Sample records for enzymatic phosphoryl transfer

  1. Common Hydrogen Bond Interactions in Diverse Phosphoryl Transfer Active Sites

    PubMed Central

    Summerton, Jean C.; Martin, Gregory M.; Evanseck, Jeffrey D.; Chapman, Michael S.

    2014-01-01

    Phosphoryl transfer reactions figure prominently in energy metabolism, signaling, transport and motility. Prior detailed studies of selected systems have highlighted mechanistic features that distinguish different phosphoryl transfer enzymes. Here, a top-down approach is developed for comparing statistically the active site configurations between populations of diverse structures in the Protein Data Bank, and it reveals patterns of hydrogen bonding that transcend enzyme families. Through analysis of large samples of structures, insights are drawn at a level of detail exceeding the experimental precision of an individual structure. In phosphagen kinases, for example, hydrogen bonds with the O3? of the nucleotide substrate are revealed as analogous to those in unrelated G proteins. In G proteins and other enzymes, interactions with O3? have been understood in terms of electrostatic favoring of the transition state. Ground state quantum mechanical calculations on model compounds show that the active site interactions highlighted in our database analysis can affect substrate phosphate charge and bond length, in ways that are consistent with prior experimental observations, by modulating hyperconjugative orbital interactions that weaken the scissile bond. Testing experimentally the inference about the importance of O3? interactions in phosphagen kinases, mutation of arginine kinase Arg280 decreases kcat, as predicted, with little impact upon KM. PMID:25238155

  2. PLD2 has both enzymatic and cell proliferation-inducing capabilities, that are differentially regulated by phosphorylation and dephosphorylation

    SciTech Connect

    Henkels, Karen M.; Short, Stephen; Peng, Hong-Juan [Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435 (United States)] [Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435 (United States); Fulvio, Mauricio Di [Department of Pharmacology and Toxicology, Wright State University School of Medicine, Dayton, OH 45435 (United States)] [Department of Pharmacology and Toxicology, Wright State University School of Medicine, Dayton, OH 45435 (United States); Gomez-Cambronero, Julian, E-mail: julian.cambronero@wright.edu [Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435 (United States)] [Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, OH 45435 (United States)

    2009-11-13

    Phospholipase D2 (PLD2) overexpression in mammalian cells results in cell transformation. We have hypothesized that this is due to an increase of de novo DNA synthesis. We show here that overexpression of PLD2-WT leads to an increased DNA synthesis, as measured by the expression levels of the proliferation markers PCNA, p27{sup KIP1} and phospho-histone-3. The enhancing effect was even higher with phosphorylation-deficient PLD2-Y179F and PLD2-Y511F mutants. The mechanism for this did not involve the enzymatic activity of the lipase, but, rather, the presence of the protein tyrosine phosphatase CD45, as silencing with siRNA for CD45 abrogated the effect. The two Y{yields}F mutants had in common a YxN consensus site that, in the phosphorylated counterparts, could be recognized by SH2-bearing proteins, such as Grb2. Even though Y179F and Y511F cannot bind Grb2, they could still find other protein partners, one of which, we have reasoned, could be CD45 itself. Affinity purified PLD2 is indeed activated by Grb2 and deactivated by CD45 in vitro. We concluded that phosphorylated PLD2, aided by Grb2, mediates lipase activity, whereas dephosphorylated PLD2 mediates an induction of cell proliferation, and the specific residues involved in this newly discovered regulation of PLD2 are Y{sup 179} and Y{sup 511}.

  3. Enzymic phosphoryl transfer to carbon and oxygen acceptors: An investigation of the biosynthesis of 2-aminoethylphosphonic acid in Tetrahymena pyriformis W. and the kinetic mechanism and cofactor controlled substrate specificity of yeast inorganic pyrophosphatase

    Microsoft Academic Search

    1987-01-01

    This research is concerned with the study of two enzymatic systems which catalyze phosphoryl transfer reactions to carbon and oxygen acceptors. The first portion of this study is concerned with the elucidation of the T. pyriformis 2-aminoethylphosphonate (AEP) biosynthetic pathway. The de novo formation of AEP from exogenously added precursors in Tetrahymena cell-free preparations was evaluated by using radioisotopic techniques

  4. Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the Basic C-Terminal Residue

    NASA Astrophysics Data System (ADS)

    Lanucara, Francesco; Chi Hoo Lee, Dave; Eyers, Claire E.

    2013-12-01

    A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H3PO4), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H3PO4 elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H3PO4 upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available.

  5. Luminescent quantum dots fluorescence resonance energy transfer-based probes for enzymatic activity and enzyme inhibitors.

    PubMed

    Shi, Lifang; Rosenzweig, Nitsa; Rosenzweig, Zeev

    2007-01-01

    The paper describes the development and characterization of analytical properties of quantum dot-based probes for enzymatic activity and for screening enzyme inhibitors. The luminescent probes are based on fluorescence resonance energy transfer (FRET) between luminescent quantum dots that serve as donors and rhodamine acceptors that are immobilized to the surface of the quantum dots through peptide linkers. Peptide-coated CdSe/ZnS quantum dots were prepared using a one-step ligand exchange process in which RGDC peptide molecules replace trioctylphosphine oxide (TOPO) molecules as the capping ligands of the quantum dots. The peptide molecules were bound to the surface of the CdSe/ZnS quantum dots through the thiol group of the peptide cysteine residue. The peptide-coated quantum dots were labeled with rhodamine to form the FRET probes. The emission quantum yield of the quantum dot FRET probes was 4-fold lower than the emission quantum yield of TOPO-capped quantum dots. However, the quantum dot FRET probes were sufficiently bright to enable quantitative enzyme and enzyme inhibition assays. The probes were used first to test the enzymatic activity of trypsin in solution based on FRET signal changes of the quantum dot-based enzymatic probes in the presence of proteolytic enzymes. For example, exposure of the quantum dot FRET probes to 500 microg/mL trypsin for 15 min resulted in 60% increase in the photoluminescence of the quantum dots and a corresponding decrease in the emission of the rhodamine molecules. These changes resulted from the release of rhodamine molecules from the surface of the quantum dots due to enzymatic cleavage of the peptide molecules. The quantum dot FRET-based probes were used to monitor the enzymatic activity of trypsin and to screen trypsin inhibitors for their inhibition efficiency. PMID:17194141

  6. Phospholemman Phosphorylation Alters Its Fluorescence Resonance Energy Transfer with the Na\\/K-ATPase Pump

    Microsoft Academic Search

    Julie Bossuyt; Sanda Despa; Jody L. Martin; Donald M. Bers

    2006-01-01

    Phospholemman (PLM) or FXYD1 is a major cardiac myo- cyte phosphorylation target upon adrenergic stimulation. Prior immunoprecipitation and functional studies suggest that phospholemman associates with the Na\\/K-pump (NKA) and mediates adrenergic Na\\/K-pump regulation. Here, we tested whether the NKA-PLM interaction is close enough to allow fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent (CFP\\/YFP) fusion pro- teins of

  7. Aluminum coordination chemistry and the inhibition of phosphoryl-transferring enzymes

    Microsoft Academic Search

    N. C. Furumo; R. E. Viola

    1986-01-01

    Aluminium ion is a potent inhibitor of the enzymes hexokinase (K\\/sub i\\/ = 0.16 ..mu..M) and glycerokinase (K\\/sub i\\/ = 4.0 ..mu..M). It has been shown that aluminum forms a complex with ATP that is 80 times more stable than the magnesium complex with ATP which is the normal substrate for phosphoryl-transferring enzymes. Kinetic studies performed on several kinases at

  8. A redox beginning: Which came first phosphoryl, acyl, or electron transfer ?. [Abstract only

    NASA Technical Reports Server (NTRS)

    Weber, Arthur L.

    1994-01-01

    Thermodynamic and kinetic information available on the synthesis of prebiotic monomers and polymers will be examined in order to illuminate the prebiotic plausibility of polymer syntheses based on (a) phosphoryl transfer that yields phosphodiester polymers, (b) acyl transfer that gives polyamides, and (c) electron transfer that produces polydisulfide or poly(thio)ester polymers. New experimental results on the oxidative polymerization of 2,3-dimercaptopropanol by ferric ions on the surface of ferric hydroxide oxide will be discussed as a chemical model of polymerization by electron transfer. This redox polymerization that yields polymers with a polydisulfide backbone was found to give oligomers up to the 15-mer from 1 mM of 2,3-dimercaptopropanol after one day at 25 C. High pressure liquid chromatography (HPLC) analysis of the oligomers was carried out on an Alltech OH-100 column eluted with acetonitrile-water.

  9. The unique N-terminal region of SRMS regulates enzymatic activity and phosphorylation of its novel substrate docking protein 1.

