3'-phosphate group from the corresponding acyl-CoA substrates. The transient kinetics for the reductive half-reaction, oxidative half-reaction and the dissociation 'off-rate' (of the reaction product from the oxidized enzyme site) were all found to be affected by deletions of the 3'-phosphate group from octanoyl-CoA and butyryl-CoA substrates. A cumulative account of these results reveals that, although the 3'-phosphate group of acyl-CoA substrates might seem 'useless' on the basis of the structural data, it has an essential functional role during HMCAD catalysis. PMID:9271097

  12. 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

  13. Various methods for determination of the degree of N-acetylation of chitin and chitosan: a review.

    PubMed

    Kasaai, Mohammad R

    2009-03-11

    Chitin, chitosan, and their derivatives have been identified as versatile biopolymers for a broad range of agriculture and food applications. Up to now, several methods have been developed to determine degree of N-acetylation, DA, for chitin and chitosan. In this article, an effort has been made to review the available literature information on the DA determination. These methods are classified into three categories: (1) spectroscopy (IR, (1)H NMR, (13)C NMR, (15)N NMR, and UV); (2) conventional (various types of titration, conductometry, potentiometry, ninhydrin assay, adsorption of free amino groups of chitosan by pictric acid); (3) destructive (elemental analysis, acid or enzymatic hydrolysis of chitin/chitosan and followed by the DA measurement by colorimetry or high performance liquid chromatography, pyrolysis-gas chromatography, and thermal analysis using differential scanning calorimetry) methods. These methods have been compared for their performances and limitations as well as their advantages and disadvantages. The use of IR and NMR spectroscopy methods provides a number of advantages. They do not need long-term procedures to prepare samples, and they provide information on the chemical structure. (1)H NMR and UV techniques are more sensitive than IR, (13)C NMR, and (15)N NMR spectroscopy. The IR technique is mostly used for a qualitative evaluation and comparison studies. Conventional methods are not applicable for highly acetylated chitin. The results of the latter methods are affected by ionic strength of the solvent, pH, and temperature of solution. In destructive methods, longer times are needed for the measurements compared to spectroscopy and conventional methods, but they are applicable for the entire range of the DA. PMID:19187020

  14. [Spectrophotometric evaluation of N-acetyl-beta-glucosaminidase in urine].

    PubMed

    Potere, C; Di Cosmo, C; Riario-Sforza, G; Di Silverio, F; Albertazzi, A; Cappelli, P

    1982-01-01

    A spectrophotometric method for the assay of N-Acetyl-beta-Glucosaminidase activity in human undiluted urines is described. The application of this method is recommended for its sensitivity (2,6 X 10(-4)M) and its rapid performance, because it represents a good alternative to current methods and essentially to the fluorimetric technique with which it has a significant statistical correlation. Estimates of normal individuals aged between 1-70 years are reported. PMID:7168631

  15. 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

  16. 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

  17. Enzymatic characterizations and activity regulations of N-acetyl-β-D-glucosaminidase from the spermary of Nile tilapia (Oreochromis niloticus).

    PubMed

    Zhang, Wei-Ni; Bai, Ding-Ping; Huang, Yi-Fan; Hu, Chong-Wei; Chen, Qing-Xi; Huang, Xiao-Hong

    2014-02-01

    N-Acetyl-β-D-glucosaminidase (NAGase) is proved to be correlated with reproduction of male animals. In this study, enzymatic characterizations of NAGase from spermary of Nile tilapia (Oreochromis niloticus) were investigated in order to further study its reproductive function in fish. Tilapia NAGase was purified to be PAGE homogeneous by the following techniques: (NH4)2SO4 fractionation (40-55%), DEAE-cellulose (DE-32) ion exchange chromatography, Sephadex G-200 gel filtration and DEAE-Sephadex (A-50). The specific activity of the purified enzyme was 4100 U/mg. The enzyme molecular weight was estimated as 118.0 kD. Kinetic studies showed that the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) by the enzyme followed Michaelis-Menten kinetics. The Michaelis-Menten constant (Km) and maximum velocity (Vm) were determined to be 0.67 mM and 23.26 μM/min, respectively. The optimum pH and optimum temperature of the enzyme for hydrolysis of pNP-NAG was to be at pH 5.7 and 55°C, respectively. The enzyme was stable in a pH range from 3.3 to 8.1 at 37°C, and inactive at temperature above 45°C. The enzyme activity was regulated by the following ions in decreasing order: Hg(2+) > Zn(2+) > Cu(2+) > Pb(2+) > Mn(2+). The IC50 of Cu(2+), Zn(2+) and Hg(2+) was 1.23, 0.28, and 0.0027 mM, respectively. However, the ions Li(+), Na(+), K(+), Mg(2+) and Ca(2+) had almost no influence on enzyme activity. In conclusion, the enzymatic characterizations of NAGase from tilapia were special to the other animals, which were correlated with its living habit; besides, CuSO4 and ZnSO4 should used very carefully as insecticides in tilapia cultivation since they both had strong regulations on the enzyme. PMID:24012383

  18. Hydrolysis and fractionation of lignocellulosic biomass

    DOEpatents

    Torget, Robert W.; Padukone, Nandan; Hatzis, Christos; Wyman, Charles E.