    PubMed

    Goel, Raghuveera K; Miah, Sayem; Black, Kristin; Kalra, Natasha; Dai, Chenlu; Lukong, Kiven E

    2013-09-01

    SRMS (Src-related tyrosine kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) belongs to a family of nonreceptor tyrosine kinases, which also includes breast tumour kinase and Fyn-related kinase. SRMS, similar to breast tumour kinase and Fyn-related kinase, harbours a Src homology 3 and Src homology 2, as well as a protein kinase domain. However, unlike breast tumour kinase and Fyn-related kinase, SRMS lacks a C-terminal regulatory tail but distinctively possesses an extended N-terminal region. Both breast tumour kinase and Fyn-related kinase play opposing roles in cell proliferation and signalling. SRMS, however, is an understudied member of this family. Although cloned in 1994, information on the biochemical, cellular and physiological roles of SRMS remains unreported. The present study is the first to explore the expression pattern of SRMS in breast cancers, its enzymatic activity and autoregulatory elements, and the characterization of docking protein 1 as its first bonafide substrate. We found that, similar to breast tumour kinase, SRMS is highly expressed in most breast cancers compared to normal mammary cell lines and tissues. We generated a series of SRMS point and deletion mutants and assessed enzymatic activity, subcellular localization and substrate recognition. We report for the first time that ectopically-expressed SRMS is constitutively active and that its N-terminal region regulates the enzymatic activity of the protein. Finally, we present evidence indicating that docking protein 1 is a direct substrate of SRMS. Our data demonstrate that, unlike members of the Src family, the enzymatic activity of SRMS is regulated by the intramolecular interactions involving the N-terminus of the enzyme and that docking protein 1 is a bona fide substrate of SRMS. PMID:23822091

  10. Transfer of Ser7 phosphorylated CENP-A from centromere to midbody during mitosis in MCF-7 cells.

    PubMed

    Liu, R-M; Tian, X-Y; Huang, X-T; Zhou, H

    2013-01-01

    Serine 7 of centromere protein A (CENP-A) is a very important mitosis-specific phosphorylation site. In this study, we demonstrate the subcellular distribution of Ser7 phosphorylated CENP-A during mitosis in MCF-7 cells. The Ser7 phosphorylation of CENP-A was observed beginning at prophase at centromeres. Upon progression of mitosis, the fluorescence signals emerged in the central region of the metaphase plate and were maintained until anaphase at centromeres. At late anaphase, the fluorescence signals moved to the midzone gradually and transferred from the centromere to the midbody completely at telophase. They were compacted into the centre of the midbody in a thin cylinder consisting of a sandglass-like "mitotic machine" with microtubules and condensed chromosome. We also found that Ser10 phosphorylated H3 and Thr11 phosphorylated H3 were co-localized at the midbody in two bell-like symmetrical bodies with Ser7 phosphorylated CENP-A during the terminal stage of cytokinesis. Midbody isolation and immunoblotting experiments also indicated that Ser7 phosphorylated CENP-A are components of the midbody. These findings suggest that Ser7 phosphorylated CENP-A acts as a chromosomal passenger protein and may play an important role in cytokinesis. PMID:23890477

  11. J. Am. Chem. SOC.1990, 112, 1951-1956 1951 Nucleophiles of High Reactivity in Phosphoryl Transfer

    E-print Network

    Herschlag, Dan

    J. Am. Chem. SOC.1990, 112, 1951-1956 1951 Nucleophiles of High Reactivity in Phosphoryl Transfer, R. G.J. Am. Chem. SOC.1%2,84, 16-24. Fina, N. J.; Edwards, J. 0. fnr.J. Chem. Kiner. 1973, 5, 1.; Plapinger, R.; Stolberg, M.; Wagner-Jauregg,T. J. Am. Chem. Soc. 1955,77,3651-3653. Green, A. L.; Saville, B

  12. Phosphoryl transfers of the phospholipase D superfamily: a quantum mechanical theoretical study.

    PubMed

    DeYonker, Nathan J; Webster, Charles Edwin

    2013-09-18

    The HKD-containing Phospholipase D superfamily catalyzes the cleavage of the headgroup of phosphatidylcholine to produce phosphatidic acid and choline. The mechanism of this cleavage process is studied theoretically. The geometric basis of our models is the X-ray crystal structure of the five-coordinate phosphohistidine intermediate from Streptomyces sp . Strain PMF (PDB Code = 1V0Y ). Hybrid ONIOM QM:QM methodology with Density Functional Theory (DFT) and semiempirical PM6 (DFT:PM6) is used to acquire thermodynamic and kinetic data for the initial phosphoryl transfer, subsequent hydrolysis, and finally, the formation of the experimentally observed ?dead-end? phosphohistidine product (PDB Code = 1V0W ). The model contains nineteen amino acid residues (including the two highly conserved HKD-motifs), four explicit water molecules, and the substrate. Via computations, the persistence of the short-lived five-coordinate phosphorane intermediate on the minutes times scale is rationalized. This five-coordinate phosphohistidine intermediate energetically exists between the hydrolysis event and ?substrate reorganization? (the reorganization of the in vitro model substrate within the active site). Computations directly support the thermodynamic favorability of the in vitro four-coordinate phosphohistidine product. In vivo, the activation energy of substrate reorganization is too high, perhaps due to a combination of substrate immobility when embedded in the lipid bilayer, as well as its larger steric bulk compared to the compound used in the in vitro substrate soaks. On this longer time scale, the enzyme will migrate along the lipid membrane toward its next substrate target, rather than promote the formation of the dead-end product. PMID:24007383

  13. Aluminum coordination chemistry and the inhibition of phosphoryl-transferring enzymes

    SciTech Connect

    Furumo, N.C.; Viola, R.E.

    1986-05-01

    Aluminium ion is a potent inhibitor of the enzymes hexokinase (K/sub i/ = 0.16 ..mu..M) and glycerokinase (K/sub i/ = 4.0 ..mu..M). It has been shown that aluminum forms a complex with ATP that is 80 times more stable than the magnesium complex with ATP which is the normal substrate for phosphoryl-transferring enzymes. Kinetic studies performed on several kinases at pH 7.0 have shown that Al-ATP is a competitive inhibitor vs. Mg-ATP with moderate K/sub i/ values (0.1-0.5 mM) for creatine kinase(CK) and myokinase(MK), and weakly competitive (K/sub i/ > 0.5 mM) with acetate, galactose, arginine and gluconate kinases. Equilibrium dialysis binding studies indicate no significant binding of aluminum ion by the enzymes, while the interaction of aluminum ion with ADP and ATP has been characterized by /sup 13/C, /sup 27/Al, and /sup 31/P NMR spectroscopy. It appears that the inhibition by aluminum is as the Al-nucleotide complex rather than direct binding of free aluminum ion by the enzyme. Kinetic studies indicate that Al/sup 3 +/ inhibition of CK and MK is pH dependent with decreased values of K/sub i/ at lower pH. At pH 6.1 K/sub i/ = 25 ..mu..M for MK (160 ..mu..M at pH 7.0) and 53 ..mu..M for CK (240 ..mu..M at pH 7.0). This may be due to an increased effective concentration of aluminum ion at lower pH.

  14. Fragmentation reactions of aromatic cation radicals: a tool for the detection of electron transfer mechanisms in biomimetic and enzymatic oxidations.

    PubMed

    Baciocchi, E

    1995-07-01

    1. Mechanistic criteria, based on the side-chain fragmentation reactions of aromatic cation radicals, involving the cleavage of a beta bond (i.e. C-H, C-Si and C-S) have been developed for the detection of electron transfer mechanisms in oxidative processes of alkylbenzenes and aromatic sulphides. 2. For benzylic oxidations, the distinction between electron transfer (ET) and hydrogen atom transfer mechanism (HAT) has been based: (a) on studies of intramolecular selectivity, which, with appropriate substrates (5-Z-1,2,3,-trimethylbenzenes and 4-Z-1,2-dimethylbenzenes, where Z = OMe, alkyl), turns out to be much higher in ET than in HAT processes; and (b) on products studies concerning the reactions of bicumyl and benzyltrimethylsilanes since in these systems, the nature of products can be significantly different for ET and HAT mechanisms. 3. These criteria have been applied to the reactions of alkylbenzenes with an NO3 radical (shown to be an ET process) as well as to the microsomal and biomimetic (by iron porphyrins in the presence of PhIO) side-chain oxidation of the same compounds, where the mechanistic probes have suggested a HAT mechanism, with the exception of the biomimetic oxidation of 4-methoxybenzyltrimethylsilane in CH2Cl2-H2O-MeOH, which probably occurs by an ET mechanism. 4. For the enzymatic and biomimetic oxidation of aromatic sulphides an oxygen transfer is suggested, since, with cumyl phenyl sulphide and 4-methoxybenzyl phenyl sulphide, these reactions lead exclusively to the corresponding sulphoxides and sulphones, whereas the same substrates, in genuine ET reactions, form cation radicals which undergo C-H and C-S bond cleavage. 5. An oxygen transfer mechanism is also likely in the biomimetic and enzymatic oxidations of sulphoxides since in these reactions 4-methoxybenzyl phenyl sulphoxide is exclusively converted to sulphone, whereas in ET reactions it forms only C-S bond cleavage products. PMID:7483664

  15. Axonal damage in the making: Neurofilament phosphorylation, proton mobility and magnetisation transfer in multiple sclerosis normal appearing white matter?

    PubMed Central

    Petzold, A.; Tozer, D.J.; Schmierer, K.

    2011-01-01

    Aims Multiple sclerosis (MS) leaves a signature on the phosphorylation and thus proton binding capacity of axonal neurofilament (Nf) proteins. The proton binding capacity in a tissue is the major determinant for exchange between bound and free protons and thus the magnetisation transfer ratio (MTR). This study investigated whether the MTR of non-lesional white matter (NLWM) was related to the brain tissue concentration of neurofilament phosphoforms. Methods Unfixed post-mortem brain slices of 12 MS patients were analysed using MTR, T1 at 1.5 T. Blocks containing NLWM were processed for embedding in paraffin and inspected microscopically. Adjacent tissue was microdissected, homogenised and specific protein levels were quantified by ELISA for the Nf heavy chain (NfH) phosphoforms, glial fibrillary acidic protein (GFAP), S100B and ferritin. Results Averaged hyperphosphorylated NfH (SMI34) but not phosphorylated NfH (SMI35) levels were different between individual patients NLWM. The concentration of hyperphosphorylated NfH-SMI34 correlated with T1 (R = 0.70, p = 0.0114) and — inversely — with MTR (R =?0.73, p = 0.0065). NfH-SMI35 was not correlated to any of the MR indices. Conclusions Post-translational modifications of axonal proteins such as phosphorylation of neurofilaments occur in NLWM and may precede demyelination. The resulting change of proton mobility influences MTR and T1. This permits the in vivo detection of these subtle tissue changes on a proteomic level in patients with MS. PMID:21958956

  16. Kinetic Isotope Effects as a Probe of Hydrogen Transfers to and from Common Enzymatic Cofactors

    PubMed Central

    Roston, Daniel; Islam, Zahidul; Kohen, Amnon

    2013-01-01

    Enzymes use a number of common cofactors as sources of hydrogen to drive biological processes, but the physics of the hydrogen transfers to and from these cofactors is not fully understood. Researchers study the mechanistically important contributions from quantum tunneling and enzyme dynamics and connect those processes to the catalytic power of enzymes that use these cofactors. Here we describe some progress that has been made in studying these reactions, particularly through the use of kinetic isotope effects (KIEs). We first discuss the general theoretical framework necessary to interpret experimental KIEs, and then describe practical uses for KIEs in the context of two case studies. The first example is alcohol dehydrogenase, which uses a nicotinamide cofactor to catalyze a hydride transfer, and the second example is thymidylate synthase, which uses a folate cofactor to catalyze both a hydride and a proton transfer. PMID:24161942

  17. Solution NMR of a 463-Residue Phosphohexomutase: Domain 4 Mobility, Substates, and Phosphoryl Transfer Defect

    SciTech Connect

    Sarma, Akella V.; Anbanandam, A.; Kelm, Allek; Mehra-Chaudhary, Ritcha; Wei, Yirui; Qin, Peiwu; Lee, Yingying; Berjanskii, Mark V.; Mick, Jacob A.; Beamer, Lesa J.; Van Doren, Steven R.

    2012-01-05

    Phosphomannomutase/phosphoglucomutase contributes to the infectivity of Pseudomonas aeruginosa, retains and reorients its intermediate by 180°, and rotates domain 4 to close the deep catalytic cleft. Nuclear magnetic resonance (NMR) spectra of the backbone of wild-type and S108C-inactivated enzymes were assigned to at least 90%. 13C secondary chemical shifts report excellent agreement of solution and crystallographic structure over the 14 ?-helices, C-capping motifs, and 20 of the 22 ?-strands. Major and minor NMR peaks implicate substates affecting 28% of assigned residues. These can be attributed to the phosphorylation state and possibly to conformational interconversions. The S108C substitution of the phosphoryl donor and acceptor slowed transformation of the glucose 1-phosphate substrate by impairing kcat. Addition of the glucose 1,6-bisphosphate intermediate accelerated this reaction by 2?3 orders of magnitude, somewhat bypassing the defect and apparently relieving substrate inhibition. The S108C mutation perturbs the NMR spectra and electron density map around the catalytic cleft while preserving the secondary structure in solution. Diminished peak heights and faster 15N relaxation suggest line broadening and millisecond fluctuations within four loops that can contact phosphosugars. 15N NMR relaxation and peak heights suggest that domain 4 reorients slightly faster in solution than domains 1?3, and with a different principal axis of diffusion. This adds to the crystallographic evidence of domain 4 rotations in the enzyme, which were previously suggested to couple to reorientation of the intermediate, substrate binding, and product release.

  18. Structural and functional analysis of the phosphoryl transfer reaction mediated by the human small C-terminal domain phosphatase, Scp1.

    PubMed

    Zhang, Mengmeng; Liu, June; Kim, Youngjun; Dixon, Jack E; Pfaff, Samuel L; Gill, Gordon N; Noel, Joseph P; Zhang, Yan

    2010-05-01

    Human small C-terminal domain phosphatase 1 (Scp1) modulates the phosphorylation state of the C-terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II), with preference for phosphorylated Ser5 in the tandem heptad repeats of the CTD. Additionally, Scp1 was identified as a conserved regulator of neuronal stem cell development. Scp1 is a member of haloacid dehalogenase (HAD) superfamily, whose catalysis depends on a Mg(2+) ion and a DXDX(T/V) motif. The first Asp of the motif is identified as the nucleophile that is subject to phosphorylation leading to a phosphoryl-aspartate intermediate. This high-energy mixed anhydride intermediate is subsequently hydrolyzed to regenerate the enzyme. In the present study, we successfully captured the phosphoryl-aspartate intermediate in the crystal structure of a Scp1D206A mutant soaked with para-nitrophenyl phosphate (pNPP), providing strong evidence for the proposed mechanism. Furthermore, steady-state kinetic analysis of a variety of Scp1 mutants revealed the importance of Asp206 in Mg(2+) coordination mediated by a water molecule. Overall, we captured the snapshots of the phosphoryl transfer reaction at each stage of Scp1-mediated catalysis. Through structural-based sequence alignment, we show that the spatial position of the D206 side chain is strictly conserved throughout HAD family. Our results strongly suggest that Asp206 and its equivalent residues in other HAD family members play important structural and possible mechanistic roles. PMID:20222012

  19. Computer simulations of enzymatic reactions: examination of linear free-energy relationships and quantum-mechanical corrections in the initial proton-transfer step of carbonic anhydrase.

    PubMed

    Warshel, A; Hwang, J K; Aqvist, J

    1992-01-01

    Computer simulation approaches can provide a powerful tool for correlating the structure of enzymes with their catalytic activity. One of the most effective ways of simulating enzymatic reactions is provided by the empirical valence bond method. The general applicability of this method has been demonstrated in several enzymatic reactions and it is reexamined here in a study of the initial proton-transfer step in the catalytic reaction of carbonic anhydrase. The simulations produce a rate constant which is in agreement with the observed kinetic data and emphasizes the importance of the electrostatic effect associated with the catalytic zinc ion. The calculations are also used to examine the validity of linear free-energy relationships (LFERs) in enzyme catalysis and to evaluate quantum-mechanical corrections of the calculated rate constant. It is found that LFERs are valid in the present case and it is argued that this reflects the fact that the protein responds linearly to the development of electrostatic forces during the reaction. It is concluded that the present approach can be used to augment experimental studies in establishing the general validity of LFERs. It is noted, however, that such relationships are much more valid for transitions between different resonance structures than for transitions between reactants and product states. PMID:1337846

  20. Modifications on the hydrogen bond network by mutations of Escherichia coli copper efflux oxidase affect the process of proton transfer to dioxygen leading to alterations of enzymatic activities

    SciTech Connect

    Kajikawa, Takao; Kataoka, Kunishige [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan)] [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan); Sakurai, Takeshi, E-mail: tsakurai@se.kanazawa-u.ac.jp [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan)] [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan)

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Proton transfer pathway to dioxygen in CueO was identified. Black-Right-Pointing-Pointer Glu506 is the key amino acid to transport proton. Black-Right-Pointing-Pointer The Ala mutation at Glu506 formed a compensatory proton transfer pathway. Black-Right-Pointing-Pointer The Ile mutation at Glu506 shut down the hydrogen bond network. -- Abstract: CueO has a branched hydrogen bond network leading from the exterior of the protein molecule to the trinuclear copper center. This network transports protons in the four-electron reduction of dioxygen. We replaced the acidic Glu506 and Asp507 residues with the charged and uncharged amino acid residues. Peculiar changes in the enzyme activity of the mutants relative to the native enzyme indicate that an acidic amino acid residue at position 506 is essential for effective proton transport. The Ala mutation resulted in the formation of a compensatory hydrogen bond network with one or two extra water molecules. On the other hand, the Ile mutation resulted in the complete shutdown of the hydrogen bond network leading to loss of enzymatic activities of CueO. In contrast, the hydrogen bond network without the proton transport function was constructed by the Gln mutation. These results exerted on the hydrogen bond network in CueO are discussed in comparison with proton transfers in cytochrome oxidase.