    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.

  19. 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

  20. Chromatin decondensed by acetylation shows an elevated radiation response

    SciTech Connect

    Nackerdien, Z.; Michie, J.; Boehm, L.

    1989-02-01

    V-79 Chinese hamster lung fibroblasts exposed to 5 mM n-sodium butyrate were irradiated with 60Co gamma rays and cell survival was determined by the cell colony assay. In a separate set of experiments the acetylated chromatin obtained from these cells was irradiated and the change of molecular weight of the DNA was evaluated by alkaline sucrose density centrifugation. At a survival level of 10(-2) to 10(-4) cells exposed to butyrate were found to be 1.3-1.4 times more radiosensitive than control cells. Exposure of isolated chromatin to 100 Gy of 60Co gamma irradiation generated 0.9 +/- 0.03 single-strand breaks (ssb) per 10 Gy per 10(8) Da and 2.0 +/- 0.3 ssb/10 Gy/10(8) Da for control and acetylated chromatin, respectively. The elevated radiation sensitivity of chromatin relaxed by acetylation is in good agreement with previous results on chromatin expanded by histone H1 depletion. Packing and accessibility of DNA in chromatin appear to be major factors which influence the radiation sensitivity. The intrinsic radiation sensitivity of chromatin in various packing states is discussed in light of the variation of radiation sensitivity of whole cells in the cell cycle which incorporates repair.

  1. 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

  2. Histone acetylation regulates osteodifferentiation of hDPSCs via DSPP.

    PubMed

    Gu, Shensheng; Liang, Jingping; Wang, Jia; Liu, Bin

    2013-01-01

    Dental pulp stem cells (DPSCs) are a unique population of precursor cells isolated from postnatal human dental pulp, with the ability to regenerate a reparative dentin-like complex. We examined the regulation of odontoblast-like differentiation of DPSCs by histone acetylation. Western blot analysis showed that histone H3 acetylation was strongly induced in osteodifferentiation medium. Inhibition of histone acetyltransferase by garcinol reversed osteodifferentiation and mineral formation. Real-time polymerase chain reaction assay indicated that the dentin sialophosphoprotein (DSPP) gene, which is mainly expressed in odontoblasts and preameloblasts in teeth and plays an important role in tooth function, was also down-regulated in garcinol-treated cells. Moreover, lentivirus-mediated knockdown of DSPP in human DPSCs was associated with significant inhibition of mineral formation, but not osteoblast differentiation. In conclusion, the results of this study suggest that DSPP positively affects mineral formation, and that odontoblast-like differentiation and maturation of DPSCs can be regulated by histone acetylation of the DSPP gene. PMID:23747867

  3. 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

  4. Selective cleavage enhanced by acetylating the side chain of lysine.

    PubMed

    Fu, Leixiaomeng; Chen, Tingting; Xue, Gaiqing; Zu, Lily; Fang, Weihai

    2013-01-01

    Selective cleavage is of great interest in mass spectrometry studies as it can help sequence identification by promoting simple fragmentation pattern of peptides and proteins. In this work, the collision-induced dissociation of peptides containing internal lysine and acetylated lysine residues were studied. The experimental and computational results revealed that multiple fragmentation pathways coexisted when the lysine residue was two amino acid residues away from N-terminal of the peptide. After acetylation of the lysine side-chain, b(n)+ ions were the most abundant primary fragment products and the Lys(Ac)-Gly amide bond became the dominant cleavage site via an oxazolone pathway. Acetylating the side-chain of lysine promoted the selective cleavage of Lys-Xxx amide bond and generated much more information of the peptide backbone sequence. The results re-evaluate the selective cleavage due to the lysine basic side-chain and provide information for studying the post-translational modification of proteins and other bio-molecules containing Lys residues. PMID:23303756

  5. 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 Tnfα, 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

  6. 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

  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. Impacts of microalgae pre-treatments for improved anaerobic digestion: thermal treatment, thermal hydrolysis, ultrasound and enzymatic hydrolysis.

    PubMed

    Ometto, Francesco; Quiroga, Gerardo; Pšenička, 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

  9. Identification of Carboxylesterase-Dependent Dabigatran Etexilate Hydrolysis

    PubMed Central

    Parker, Robert B.; Herring, Vanessa L.; Hu, Zhe-Yi

    2014-01-01

    Dabigatran etexilate (DABE) is an oral prodrug that is rapidly converted to the active thrombin inhibitor, dabigatran (DAB), by serine esterases. The aims of the present study were to investigate the in vitro kinetics and pathway of DABE hydrolysis by human carboxylesterase enzymes, and the effect of alcohol on these transformations. The kinetics of DABE hydrolysis in two human recombinant carboxylesterase enzymes (CES1 and CES2) and in human intestinal microsomes and human liver S9 fractions were determined. The effects of alcohol (a known CES1 inhibitor) on the formation of DABE metabolites in carboxylesterase enzymes and human liver S9 fractions were also examined. The inhibitory effect of bis(4-nitrophenyl) phosphate on the carboxylesterase-mediated metabolism of DABE and the effect of alcohol on the hydrolysis of a classic carboxylesterase substrate (cocaine) were studied to validate the in vitro model. The ethyl ester of DABE was hydrolyzed exclusively by CES1 to M1 (Km 24.9 ± 2.9 μM, Vmax 676 ± 26 pmol/min per milligram protein) and the carbamate ester of DABE was exclusively hydrolyzed by CES2 to M2 (Km 5.5 ± 0.8 μM; Vmax 71.1 ± 2.4 pmol/min per milligram protein). Sequential hydrolysis of DABE in human intestinal microsomes followed by hydrolysis in human liver S9 fractions resulted in complete conversion to DAB. These results suggest that after oral administration of DABE to humans, DABE is hydrolyzed by intestinal CES2 to the intermediate M2 metabolite followed by hydrolysis of M2 to DAB in the liver by CES1. Carboxylesterase-mediated hydrolysis of DABE was not inhibited by alcohol. PMID:24212379

  10. Hydrolysis of cisplatin--a first-principles metadynamics study.

    PubMed

    Lau, Justin Kai-Chi; Ensing, Bernd

    2010-09-21

    Cisplatin, or cis-[Pt(NH(3))(2)Cl(2)], was the first member of a new revolutionary class of anticancer drugs that is still used today for the treatment of a wide variety of cancers. The mode of action of cisplatin starts inside the cell with the hydrolysis of Pt-Cl bonds to form a Pt-aqua complex. The solvent environment plays an essential role in many biochemical processes in general, and is expected to have a particular strong effect on the activation (hydrolysis) of cisplatin and cisplatin derivatives. To investigate these solvent effects, we have studied the explicit solvent structures during cisplatin hydrolysis by means of Car-Parrinello molecular dynamics simulations. Since hydrolysis is an activated process, and thus a rare event on the simulation timescale, we have applied the metadynamics sampling technique to map out the free energy landscape from which the reaction mechanism and activation free energy are obtained. Our simulations show that hydrogen bonding between solvent water molecules and metal complexes in the hydrolyzed product systems is stronger than that in the reactant cisplatin system. In addition, the free energy profiles from our metadynamics simulations for the cisplatin hydrolysis shows that the second hydrolysis of cisplatin is thermodynamically favourable, which is in good agreement with experimental results and previous static density functional theory calculations. The reactant channels for both hydrolysis steps are rather wide and flat, indicative of a continuous spectrum of allowed mechanisms with no strong preference for either concerted dissociative or concerted associative pathways. Three or five coordinated metastable intermediates do not exist in aqueous solution. PMID:20582358

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.)

  18. Linking hydrolysis performance to Trichoderma reesei cellulolytic enzyme profile.

    PubMed

    Lehmann, Linda; Rønnest, Nanna P; Jørgensen, Christian I; Olsson, Lisbeth; Stocks, Stuart M; Jørgensen, Henrik S; Hobley, Timothy

    2016-05-01

    Trichoderma reesei expresses a large number of enzymes involved in lignocellulose hydrolysis and the mechanism of how these enzymes work together is too complex to study by traditional methods, for example, by spiking with single enzymes and monitoring hydrolysis performance. In this study, a multivariate approach, partial least squares regression, was used to see whether it could help explain the correlation between enzyme profile and hydrolysis performance. Diverse enzyme mixtures were produced by T. reesei Rut-C30 by exploiting various fermentation conditions and used for hydrolysis of washed pretreated corn stover as a measure of enzyme performance. In addition, the enzyme mixtures were analyzed by liquid chromatography-tandem mass spectrometry to identify and quantify the different proteins. A multivariate model was applied for the prediction of enzyme performance based on the combination of different proteins present in an enzyme mixture. The multivariate model was used for identification of candidate proteins that are correlated to enzyme performance on pretreated corn stover. A very large variation in hydrolysis performance was observed and this was clearly caused by the difference in fermentation conditions. Besides β-glucosidase, the multivariate model identified several xylanases, Cip1 and Cip2, as relevant proteins to study further. Biotechnol. Bioeng. 2016;113: 1001-1010. © 2015 Wiley Periodicals, Inc. PMID:26524197

  19. Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes.