  1. Rate-promoting vibrations and coupled hydrogen-electron transfer reactions in the condensed phase: A model for enzymatic catalysis

    Microsoft Academic Search

    Joshua S. Mincer; Steven D. Schwartz

    2004-01-01

    A model is presented for coupled hydrogen-electron transfer reactions in condensed phase in the presence of a rate promoting vibration. Large kinetic isotope effects (KIEs) are found when the hydrogen is substituted with deuterium. While these KIEs are essentially temperature independent, reaction rates do exhibit temperature dependence. These findings agree with recent experimental data for various enzyme-catalyzed reactions, such as

  2. Rate-promoting vibrations and coupled hydrogen–electron transfer reactions in the condensed phase: A model for enzymatic catalysis

    Microsoft Academic Search

    Joshua S. Mincer; Steven D. Schwartz

    2004-01-01

    A model is presented for coupled hydrogen–electron transfer reactions in condensed phase in the presence of a rate promoting vibration. Large kinetic isotope effects (KIEs) are found when the hydrogen is substituted with deuterium. While these KIEs are essentially temperature independent, reaction rates do exhibit temperature dependence. These findings agree with recent experimental data for various enzyme-catalyzed reactions, such as

  3. Rate-promoting vibrations and coupled hydrogen-electron transfer reactions in the condensed phase: A model for enzymatic catalysis

    NASA Astrophysics Data System (ADS)

    Mincer, Joshua S.; Schwartz, Steven D.

    2004-04-01

    A model is presented for coupled hydrogen-electron transfer reactions in condensed phase in the presence of a rate promoting vibration. Large kinetic isotope effects (KIEs) are found when the hydrogen is substituted with deuterium. While these KIEs are essentially temperature independent, reaction rates do exhibit temperature dependence. These findings agree with recent experimental data for various enzyme-catalyzed reactions, such as the amine dehydrogenases and soybean lipoxygenase. Consistent with earlier results, turning off the promoting vibration results in an increased KIE. Increasing the barrier height increases the KIE, while increasing the rate of electron transfer decreases it. These results are discussed in light of other views of vibrationally enhanced tunneling in enzymes.

  4. On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer.

    PubMed

    McCormick, Nicole E; Jakeman, David L

    2015-06-01

    Phosphoenolpyruvate synthase (PEPs) catalyzes the conversion of pyruvate to phosphoenolpyruvate (PEP) using a two-step mechanism invoking a phosphorylated-His intermediate. Formation of PEP is an initial step in gluconeogenesis, and PEPs is essential for growth of Escherichia coli on 3-carbon sources such as pyruvate. The production of PEPs has also been linked to bacterial virulence and antibiotic resistance. As such, PEPs is of interest as a target for antibiotic development, and initial investigations of PEPs have indicated inhibition by sodium fluoride. Similar inhibition has been observed in a variety of phospho-transfer enzymes through the formation of metal fluoride complexes within the active site. Herein we quantify the inhibitory capacity of sodium fluoride through a coupled spectrophotometric assay. The observed inhibition provides indirect evidence for the formation of a MgF3(-) complex within the enzyme active site and insight into the phospho-transfer mechanism of PEPs. The effect of AlCl3 on PEPs enzyme activity was also assessed and found to decrease substrate binding and turnover. PMID:25707819

  5. Electron transfer flavoprotein domain II orientation monitored using double electron-electron resonance between an enzymatically reduced, native FAD cofactor, and spin labels

    PubMed Central

    Swanson, Michael A; Kathirvelu, Velavan; Majtan, Tomas; Frerman, Frank E; Eaton, Gareth R; Eaton, Sandra S

    2011-01-01

    Human electron transfer flavoprotein (ETF) is a soluble mitochondrial heterodimeric flavoprotein that links fatty acid ?-oxidation to the main respiratory chain. The crystal structure of human ETF bound to medium chain acyl-CoA dehydrogenase indicates that the flavin adenine dinucleotide (FAD) domain (?II) is mobile, which permits more rapid electron transfer with donors and acceptors by providing closer access to the flavin and allows ETF to accept electrons from at least 10 different flavoprotein dehydrogenases. Sequence homology is high and low-angle X-ray scattering is identical for Paracoccus denitrificans (P. denitrificans) and human ETF. To characterize the orientations of the ?II domain of P. denitrificans ETF, distances between enzymatically reduced FAD and spin labels in the three structural domains were measured by double electron-electron resonance (DEER) at X- and Q-bands. An FAD to spin label distance of 2.8 ± 0.15 nm for the label in the FAD-containing ?II domain (A210C) agreed with estimates from the crystal structure (3.0 nm), molecular dynamics simulations (2.7 nm), and rotamer library analysis (2.8 nm). Distances between the reduced FAD and labels in ?I (A43C) were between 4.0 and 4.5 ± 0.35 nm and for ?III (A111C) the distance was 4.3 ± 0.15 nm. These values were intermediate between estimates from the crystal structure of P. denitrificans ETF and a homology model based on substrate-bound human ETF. These distances suggest that the ?II domain adopts orientations in solution that are intermediate between those which are observed in the crystal structures of free ETF (closed) and ETF bound to a dehydrogenase (open). PMID:21308847

  6. Tapping a bacterial enzymatic pathway for the preparation and manipulation of synthetic nanomaterials.

    PubMed

    Ku, Ti-Hsuan; Sahu, Swagat; Kosa, Nicolas M; Pham, Kevin M; Burkart, Michael D; Gianneschi, Nathan C

    2014-12-17

    We present a spherical micelle generated in a three-step sequence in which a farnesyl-pantetheine conjugate is phosphorylated, adenylated, and phosphorylated once more to generate a farnesyl-CoA amphiphile that self-assembles into spherical micelles. A sphere-to-fibril morphological switch is achieved by enzymatically transferring the farnesyl group of the farnesyl-CoA micelle onto a peptide via phosphopantetheinyl transferase to generate a peptide amphiphile. Each step in the sequence is followed with characterization by HPLC, MS, TEM, and DLS. This system offers an entry into cofactor-mediated peptide decoration by extending the principles of bioresponsive polymeric materials to sequential enzyme cascades. PMID:25468257

  7. Extrachromosomal oligomycin-resistant mutants of the petite-negative yeast Kluyveromyces lactis. Properties of mitochondrial ATPase and cross-resistance to inhibitors of phosphoryl transfer reactions.

    PubMed

    Brunner, A; Tuena de Cobos, A

    1980-01-01

    The mitochondrial ATPase from oligomycin-resistant mutants which map on different regions of an extrachromosomal DNA (01 and 011 class mutants) showed an increased resistance to oligomycin and venturicidin when assayed in vitro as compared to the sensitive strains. The resistance to oligomycin of the isolated mitochondrial ATPase from 01 class mutants was higher than that of the 011 class mutants. Cross resistance of the oligomycin-resistant mutants to the antibiotics peliomycin and ossamycin, which also inhibit phosphoryl transfer reactions in mitochondria (Walter et al., 1967), was observed, 01 mutants being more resistant to ossamycin than 011 class mutants. At the concentrations of peliomycin studied, no difference in sensitivity among both groups of oligomycin-resistant mutants could be detected. Mitochondrial respiration and isolated mitochondrial ATPase activity are sensitive to venturicidin, suggesting that the previously observed (Brunner et al., 1977) in vivo venturicidin resistance of K. lactis is probably due to an impairment of the influx of the drug at the level of the plasma membrane. PMID:6446648

  8. Normal kinetics of intestinal glucose absorption in the absence of GLUT2: Evidence for a transport pathway requiring glucose phosphorylation and transfer into the endoplasmic reticulum

    PubMed Central

    Stümpel, Frank; Burcelin, Rémy; Jungermann, Kurt; Thorens, Bernard

    2001-01-01

    Glucose is absorbed through the intestine by a transepithelial transport system initiated at the apical membrane by the cotransporter SGLT-1; intracellular glucose is then assumed to diffuse across the basolateral membrane through GLUT2. Here, we evaluated the impact of GLUT2 gene inactivation on this transepithelial transport process. We report that the kinetics of transepithelial glucose transport, as assessed in oral glucose tolerance tests, was identical in the presence or absence of GLUT2; that the transport was transcellular because it could be inhibited by the SGLT-1 inhibitor phlorizin, and that it could not be explained by overexpression of another known glucose transporter. By using an isolated intestine perfusion system, we demonstrated that the rate of transepithelial transport was similar in control and GLUT2?/? intestine and that it was increased to the same extent by cAMP in both situations. However, in the absence, but not in the presence, of GLUT2, the transport was inhibited dose-dependently by the glucose-6-phosphate translocase inhibitor S4048. Furthermore, whereas transport of [14C]glucose proceeded with the same kinetics in control and GLUT2?/? intestine, [14C]3-O-methylglucose was transported in intestine of control but not of mutant mice. Together our data demonstrate the existence of a transepithelial glucose transport system in GLUT2?/? intestine that requires glucose phosphorylation and transfer of glucose-6-phosphate into the endoplasmic reticulum. Glucose may then be released out of the cells by a membrane traffic-based pathway similar to the one we previously described in GLUT2-null hepatocytes. PMID:11562503

  9. Normal kinetics of intestinal glucose absorption in the absence of GLUT2: evidence for a transport pathway requiring glucose phosphorylation and transfer into the endoplasmic reticulum.