    PubMed

    Qing, Qing; Yang, Bin; Wyman, Charles E

    2010-12-01

    Typically, the enzymatic hydrolysis rate of lignocellulosic biomass is fast initially but then slows down more rapidly than can be explained by just consumption of substrate. Although several factors including enzyme inhibition, enzyme deactivation, a drop in substrate reactivity, or nonproductive binding of enzyme to lignin could be responsible for this loss of effectiveness, we recently reported evidence that xylose, xylan, and xylooligomers dramatically decrease conversion rates and yields, but clarification was still needed for the magnitude of their effect. Therefore, in this study, xylan and various xylooligomers were added to Avicel hydrolysis at low enzyme loadings and found to have a greater effect than adding equal amounts of xylose derived from these materials or when added separately. Furthermore, xylooligomers were more inhibitory than xylan or xylose in terms of a decreased initial hydrolysis rate and a lower final glucose yield even for a low concentration of 1.67 mg/ml. At a higher concentration of 12.5mg/ml, xylooligomers lowered initial hydrolysis rates of Avicel by 82% and the final hydrolysis yield by 38%. Mixed DP xylooligomers showed strong inhibition on cellulase enzymes but not on beta-glucosidase enzymes. By tracking the profile change of xylooligomers, a large portion of the xylooligomers was found to be hydrolyzed by Spezyme CP enzyme preparations, indicating competitive inhibition by mixed xylooligomers. A comparison among glucose sugars and xylose sugars also showed that xylooligomers were more powerful inhibitors than well-established glucose and cellobiose. PMID:20708404

  20. Enzymatic Hydrolysis of Hydrotropic Pulps at Different Substrate Loadings.

    PubMed

    Denisova, Marina N; Makarova, Ekaterina I; Pavlov, Igor N; Budaeva, Vera V; Sakovich, Gennady V

    2016-03-01

    Enzymatic hydrolysis of cellulosic raw materials to produce nutrient broths for microbiological synthesis of ethanol and other valuable products is an important field of modern biotechnology. Biotechnological processing implies the selection of an effective pretreatment technique for raw materials. In this study, the hydrotropic treatment increased the reactivity of the obtained substrates toward enzymatic hydrolysis by 7.1 times for Miscanthus and by 7.3 times for oat hulls. The hydrotropic pulp from oat hulls was more reactive toward enzymatic hydrolysis compared to that from Miscanthus, despite that the substrates had similar compositions. As the initial substrate loadings were raised during enzymatic hydrolysis of the hydrotropic Miscanthus and oat hull pulps, the concentration of reducing sugars increased by 34 g/dm(3) and the yield of reducing sugars decreased by 31 %. The findings allow us to predict the efficiency of enzymatic hydrolysis of hydrotropic pulps from Miscanthus and oat hulls when scaling up the process by volume. PMID:26634840

  1. Sanfilippo syndrome type C: mutation spectrum in the heparan sulfate acetyl-CoA: alpha-glucosaminide N-acetyltransferase (HGSNAT) gene.

    PubMed

    Feldhammer, Matthew; Durand, Stéphanie; Mrázová, Lenka; Boucher, Renée-Myriam; Laframboise, Rachel; Steinfeld, Robert; Wraith, James E; Michelakakis, Helen; van Diggelen, Otto P; Hrebícek, Martin; Kmoch, Stanislav; Pshezhetsky, Alexey V

    2009-06-01

    Mucopolysaccharidosis (MPS) type IIIC or Sanfilippo syndrome type C is a rare autosomal recessive disorder caused by the deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA (AcCoA): alpha-glucosaminide N-acetyltransferase (HGSNAT; EC 2.3.1.78), which catalyzes transmembrane acetylation of the terminal glucosamine residues of heparan sulfate prior to their hydrolysis by alpha-N-acetylglucosaminidase. Lysosomal storage of undegraded heparan sulfate in the cells of affected patients leads to neuronal death, causing neurodegeneration and severely impaired development accompanied by mild visceral and skeletal abnormalities, including mild dwarfism, coarse facies, and joint stiffness. To date, 50 HGSNAT mutations have been identified in MPS IIIC patients: 40 were previously published and 10 novel mutations are reported here. The mutations span the entire structure of the gene and include 13 splice-site mutations, 11 insertions and deletions, 8 nonsense mutations, and 18 missense mutations (http://chromium.liacs.nl/LOVD2/home.php?select_db=HGSNAT). In addition, four polymorphisms result in amino acid changes that do not affect activity of the enzyme. In this work we discuss the spectrum of MPS IIIC mutations, their clinical presentation and distribution within the patient population, and speculate how the mutations may affect the structure and function of HGSNAT. PMID:19479962

  2. 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

  3. 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

  4. 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

  5. Determining yields in high solids enzymatic hydrolysis of biomass.

    PubMed

    Kristensen, Jan B; Felby, Claus; Jørgensen, Henning

    2009-05-01

    As technologies for utilizing biomass for fuel and chemical production continue to improve, enzymatic hydrolysis can be run at still higher solids concentrations. For hydrolyses that initially contain little or no free water (10-40% total solids, w/w), the saccharification of insoluble polymers into soluble sugars involves changes of volume, density, and proportion of insoluble solids. This poses a new challenge when determining the degree of hydrolysis (conversion yield). Experiments have shown that calculating the yield from the resulting sugar concentration in the supernatant of the slurry and using the assumed initial volume leads to significant overestimations of the yield. By measuring the proportion of insoluble solids in the slurry as well as the sugar concentration and specific gravity of the aqueous phase, it is possible to precisely calculate the degree of conversion. The discrepancies between the different ways of calculating yields are demonstrated along with a nonlaborious method for approximating yields in high solids hydrolysis. PMID:18836690

  6. 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

  7. Calculating sugar yields in high solids hydrolysis of biomass.

    PubMed

    Zhu, Yongming; Malten, Marco; Torry-Smith, Mads; McMillan, James D; Stickel, Jonathan J

    2011-02-01

    Calculation of true sugar yields in high solids enzymatic hydrolysis of biomass is challenging due to the varying liquid density and liquid volume resulting from solid solubilization. Ignoring these changes in yield calculations can lead to significant errors. In this paper, a mathematical method was developed for the estimation of liquid volume change and thereafter the sugar yield. The information needed in the calculations include the compositions of the substrate, initial solids loading, initial liquid density, and sugar concentrations before and after hydrolysis. All of these variables are measurable with conventional laboratory procedures. This method was validated experimentally for enzymatic hydrolysis of dilute sulfuric acid pretreated corn stover at solid loadings up to 23% (w/w). The maximum relative error of predicted glucose yield from the true value was less than 4%. Compared to other methods reported in the literature, this method is relatively easy to use and provides good accuracy. PMID:21109427

  8. [Electrophysiologic changes in peripheral nerves in patients with 2 acetylation types of diabetes mellitus].

    PubMed

    Kopeć, A; Rowińska-Marcińska, K; Stryjek-Kamińska, D; Malczewski, B

    1986-01-01

    The purpose of the work was checking whether possible electrophysiological changes in peripheral nerves in diabetes were connected with acetylation phenotype. The authors determined the conduction velocity in peripheral nerves (medianus, peroneal, femoral, facial and sural) in 49 diabetics, divided into two groups depending on the acetylation phenotype determined by Evans method. The results failed to demonstrate differences in the frequency of neuropathy between both groups of acetylators. PMID:3600972

  9. 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.

  10. Acetylation of Histones and Transcription-Related Factors

    PubMed Central

    Sterner, David E.; Berger, Shelley L.

    2000-01-01

    The state of chromatin (the packaging of DNA in eukaryotes) has long been recognized to have major effects on levels of gene expression, and numerous chromatin-altering strategies—including ATP-dependent remodeling and histone modification—are employed in the cell to bring about transcriptional regulation. Of these, histone acetylation is one of the best characterized, as recent years have seen the identification and further study of many histone acetyltransferase (HAT) proteins and their associated complexes. Interestingly, most of these proteins were previously shown to have coactivator or other transcription-related functions. Confirmed and putative HAT proteins have been identified from various organisms from yeast to humans, and they include Gcn5-related N-acetyltransferase (GNAT) superfamily members Gcn5, PCAF, Elp3, Hpa2, and Hat1: MYST proteins Sas2, Sas3, Esa1, MOF, Tip60, MOZ, MORF, and HBO1; global coactivators p300 and CREB-binding protein; nuclear receptor coactivators SRC-1, ACTR, and TIF2; TATA-binding protein-associated factor TAFII250 and its homologs; and subunits of RNA polymerase III general factor TFIIIC. The acetylation and transcriptional functions of these HATs and the native complexes containing them (such as yeast SAGA, NuA4, and possibly analogous human complexes) are discussed. In addition, some of these HATs are also known to modify certain nonhistone transcription-related proteins, including high-mobility-group chromatin proteins, activators such as p53, coactivators, and general factors. Thus, we also detail these known factor acetyltransferase (FAT) substrates and the demonstrated or potential roles of their acetylation in transcriptional processes. PMID:10839822

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. Aspirin acetylates wild type and mutant p53 in colon cancer cells: identification of aspirin acetylated sites on recombinant p53.

    PubMed

    Ai, Guoqiang; Dachineni, Rakesh; Kumar, D Ramesh; Marimuthu, Srinivasan; Alfonso, Lloyd F; Bhat, G Jayarama

    2016-05-01

    Aspirin's ability to inhibit cell proliferation and induce apoptosis in cancer cell lines is considered to be an important mechanism for its anti-cancer effects. We previously demonstrated that aspirin acetylated the tumor suppressor protein p53 at lysine 382 in MDA-MB-231 human breast cancer cells. Here, we extended these observations to human colon cancer cells, HCT 116 harboring wild type p53, and HT-29 containing mutant p53. We demonstrate that aspirin induced acetylation of p53 in both cell lines in a concentration-dependent manner. Aspirin-acetylated p53 was localized to the nucleus. In both cell lines, aspirin induced p21(CIP1). Aspirin also acetylated recombinant p53 (rp53) in vitro suggesting that it occurs through a non-enzymatic chemical reaction. Mass spectrometry analysis and immunoblotting identified 10 acetylated lysines on rp53, and molecular modeling showed that all lysines targeted by aspirin are surface exposed. Five of these lysines are localized to the DNA-binding domain, four to the nuclear localization signal domain, and one to the C-terminal regulatory domain. Our results suggest that aspirin's anti-cancer effect may involve acetylation and activation of wild type and mutant p53 and induction of target gene expression. This is the first report attempting to characterize p53 acetylation sites targeted by aspirin. PMID:26596838

  16. Epigenetic Readers of Lysine Acetylation Regulate Cocaine-Induced Plasticity

    PubMed Central

    Sartor, Gregory C.; Powell, Samuel K.; Brothers, Shaun P.