    PubMed

    Stümpel, F; Burcelin, R; Jungermann, K; Thorens, B

    2001-09-25

    Glucose is absorbed through the intestine by a transepithelial transport system initiated at the apical membrane by the cotransporter SGLT-1; intracellular glucose is then assumed to diffuse across the basolateral membrane through GLUT2. Here, we evaluated the impact of GLUT2 gene inactivation on this transepithelial transport process. We report that the kinetics of transepithelial glucose transport, as assessed in oral glucose tolerance tests, was identical in the presence or absence of GLUT2; that the transport was transcellular because it could be inhibited by the SGLT-1 inhibitor phlorizin, and that it could not be explained by overexpression of another known glucose transporter. By using an isolated intestine perfusion system, we demonstrated that the rate of transepithelial transport was similar in control and GLUT2(-/-) intestine and that it was increased to the same extent by cAMP in both situations. However, in the absence, but not in the presence, of GLUT2, the transport was inhibited dose-dependently by the glucose-6-phosphate translocase inhibitor S4048. Furthermore, whereas transport of [(14)C]glucose proceeded with the same kinetics in control and GLUT2(-/-) intestine, [(14)C]3-O-methylglucose was transported in intestine of control but not of mutant mice. Together our data demonstrate the existence of a transepithelial glucose transport system in GLUT2(-/-) intestine that requires glucose phosphorylation and transfer of glucose-6-phosphate into the endoplasmic reticulum. Glucose may then be released out of the cells by a membrane traffic-based pathway similar to the one we previously described in GLUT2-null hepatocytes. PMID:11562503

  10. Quantitation of changes in protein phosphorylation: A simple method based on stable isotope labeling and mass spectrometry

    Microsoft Academic Search

    Débora Bonenfant; Tobias Schmelzle; Estela Jacinto; José L. Crespo; Thierry Mini; Michael N. Hall; Paul Jenoe

    2003-01-01

    Reversible protein phosphorylation plays an important role in many cellular processes. However, a simple and reliable method to measure changes in the extent of phosphorylation is lacking. Here, we present a method to quantitate the changes in phosphorylation occurring in a protein in response to a stimulus. The method consists of three steps: (i) enzymatic digestion in H216O or isotopically

  11. Biofuel Cells: Enhanced Enzymatic Bioelectrocatalysis

    NASA Astrophysics Data System (ADS)

    Meredith, Matthew T.; Minteer, Shelley D.

    2012-07-01

    Enzymatic biofuel cells represent an emerging technology that can create electrical energy from biologically renewable catalysts and fuels. A wide variety of redox enzymes have been employed to create unique biofuel cells that can be used in applications such as implantable power sources, energy sources for small electronic devices, self-powered sensors, and bioelectrocatalytic logic gates. This review addresses the fundamental concepts necessary to understand the operating principles of biofuel cells, as well as recent advances in mediated electron transfer- and direct electron transfer-based biofuel cells, which have been developed to create bioelectrical devices that can produce significant power and remain stable for long periods.

  12. Requirements for Phosphorylation of MAP Kinase During Meiosis in Xenopus Oocytes

    Microsoft Academic Search

    James Posada; Jonathan A. Cooper

    1992-01-01

    Mitogen-activated protein (MAP) kinases are activated in response to a variety of extracellular stimuli by phosphorylation on tyrosine and threonine residues. Xp42 is a Xenopus laevis MAP kinase that is activated during oocyte maturation. Modified forms of Xp42 that lacked enzymatic activity or either of the phosphorylation sites were expressed in Xenopus oocytes. When meiotic maturation was induced with progesterone,

  13. The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu

    PubMed Central

    De Gieter, Steven; van Nuland, Nico A.J.; Loris, Remy; Zenkin, Nikolay

    2013-01-01

    Fic proteins are ubiquitous in all domains of life and play critical roles in multiple cellular processes through AMPylation of (transfer of AMP to) target proteins. Doc from the doc/phd toxin/antitoxin module is a member of the Fic family and inhibits bacterial translation by an unknown mechanism. Here we show that, in contrast to the predicted AMPylating activity, Doc is a new type of kinase that inhibits bacterial translation by phosphorylating the conserved threonine (Thr382) of the translation elongation factor EF-Tu, rendering it unable to bind aminoacylated tRNAs. We provide evidence that EF-Tu phosphorylation diverged from AMPylation by antiparallel binding of the NTP relative to the catalytic residues of the conserved Fic catalytic core of Doc. The results bring insights into the mechanism and role of phosphorylation of EF-Tu in bacterial physiology as well as represent an example of catalytic plasticity of enzymes and a mechanism for the evolution of new enzymatic activities. PMID:24141193

  14. Toward a systems-level view of dynamic phosphorylation networks

    PubMed Central

    Newman, Robert H.; Zhang, Jin; Zhu, Heng

    2014-01-01

    To better understand how cells sense and respond to their environment, it is important to understand the organization and regulation of the phosphorylation networks that underlie most cellular signal transduction pathways. These networks, which are composed of protein kinases, protein phosphatases and their respective cellular targets, are highly dynamic. Importantly, to achieve signaling specificity, phosphorylation networks must be regulated at several levels, including at the level of protein expression, substrate recognition, and spatiotemporal modulation of enzymatic activity. Here, we briefly summarize some of the traditional methods used to study the phosphorylation status of cellular proteins before focusing our attention on several recent technological advances, such as protein microarrays, quantitative mass spectrometry, and genetically-targetable fluorescent biosensors, that are offering new insights into the organization and regulation of cellular phosphorylation networks. Together, these approaches promise to lead to a systems-level view of dynamic phosphorylation networks. PMID:25177341

  15. Phosphorylation of Thylakoid Proteins

    Microsoft Academic Search

    Alexander V. Vener

    Application of novel techniques for the characterization of in vivo protein phosphorylation has revealed sixteen distinct\\u000a phosphorylation sites in ten integral and two peripheral proteins in photosynthetic thylakoid membranes. In addition to phosphorylation\\u000a of the photosystem II (PS II) proteins D1, D2, CP43, and PsbH, and the light-harvesting antenna polypeptides LHCII and CP29,\\u000a phosphorylation has been found in photosystem I

  16. Suppression of Akt1 phosphorylation by adenoviral transfer of the PTEN gene inhibits hypoxia-induced proliferation of rat pulmonary arterial smooth muscle cells

    SciTech Connect

    Luo, Chunxia [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)] [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Yi, Bin, E-mail: yibin1974@163.com [Department of Anesthesia, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China) [Department of Anesthesia, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Bai, Li [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China)] [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Xia, Yongzhi [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)] [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Wang, Guansong; Qian, Guisheng [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China)] [Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 (China); Feng, Hua [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)] [Department of Neurosurgery, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China)

    2010-07-02

    Recent findings identify the role of proliferation of pulmonary artery smooth muscle cells (PASMCs) in pulmonary vascular remodeling. Phosphoinositide 3 kinase (PI3K) and serine/threonine kinase (Akt) proteins are expressed in vascular smooth muscle cells. In addition, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been identified as a negative regulator of cytokine signaling that inhibits the PI3K-Akt pathway. However, little is known about the role of PTEN/Akt signaling in hypoxia-associated vascular remodeling. In this study, we found that hypoxia-induced the expression of Akt1 mRNA and phosphorylated protein by at least twofold in rat PASMCs. Phospho-PTEN significantly decreased in the nuclei of PASMCs after hypoxic stimulation. After forcing over-expression of PTEN by adenovirus-mediated PTEN (Ad-PTEN) transfection, the expression of phospho-Akt1 was significantly suppressed in PASMCs at all time-points measured. Additionally, we showed here that hypoxia increased proliferation of PASMCs by nearly twofold and over-expression of PTEN significantly inhibited hypoxia-induced PASMCs proliferation. These findings suggest that phospho-PTEN loss in the nuclei of PASMCs under hypoxic conditions may be the major cause of aberrant activation of Akt1 and may, therefore, play an important role in hypoxia-associated pulmonary arterial remodeling. Finally, the fact that transfection with Ad-PTEN inhibits the phosphorylation of Akt1 in PASMCs suggests a potential therapeutic effect on hypoxia-associated pulmonary arterial remodeling.

  17. SOLID-PHASE ASSAY FOR THE PHOSPHORYLATION OF PROTEINS BLOTTED ON NITROCELLULOSE MEMBRANE FILTERS

    EPA Science Inventory

    A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters and the blotted polypeptides are phosphorylated with ...

  18. Regulation of protein phosphorylation in oat mitochondria

    SciTech Connect

    Pike, C.; Kopeck, K.; Sceppa, E. (Franklin and Marshall College, Lancaster, PA (USA))

    1989-04-01

    We sought to identify phosphorylated proteins in isolated oat mitocchondria and to characterize the enzymatic and regulatory properties of the protein kinase(s). Mitochondria from oats (Avena sativa L. cv. Garry) were purified on Percoll gradients. Mitochondria were incubated with {sup 32}P-{gamma}-ATP; proteins were separated by SDS-PAGE. A small number of bands was detected on autoradiograms, most prominently at 70 kD and 42 kD; the latter band has been tentatively identified as a subunit of the pyruvate dehydrogenase complex, a well-known phosphoprotein. The protein kinase(s) could also phosphorylate casein, but not histone. Spermine enhanced the phosphorylation of casein and inhibited the phosphorylation of the 42 kD band. These studies were carried out on both intact and burst mitochondria. Control by calcium and other ions was investigated. The question of the action of regulators on protein kinase or protein phosphatase was studied by the use of {sup 35}S-adenosine thiotriphosphate.