    2015-01-01

    Epigenetic processes that regulate histone acetylation play an essential role in behavioral and molecular responses to cocaine. To date, however, only a small fraction of the mechanisms involved in the addiction-associated acetylome have been investigated. Members of the bromodomain and extraterminal (BET) family of epigenetic “reader” proteins (BRD2, BRD3, BRD4, and BRDT) bind acetylated histones and serve as a scaffold for the recruitment of macromolecular complexes to modify chromatin accessibility and transcriptional activity. The role of BET proteins in cocaine-induced plasticity, however, remains elusive. Here, we used behavioral, pharmacological, and molecular techniques to examine the involvement of BET bromodomains in cocaine reward. Of the BET proteins, BRD4, but not BRD2 or BRD3, was significantly elevated in the nucleus accumbens (NAc) of mice and rats following repeated cocaine injections and self-administration. Systemic and intra-accumbal inhibition of BRD4 with the BET inhibitor, JQ1, attenuated the rewarding effects of cocaine in a conditioned place preference procedure but did not affect conditioned place aversion, nor did JQ1 alone induce conditioned aversion or preference. Investigating the underlying mechanisms, we found that repeated cocaine injections enhanced the binding of BRD4, but not BRD3, to the promoter region of Bdnf in the NAc, whereas systemic injection of JQ1 attenuated cocaine-induced expression of Bdnf in the NAc. JQ1 and siRNA-mediated knockdown of BRD4 in vitro also reduced expression of Bdnf. These findings indicate that disrupting the interaction between BET proteins and their acetylated lysine substrates may provide a new therapeutic avenue for the treatment of drug addiction. SIGNIFICANCE STATEMENT Proteins involved in the “readout” of lysine acetylation marks, referred to as BET bromodomain proteins (including BRD2, BRD3, BRD4, and BRDT), have been shown to be key regulators of chromatin dynamics and disease, and BET inhibitors are currently being studied in several clinical trials. However, their role in addiction-related phenomena remains unknown. In the current studies, we revealed that BRD4 is elevated in the nucleus accumbens and recruited to promoter regions of addiction-related genes following repeated cocaine administration, and that inhibition of BRD4 attenuates transcriptional and behavioral responses to cocaine. Together, these studies reveal that BET inhibitors may have therapeutic utility in the treatment of cocaine addiction. PMID:26558777

  17. 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

  18. Enzyme-Coupled Assay for β-Xylosidase Hydrolysis of Natural Substrates

    PubMed Central

    Wagschal, Kurt; Franqui-Espiet, Diana; Lee, Charles C.; Robertson, George H.; Wong, Dominic W. S.

    2005-01-01

    We describe here a new enzyme-coupled assay for the quantitation of d-xylose using readily available enzymes that allows kinetic evaluation of hemicellulolytic enzymes using natural xylooligosaccharide substrates. Hydrogen peroxide is generated as an intermediary analyte, which allows flexibility in the choice of the chromophore or fluorophore used as the final reporter. Thus, we present d-xylose quantitation results for solution-phase assays performed with both the fluorescent reporter resorufin, generated from N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), whose corresponding radical cation has an absorbance maximum at ∼400 nm. We also describe a useful solid-phase variation of the assay performed with the peroxidase substrate 3,3′-diaminobenzidine tetrahydrochloride, which produces an insoluble brown precipitate. In addition, kinetic parameters for hydrolysis of the natural substrates xylobiose and xylotriose were obtained using this assay for a glycosyl hydrolase family 39 β-xylosidase from Thermoanaerobacterium sp. strain JW/SL YS485 (Swiss-Prot accession no. O30360). At higher xylobiose substrate concentrations the enzyme showed an increase in the rate indicative of transglycosylation, while for xylotriose marked substrate inhibition was observed. At lower xylobiose concentrations kcat was 2.7 ± 0.4 s−1, Km was 3.3 ± 0.7 mM, and kcat/Km was 0.82 ± 0.21 mM−1 · s−1. Nonlinear curve fitting to a substrate inhibition model showed that for xylotriose Ki was 1.7 ± 0.1 mM, kcat was 2.0 ± 0.1 s−1, Km was 0.144 ± 0.011 mM, and kcat/Km was 14 ± 1.3 mM−1 · s−1. PMID:16151120

  19. 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

  20. 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.

  1. Preparation of radioactive acetyl-l-carnitine by an enzymatic exchange reaction

    SciTech Connect

    Emaus, R.; Bieber, L.L.

    1982-01-15

    A rapid method for the preparation of (1-/sup 14/C)acetyl-L-carnitine is described. The method involves exchange of (1-/sup 14/C)acetic acid into a pool of unlabeled acetyl-L-carnitine using the enzymes acetyl-CoA synthetase and carnitine acetyltransferase. After isotopic equilibrium is attained, radioactive acetylcarnitine is separated from the other reaction components by chromatography on Dowex 1 (C1/sup -/) anion exchange resin. One of the procedures used to verify the product (1-/sup 14/C)acetyl-L-carnitine can be used to synthesize (3S)-(5-/sup 14/C)citric acid.

  2. 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

  3. 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

  4. 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

  5. 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

  6. Characterization of Maize Acetyl-Coenzyme A Carboxylase.

    PubMed

    Egli, M. A.; Gengenbach, B. G.; Gronwald, J. W.; Somers, D. A.; Wyse, D. L.

    1993-02-01

    Maize (Zea mays L.) leaf acetyl-CoA carboxylase (ACCase) was purified about 500-fold by ammonium sulfate fractionation and gel filtration and blue Sepharose affinity and anion-exchange chromatography. Most ACCase activity (85%) recovered from the anion-exchange column was found in a highly purified fraction (specific activity 5.5 [mu]mol acid-stable product min-1 mg-1) that consisted primarily of a single 227-kD biotinylated polypeptide. The fraction represented 29% of the original activity and was designated ACCase I. A second partially purified ACCase activity (ACCase II) eluted earlier during anion-exchange chromatography, contained a single biotinylated polypeptide of 219 kD, was poorly recognized by antiserum raised against the ACCase I polypeptide, and was less inhibited by the herbicides haloxyfop or sethoxydim than was ACCase I. ACCase I and II both utilized propionyl-CoA as substrate about 50% as effectively as acetyl-CoA, and neither utilized methylcrotonyl-CoA. Immunoprecipitation with antiserum and protein blotting of crude extracts of leaf, embryo, and endosperm tissue and suspension cells indicated that most ACCase activity in these tissues was immunologically similar and consisted of ACCase I. Only leaves contained significant amounts of the ACCase II polypeptide; however, no ACCase II polypeptide was found in isolated mesophyll chloroplasts. The ACCase I and II polypeptides appear to be subunits of distinct ACCase isoforms. PMID:12231704

  7. Characterization of Maize Acetyl-Coenzyme A Carboxylase.

    PubMed Central

    Egli, M. A.; Gengenbach, B. G.; Gronwald, J. W.; Somers, D. A.; Wyse, D. L.

    1993-01-01

    Maize (Zea mays L.) leaf acetyl-CoA carboxylase (ACCase) was purified about 500-fold by ammonium sulfate fractionation and gel filtration and blue Sepharose affinity and anion-exchange chromatography. Most ACCase activity (85%) recovered from the anion-exchange column was found in a highly purified fraction (specific activity 5.5 [mu]mol acid-stable product min-1 mg-1) that consisted primarily of a single 227-kD biotinylated polypeptide. The fraction represented 29% of the original activity and was designated ACCase I. A second partially purified ACCase activity (ACCase II) eluted earlier during anion-exchange chromatography, contained a single biotinylated polypeptide of 219 kD, was poorly recognized by antiserum raised against the ACCase I polypeptide, and was less inhibited by the herbicides haloxyfop or sethoxydim than was ACCase I. ACCase I and II both utilized propionyl-CoA as substrate about 50% as effectively as acetyl-CoA, and neither utilized methylcrotonyl-CoA. Immunoprecipitation with antiserum and protein blotting of crude extracts of leaf, embryo, and endosperm tissue and suspension cells indicated that most ACCase activity in these tissues was immunologically similar and consisted of ACCase I. Only leaves contained significant amounts of the ACCase II polypeptide; however, no ACCase II polypeptide was found in isolated mesophyll chloroplasts. The ACCase I and II polypeptides appear to be subunits of distinct ACCase isoforms. PMID:12231704

  8. The dynamic organization of fungal acetyl-CoA carboxylase.

    PubMed

    Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

    2016-01-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control. PMID:27073141

  9. CCR2 chemokines bind selectively to acetylated heparan sulfate octasaccharides.

    PubMed

    Schenauer, Matthew R; Yu, Yonghao; Sweeney, Matthew D; Leary, Julie A

    2007-08-31

    Chemokines participate in well documented interactions with glycosaminoglycans (GAGs). Although many chemokine amino acid residues involved in binding have been identified, much less is known about the bound regions of GAG. Heparan sulfate (HS) is the predominant cell surface GAG, and its heterogeneous nature offers proteins a variety of structural motifs with which to interact. In the present study, we describe the interactions of three CC chemokines, MCP-1/CCL2, MCP-2/CCL8, and MCP-3/CCL7, with HS-derived oligosaccharides. To this end, we generated and characterized a complex HS octasaccharide library containing 17 different octasaccharide compositions based on acetyl and sulfate group content. Electrospray ionization mass spectrometry was used to detect chemokine-HS octasaccharide complexes in the bound state, and an affinity purification protocol was used to select and identify chemokine-binding octasaccharides from the complex mixture. The results indicate that HS octasaccharide sulfation is the foremost requirement for chemokine binding. However, within octasaccharides of constant charge density, acetylation is also observed to augment binding, suggesting that there may be as yet undiscovered specificity in the chemokine-HS interaction. PMID:17597064