  19. Enzymatic modification of schizophyllan

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An enzymatic method was developed for the progressive modification of the polysaccharide schizophyllan. Fungal strains Hypocrea nigricans NRRL 62555, Penicillium crustosum NRRL 62558, and Penicillium simplicissimum NRRL 62550 were previously identified as novel sources of ß-endoglucanase with specif...

  20. Fractalkine signaling and Tau hyper-phosphorylation are associated with autophagic alterations in lentiviral Tau and A?1–42 gene transfer models

    PubMed Central

    Hebron, Michaeline L.; Algarzae, Norah K.; Lonskaya, Irina; Moussa, Charbel

    2013-01-01

    Tau hyper-phosphorylation (p-Tau) and neuro-inflammation are hallmarks of neurodegeneration. Previous findings suggest that microglial activation via CX3CL1 promotes p-Tau. We examined inflammation and autophagic p-Tau clearance in lentiviral Tau and mutant P301L expressing rats and used lentiviral A?1–42 to induce p-Tau. Lentiviral Tau or P301L expression significantly increased caspase-3 activity and TNF-?, but CX3CL1 was significantly higher in animals expressing Tau compared to P301L. Lentiviral A?1–42 induced p-Tau 4 weeks post-injection, and increased caspase-3 activation (8-fold) and TNF-? levels. Increased levels of ADAM-10/17 were also detected with p-Tau. IL-6 levels were increased but CX3CL1 did not change in the absence of p-Tau (2 weeks); however, p- Tau reversed these effects, which were associated with increased microglial activity. We observed changes in autophagic markers, including accumulation of autophagic vacuoles (AVs) and p-Tau accumulation in autophagosomes but not lysosomes, suggesting alteration of autophagy. Taken together, microglial activation may promote p-Tau independent of total Tau levels via CX3CL1 signaling, which seems to depend on interaction with inflammatory markers, mainly IL-6. The simultaneous change in autophagy and CX3CL1 signaling suggests communication between microglia and neurons, raising the possibility that accumulation of intraneuronal amyloid, due to lack of autophagic clearance, may lead microglia activation to promote p-Tau as a tag for phagocytic degradation. PMID:23333589

  1. Isolation of 3-phosphohistidine from phosphorylated pyruvate, phosphate dikinase.

    PubMed Central

    Spronk, A M; Yoshida, H; Wood, H G

    1976-01-01

    Pyruvate, phosphate dikinase (EC 2-7-9-1) catalyzes formation of phosphoenolpyruvate, AMP, and inorganic pyrophosphate from pyruvate, ATP, and orthophosphate. A pyrophosphoryl and phosphoryl form of the enzyme is involved in this transfer. The [32P]phosphoryl form of pyruvate, phosphate dikinase was prepared with enzyme isolated from Bacteroides symbiosus. The [32P]phosphoryl enzyme was found to have properties corresponding to a phosphoramidate linkage and this was confirmed by isolation of 3-[32P]phosphohistidine from alkaline hydrolysates of the enzyme. The histidyl residue is considered to be the pyrophosphoryl- and phosphoryl-carrier between the three substrate sites of this enzyme. PMID:12506

  2. Enzymatic characterization of recombinant nitrate reductase expressed and purified from Neurospora crassa.

    PubMed

    Ringel, Phillip; Probst, Corinna; Dammeyer, Thorben; Buchmeier, Sabine; Jänsch, Lothar; Wissing, Josef; Tinnefeld, Philip; Mendel, Ralf R; Jockusch, Brigitte M; Kruse, Tobias

    2015-07-01

    We established an expression and purification procedure for recombinant protein production in Neurospora crassa (N. crassa). This Strep-tag® based system was successfully used for purifying recombinant N. crassa nitrate reductase (NR), whose enzymatic activity was compared to recombinant N. crassa NR purified from Escherichia coli. The purity of the two different NR preparations was similar but NR purified from N. crassa showed a significantly higher nitrate turnover rate. Two phosphorylation sites were identified for NR purified from the endogenous expression system. We conclude that homologous expression of N. crassa NR yields a higher active enzyme and propose that NR phosphorylation causes enhanced enzymatic activity. PMID:25914160

  3. Enzymatic Synthesis of Magnetic Nanoparticles

    PubMed Central

    Kolhatkar, Arati G.; Dannongoda, Chamath; Kourentzi, Katerina; Jamison, Andrew C.; Nekrashevich, Ivan; Kar, Archana; Cacao, Eliedonna; Strych, Ulrich; Rusakova, Irene; Martirosyan, Karen S.; Litvinov, Dmitri; Lee, T. Randall; Willson, Richard C.

    2015-01-01

    We report the first in vitro enzymatic synthesis of paramagnetic and antiferromagnetic nanoparticles toward magnetic ELISA reporting. With our procedure, alkaline phosphatase catalyzes the dephosphorylation of l-ascorbic-2-phosphate, which then serves as a reducing agent for salts of iron, gadolinium, and holmium, forming magnetic precipitates of Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5. The nanoparticles were found to be paramagnetic at 300 K and antiferromagnetic under 25 K. Although weakly magnetic at 300 K, the room-temperature magnetization of the nanoparticles found here is considerably greater than that of analogous chemically-synthesized LnxFeyOz (Ln = Gd, Ho) samples reported previously. At 5 K, the nanoparticles showed a significantly higher saturation magnetization of 45 and 30 emu/g for Fe45±14Gd5±2O50±15 and Fe42±4Ho6±4O52±5, respectively. Our approach of enzymatically synthesizing magnetic labels reduces the cost and avoids diffusional mass-transfer limitations associated with pre-synthesized magnetic reporter particles, while retaining the advantages of magnetic sensing. PMID:25854425

  4. Enzymatic DNA molecules

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F. (Inventor); Breaker, Ronald R. (Inventor)

    1998-01-01

    The present invention discloses deoxyribonucleic acid enzymes--catalytic or enzymatic DNA molecules--capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.

  5. Printing enzymatic reactions.

    PubMed

    Tian, Junfei; Shen, Wei

    2011-02-01

    We used relief and planographic printing methods to print the catalytic effect of an enzyme, but not the enzyme molecules, onto paper. Printing enzymatic reactions have applications in bioactive papers, low-cost diagnostics, anti-counterfeiting devices and advanced packaging materials. These methods can create novel printing effects on commodity surfaces for advanced applications. PMID:21109893

  6. Screening for protein phosphorylation using nanoscale reactions on microdroplet arrays.

    PubMed

    Küster, Simon K; Pabst, Martin; Zenobi, Renato; Dittrich, Petra S

    2015-01-26

    We present a novel and straightforward screening method to detect protein phosphorylations in complex protein mixtures. A proteolytic digest is separated by a conventional nanoscale liquid chromatography (nano-LC) separation and the eluate is immediately compartmentalized into microdroplets, which are spotted on a microarray MALDI plate. Subsequently, the enzyme alkaline phosphatase is applied to every second microarray spot to remove the phosphate groups from phosphorylated peptides, which results in a mass shift of n×-80?Da. The MALDI-MS scan of the microarray is then evaluated by a software algorithm to automatically identify the phosphorylated peptides by exploiting the characteristic chromatographic peak profile induced by the phosphatase treatment. This screening method does not require extensive MS/MS experiments or peak list evaluation and can be easily extended to other enzymatic or chemical reactions. PMID:25504774

  7. Phosphorylation of AMPA receptors

    Microsoft Academic Search

    John Q. Wang; Anish Arora; Lu Yang; Nikhil K. Parelkar; Guochi Zhang; Xianyu Liu; Eun Sang Choe; Limin Mao

    2005-01-01

    The ionotropic ?-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor is densely distributed in the mammalian\\u000a brain and is primarily involved in mediating fast excitatory synaptic transmission. Recent studies in both heterologous expression\\u000a systems and cultured neurons have shown that the AMPA receptor can be phosphorylated on their subunits (GluR1, GluR2, and\\u000a GluR4). All phosphorylation sites reside at serine, threonine, or tyrosine on the

  8. Graphene based enzymatic bioelectrodes and biofuel cells

    NASA Astrophysics Data System (ADS)

    Karimi, Anahita; Othman, Ali; Uzunoglu, Aytekin; Stanciu, Lia; Andreescu, Silvana

    2015-04-01

    The excellent electrical conductivity and ease of functionalization make graphene a promising material for use in enzymatic bioelectrodes and biofuel cells. Enzyme based biofuel cells have attracted substantial interest due to their potential to harvest energy from organic materials. This review provides an overview of the functional properties and applications of graphene in the construction of biofuel cells as alternative power sources. The review covers the current state-of-the-art research in graphene based nanomaterials (physicochemical properties and surface functionalities), the role of these parameters in enhancing electron transfer, the stability and activity of immobilized enzymes, and how enhanced power density can be achieved. Specific examples of enzyme immobilization methods, enzyme loading, stability and function on graphene, functionalized graphene and graphene based nanocomposite materials are discussed along with their advantages and limitations. Finally, a critical evaluation of the performance of graphene based enzymatic biofuel cells, the current status, challenges and future research needs are provided.

  9. Struvite and prebiotic phosphorylation.

    NASA Technical Reports Server (NTRS)

    Handschuh, G. J.; Orgel, L. E.