  10. Acetyl-L-carnitine in neuropathic pain: experimental data.

    PubMed

    Chiechio, Santina; Copani, Agata; Gereau, Robert W; Nicoletti, Ferdinando

    2007-01-01

    Acetyl-L-carnitine (ALC) has gained clinical interest for its analgesic effect in different forms of neuropathies associated with chronic pain, such as diabetic and HIV-related peripheral neuropathies. The antinociceptive effect of ALC has been confirmed in several experimental models of neuropathic pain, including streptozotocin- and chemotherapy-induced neuropathy, and the sciatic nerve chronic constriction injury model. In these models, prophylactic administration of ALC has proven to be effective in preventing the development of neuropathic pain. In addition, ALC is known to produce a strong antinociceptive effect when given after neuropathic pain has been established. ALC can also improve the function of peripheral nerves by increasing nerve conduction velocity, reducing sensory neuronal loss, and promoting nerve regeneration. Analgesia requires repeated administrations of ALC, suggesting that the drug regulates neuroplasticity across the pain neuraxis. Recent evidence indicates that ALC regulates processes that go beyond its classical role in energy metabolism. These processes involve the activation of muscarinic cholinergic receptors in the forebrain, and an increased expression of type-2 metabotropic glutamate (mGlu2) receptors in dorsal root ganglia neurons. Induction of mGlu2 receptors is mediated by acetylation mechanisms that involve transcription factors of the nuclear factor (NF)-kappaB family. PMID:17696591

  11. The dynamic organization of fungal acetyl-CoA carboxylase

    NASA Astrophysics Data System (ADS)

    Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

    2016-04-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.

  12. The dynamic organization of fungal acetyl-CoA carboxylase

    PubMed Central

    Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

    2016-01-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control. PMID:27073141

  13. Two Arabidopsis Proteins Synthesize Acetylated Xylan in Vitro

    PubMed Central

    Urbanowicz, Breeanna R.; Peña, 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

  14. 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

  15. Microtubule acetylation promotes kinesin-1 binding and transport.

    PubMed

    Reed, Nathan A; Cai, Dawen; Blasius, T Lynne; Jih, Gloria T; Meyhofer, Edgar; Gaertig, Jacek; Verhey, Kristen J

    2006-11-01

    Long-distance intracellular delivery is driven by kinesin and dynein motor proteins that ferry cargoes along microtubule tracks . Current models postulate that directional trafficking is governed by known biophysical properties of these motors-kinesins generally move to the plus ends of microtubules in the cell periphery, whereas cytoplasmic dynein moves to the minus ends in the cell center. However, these models are insufficient to explain how polarized protein trafficking to subcellular domains is accomplished. We show that the kinesin-1 cargo protein JNK-interacting protein 1 (JIP1) is localized to only a subset of neurites in cultured neuronal cells. The mechanism of polarized trafficking appears to involve the preferential recognition of microtubules containing specific posttranslational modifications (PTMs) by the kinesin-1 motor domain. Using a genetic approach to eliminate specific PTMs, we show that the loss of a single modification, alpha-tubulin acetylation at Lys-40, influences the binding and motility of kinesin-1 in vitro. In addition, pharmacological treatments that increase microtubule acetylation cause a redirection of kinesin-1 transport of JIP1 to nearly all neurite tips in vivo. These results suggest that microtubule PTMs are important markers of distinct microtubule populations and that they act to control motor-protein trafficking. PMID:17084703

  16. 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

  17. 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

  18. 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.

  19. 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

  20. Acetylation of α-tubulin on Lys40 is a widespread post-translational modification in angiosperms.

    PubMed

    Nakagawa, Umihito; Suzuki, Daisuke; Ishikawa, Mai; Sato, Hideki; Kamemura, Kazuo; Imamura, Aya

    2013-01-01

    Acetylation of α-tubulin on Lys(40) is thought to be a modification that regulates the dynamic instability of microtubules, but little is known about the occurrence of α-tubulin acetylation in plants. Here we report on a growth stage-dependent change in levels of α-tubulin acetylation and the organ distribution of acetylated α-tubulin in Arabidopsis thaliana plants. Widespread occurrence of α-tubulin acetylation in the leaves of 15 species (20 cultivars) of angiosperms was also confirmed. Our data indicate that acetylated α-tubulin is widespread in many angiosperms, but levels can differ, sometimes considerably, among different organs and developmental stages. PMID:23832354