    1973-01-01

    Struvite rather than apatite or amorphous calcium phosphate is precipitated when phosphate is added to seawater containing more than 0.01M NH4+ ions. Struvite may have precipitated from evaporating seawater on the primitive earth, and may have been important for prebiotic phosphorylation.

  10. Oxidative phosphorylation revisited.

    PubMed

    Nath, Sunil; Villadsen, John

    2015-03-01

    The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic explanation for the energy coupling and ATP synthesis carried out in mitochondria and chloroplast thylakoids. The mechanism does not suffer from the flaws in Mitchell's chemiosmotic theory that have been pointed out in many studies since its first appearance 50 years ago, when it was hailed as a ground-breaking mechanistic explanation of what is perhaps the most important process in cellular energetics. The new findings fit very well with the predictions of Nath's torsional mechanism of energy transduction and ATP synthesis. It is argued that this mechanism, based on at least 15 years of experimental and theoretical work by Sunil Nath, constitutes a fundamentally different theory of the energy conversion process that eliminates all the inconsistencies in Mitchell's chemiosmotic theory pointed out by other authors. It is concluded that the energy-transducing complexes in oxidative phosphorylation and photosynthesis are proton-dicarboxylic acid anion cotransporters and not simply electrogenic proton translocators. These results necessitate revision of previous theories of biological energy transduction, coupling, and ATP synthesis. The novel molecular mechanism is extended to cover ATP synthesis in prokaryotes, in particular to alkaliphilic and haloalkaliphilic bacteria, essentially making it a complete theory addressing mechanistic, kinetic, and thermodynamic details. Finally, based on the new interpretation of oxidative phosphorylation, quantitative values for the P/O ratio, the amount of ATP generated per redox package of the reduced substrates, are calculated and compared with experimental values for fermentation on different substrates. It is our hope that the presentation of oxidative phosphorylation and photophosphorylation from a wholly new perspective will rekindle scientific discussion of a key process in bioenergetics and catalyze new avenues of research in a truly interdisciplinary field. PMID:25384602

  11. Determining in vivo Phosphorylation Sites using Mass Spectrometry

    PubMed Central

    Breitkopf, Susanne B.; Asara, John M.

    2012-01-01

    Phosphorylation is the most studied protein post-translational modification (PTM) in biological systems since it controls cell growth, proliferation, survival, etc. High resolution/high mass accuracy mass spectrometers are used to identify protein phosphorylation sites due to their speed, sensitivity, selectivity and throughput. The protocol described here focuses on two common strategies: 1) Identifying phosphorylation sites from individual proteins and small protein complexes, and 2) Identifying global phosphorylation sites from whole cell and tissue extracts. For the first, endogenous or epitope tagged proteins are typically immunopurified (IP) from cell lysates, purified via gel electrophoresis or precipitation and enzymatically digested into peptides. Samples can be optionally enriched for phosphopeptides using immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) and then analyzed by microcapillary liquid chromatography/tandem mass spectrometry (LC-MS/MS). Global phosphorylation site analyses that capture pSer/pThr/pTyr sites from biological sources sites are more resource and time-consuming and involve digesting the whole cell lysate, followed by peptide fractionation by strong cation exchange chromatography (SCX), phosphopeptide enrichment by IMAC or TiO2 and LC-MS/MS. Alternatively, one can fractionate the protein lysate by SDS-PAGE, followed by digestion, phosphopeptide enrichment and LC-MS/MS. One can also IP only phospho-tyrosine peptides using a pTyr antibody followed by LC-MS/MS. PMID:22470061

  12. Structural basis of enzymatic benzene ring reduction.

    PubMed

    Weinert, Tobias; Huwiler, Simona G; Kung, Johannes W; Weidenweber, Sina; Hellwig, Petra; Stärk, Hans-Joachim; Biskup, Till; Weber, Stefan; Cotelesage, Julien J H; George, Graham N; Ermler, Ulrich; Boll, Matthias

    2015-08-01

    In chemical synthesis, the widely used Birch reduction of aromatic compounds to cyclic dienes requires alkali metals in ammonia as extremely low-potential electron donors. An analogous reaction is catalyzed by benzoyl-coenzyme A reductases (BCRs) that have a key role in the globally important bacterial degradation of aromatic compounds at anoxic sites. Because of the lack of structural information, the catalytic mechanism of enzymatic benzene ring reduction remained obscure. Here, we present the structural characterization of a dearomatizing BCR containing an unprecedented tungsten cofactor that transfers electrons to the benzene ring in an aprotic cavity. Substrate binding induces proton transfer from the bulk solvent to the active site by expelling a Zn(2+) that is crucial for active site encapsulation. Our results shed light on the structural basis of an electron transfer process at the negative redox potential limit in biology. They open the door for biological or biomimetic alternatives to a basic chemical synthetic tool. PMID:26120796

  13. Enzymatic cascade bioreactor

    DOEpatents

    Simmons, Blake A. (San Francisco, CA); Volponi, Joanne V. (Livermore, CA); Ingersoll, David (Albuquerque, NM); Walker, Andrew (Woodinville, WA)

    2007-09-04

    Disclosed is an apparatus and method for continuously converting sucrose to .beta.-D-glucose. The method comprises a three stage enzymatic reactor in which an aqueous solution of sucrose is first converted into a solution of fructose and .alpha.-D-glucose by passing it through a porous, packed column containing an inert media on which invertase is immobilized. This solution is then sent through a second packed column containing glucose isomerase and finally a third packed column containing mutarotase. Solution temperature and pH are adjusted to maximize glucose output.

  14. Enzymatic conversion of proteins to glycoproteins.

    PubMed Central

    Pless, D D; Lennarz, W J

    1977-01-01

    The enzymatic transfer of the oligosaccharide moiety from an oligosaccharide-lipid to denatured forms of three secretory proteins--ovalbumin, alpha-lactalbumin, and ribonuclease A--has been demonstrated utilizing a membrane fraction from hen oviduct. Based on a survey of 10 proteins denatured by sulfitolysis, the presence of the tripeptide sequence -Asn-X-Thr-Ser- (X represents a variable amino acid) appears to be necessary but not sufficient for the protein to serve as acceptor in vitro. The results of this investigation also suggest that unfolding of the polypeptide chain is required in order to expose sites for carbohydrate attachment. PMID:264667

  15. Phosphorylation by PINK1 Releases the UBL Domain and Initializes the Conformational Opening of the E3 Ubiquitin Ligase Parkin

    PubMed Central

    Moussaud-Lamodière, Elisabeth L.; Dourado, Daniel F. A. R.; Flores, Samuel C.; Springer, Wolfdieter

    2014-01-01

    Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased) molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin activators through targeted drug design. PMID:25375667

  16. Phosphatidate-dependent protein phosphorylation.

    PubMed Central

    Bocckino, S B; Wilson, P B; Exton, J H

    1991-01-01

    Phosphatidate-dependent protein phosphorylation was observed in soluble extracts from rat liver, brain, lung, and testis. The phosphorylation was stimulated by free Ca2+ in the range of 360-800 nM. Incubation mixtures containing phosphatidate provided markedly different profiles of protein phosphorylation from those with phosphatidylserine plus 1,2-diolein. Phosphatidate-dependent phosphorylation of a 30-kDa protein in the soluble fraction from heart was also observed. This phosphorylation did not require Ca2+. Soluble fractions from liver, testis, brain, and lung phosphorylated the 30-kDa heart protein in a phosphatidate-dependent Ca(2+)-independent manner. We propose that part of the action of phosphatidate in cells may be mediated by a protein kinase(s). Images PMID:2068102

  17. Phosphorylation of Yellow Fever Virus NS5 alters methyltransferase activity

    PubMed Central

    Bhattacharya, Dipankar; Hoover, Spencer; Falk, Shaun P.; Weisblum, Bernard; Vestling, Martha; Striker, Rob

    2008-01-01

    Serine/threonine phosphorylation of the nonstructural protein 5 (NS5) is conserved feature of flaviviruses, but the kinase(s) responsible and function(s) remain unknown. Mass spectrometry was used to characterize phosphorylated residues of yellow fever virus (YFV) NS5 expressed in mammalian cells. Multiple different phosphopeptides were detected. Mutational and additional mass spectrometry data implicated serine 56 (S56), a conserved residue near the active site in the NS5 methyltransferase domain, as one of the phosphorylation sites. Methyltransferase activity is required to form a methylated RNA cap structure and for translation of the YFV polyprotein. We show the 2’-O- methylation reaction requires the hydroxyl side chain of S56, and replacement with a negative charge inhibits enzymatic activity. Furthermore mutational alteration of S56, S56A or S56D, prevents amplification in a viral replicon system. Collectively our data suggest phosphorylation of NS5 S56 may act to shut down capping in the viral life cycle. PMID:18757072

  18. Enzymatically active ultrathin pepsin membranes.

    PubMed

    Raaijmakers, Michiel J T; Schmidt, Thomas; Barth, Monika; Tutus, Murat; Benes, Nieck E; Wessling, Matthias

    2015-05-11

    Enzymatically active proteins enable efficient and specific cleavage reactions of peptide bonds. Covalent coupling of the enzymes permits immobilization, which in turn reduces autolysis-induced deactivation. Ultrathin pepsin membranes were prepared by facile interfacial polycondensation of pepsin and trimesoyl chloride. The pepsin membrane allows for simultaneous enzymatic conversion and selective removal of digestion products. The large water fluxes through the membrane expedite the transport of large molecules through the pepsin layers. The presented method enables the large-scale production of ultrathin, cross-linked, enzymatically active membranes. PMID:25779668

  19. Enzymatic and non-enzymatic mechanisms of dimesna metabolism.

    PubMed

    Cutler, Murray J; Velenosi, Thomas J; Bodalia, Ankur; House, Andrew A; Urquhart, Bradley L; Freeman, David J

    2015-03-01

    The chemical reduction of the disulfide homodimer dimesna to its constituent mesna moieties is essential for its mitigation of nephrotoxicity associated with cisplatin and ifosfamide anticancer therapies and enhancement of dialytic clearance of the cardiovascular risk factor homocysteine. The objective of this study was to investigate potential enzymatic and non-enzymatic mechanisms of intracellular dimesna reduction. Similar to endogenous intracellular disulfides, dimesna undergoes thiol-disulfide exchange with thiolate anion-forming sulfhydryl groups via the two-step SN2 reaction. Determination of equilibrium constants of dimesna reduction when mixed with cysteine or glutathione provided a mechanistic explanation for dramatic cysteine and homocysteine depletion, but sparing of the endogenous antioxidant glutathione, previously observed during mesna therapy. Dimesna was reduced by recombinant enzymes of the thioredoxin system; however, oxidation of NADPH by the glutaredoxin system was only observed in the presence of combined dimesna and reduced glutathione, suggesting formation of oxidized glutathione following an initial non-enzymatic reduction of dimesna. Production of mesna by enzymatic and non-enzymatic mechanisms in HeLa cell lysate following dimesna incubation was demonstrated by a loss in mesna production following protein denaturation and prediction of residual non-enzymatic mesna production by mathematical modeling of thiol-disulfide exchange reactions. Reaction modeling also revealed that mixed disulfides make up a significant proportion of intracellular thiols, supporting their role in providing additional nephroprotection, independent of direct platinum conjugation. PMID:25488427

  20. Homogeneous, Heterogeneous, and Enzymatic Catalysis.

    ERIC Educational Resources Information Center

    Oyama, S. Ted; Somorjai, Gabor A.

    1988-01-01

    Discusses three areas of catalysis: homegeneous, heterogeneous, and enzymatic. Explains fundamentals and economic impact of catalysis. Lists and discusses common industrial catalysts. Provides a list of 107 references. (MVL)

  1. How Phosphotransferase System-Related Protein Phosphorylation Regulates Carbohydrate Metabolism in Bacteria†

    PubMed Central

    Deutscher, Josef; Francke, Christof; Postma, Pieter W.

    2006-01-01

    The phosphoenolpyruvate(PEP):carbohydrate phosphotransferase system (PTS) is found only in bacteria, where it catalyzes the transport and phosphorylation of numerous monosaccharides, disaccharides, amino sugars, polyols, and other sugar derivatives. To carry out its catalytic function in sugar transport and phosphorylation, the PTS uses PEP as an energy source and phosphoryl donor. The phosphoryl group of PEP is usually transferred via four distinct proteins (domains) to the transported sugar bound to the respective membrane component(s) (EIIC and EIID) of the PTS. The organization of the PTS as a four-step phosphoryl transfer system, in which all P derivatives exhibit similar energy (phosphorylation occurs at histidyl or cysteyl residues), is surprising, as a single protein (or domain) coupling energy transfer and sugar phosphorylation would be sufficient for PTS function. A possible explanation for the complexity of the PTS was provided by the discovery that the PTS also carries out numerous regulatory functions. Depending on their phosphorylation state, the four proteins (domains) forming the PTS phosphorylation cascade (EI, HPr, EIIA, and EIIB) can phosphorylate or interact with numerous non-PTS proteins and thereby regulate their activity. In addition, in certain bacteria, one of the PTS components (HPr) is phosphorylated by ATP at a seryl residue, which increases the complexity of PTS-mediated regulation. In this review, we try to summarize the known protein phosphorylation-related regulatory functions of the PTS. As we shall see, the PTS regulation network not only controls carbohydrate uptake and metabolism but also interferes with the utilization of nitrogen and phosphorus and the virulence of certain pathogens. PMID:17158705

  2. Enzymatic hydrolysis of molasses.

    PubMed

    Najafpour, Ghasem D; Shan, Cheong Poi

    2003-01-01

    Kinetic studies of the enzymatic hydrolysis of molasses were conducted using glucoamylase. Central Sugar Refinery SDN BHD contains 13-20% glucose. The molasses was diluted and the kinetic experiments were conducted at 67 degrees C with 100-1000 mg/l of glucoamylase. The glucose contents of the molasses were enhanced after hydrolysis of molasses solution with 1000 mg/l glucoamylase. A Lineweaver-Burk plot was obtained based on enzyme kinetic data. The rate constant, Km and maximum reaction rate, Vmax for 500 mg/l of glucoamylase were 100 mmol/l (18 g/l) and 5 mmol/l min (0.9 g/l min), respectively. The maximum reaction rate, Vmax for 1000 mg/l of glucoamylase was doubled, to 100 mmol/l (18 g/l) and the rate constant, Km was the same for 500 mg/l of glucoamylase. The substrate inhibition model was noncompetitive based on the resulting Lineweaver-Burk plot for enzyme concentration of 500 and 1000 mg/l. PMID:12421015

  3. Phosphoproteomic Analysis of the Mouse Brain Cytosol Reveals a Predominance of Protein Phosphorylation in Regions of Intrinsic Sequence Disorder

    Microsoft Academic Search

    Mark O. Collins; Lu Yu; Iain Campuzano; Seth G. N. Grant; Jyoti S. Choudhary

    2008-01-01

    We analyzed the mouse forebrain cytosolic phosphopro- teome using sequential (protein and peptide) IMAC puri- fications, enzymatic dephosphorylation, and targeted tan- dem mass spectrometry analysis strategies. In total, using complementary phosphoenrichment and LC- MS\\/MS strategies, 512 phosphorylation sites on 540 non- redundant phosphopeptides from 162 cytosolic phospho- proteins were characterized. Analysis of protein domains and amino acid sequence composition of

  4. Phosphorylation of chicken growth hormone

    SciTech Connect

    Aramburo, C.; Montiel, J.L. (Universidad Nacional Autonoma de Mexico (Mexico)); Donoghue, D.; Scanes, C.G. (Rutgers Univ., New Brunswick, NJ (USA)); Berghman, L.R. (Laboratory for Neuroendocrinology and Immunological Biotechnology, Louvain (Belgium))

    1990-01-01

    The possibility that chicken growth hormone (cGH) can be phosphorylated has been examined. Both native and biosynthetic cGH were phosphorylated by cAMP-dependent protein kinase (and {gamma}-{sup 32}P-ATP). The extent of phosphorylation was however less than that observed with ovine prolactin. Under the conditions employed, glycosylated cGH was not phosphorylated. Chicken anterior pituitary cells in primary culture were incubated in the presence of {sup 32}P-phosphate. Radioactive phosphate was incorporated in vitro into the fraction immunoprecipitable with antisera against cGH. Incorporation was increased with cell number and time of incubation. The presence of GH releasing factor (GRF) increased the release of {sup 32}P-phosphate labeled immunoprecipitable GH into the incubation media but not content of immunoprecipitable GH in the cells. The molecular weight of the phosphorylated immunoreactive cGH in the cells corresponded to cGH dimer.

  5. Cysteine phosphorylation of the glucose transporter of Escherichia coli.

    PubMed

    Meins, M; Jenö, P; Müller, D; Richter, W J; Rosenbusch, J P; Erni, B

    1993-06-01

    The glucose transporter (IIBCGlc/IIAGlc complex) of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported sugar. The IIAGlc subunit transfers the phosphoryl group from the phosphoryl carrier protein P-HPr to the IIBCGlc subunit. IIBCGlc translocates and phosphorylates glucose. The site of IIBCGlc phosphorylation is cysteine 421 as shown by mass spectrometric and biochemical analyses of phosphorylated peptides. Site-directed mutagenesis of Cys421 (C421S) afforded a stable but completely inactive protein (Nuoffer, C., Zanolari, B., and Erni, B. (1988) J. Biol. Chem. 263, 6647-6655). Cys421 is located in the C-terminal cytoplasmic domain of the IIBCGlc subunit in a sequence context (LDACITRL) which is well conserved in other transporters of the bacterial phosphotransferase system. Phosphocysteine has been shown previously to be the catalytic intermediate of the mannitol transporter (Pas, H. H., Meyer, G. H., Kruizinga, W. H., Tamminga, K. S., van Weeghel, R. P., and Robillard, G. T. PMID:8505292

  6. Oxidative and Photosynthetic Phosphorylation Mechanisms

    ERIC Educational Resources Information Center

    Wang, Jui H.

    1970-01-01

    Proposes a molecular mechanism for the coupling of phosphorylation to electron transport in both mitochondria and chloroplasts. Justifies the proposed reaction schemes in terms of thermodynamics and biochemical data. Suggests how areobic respiration could have evolved. (EB)

  7. IGF1 regulates PKM2 function through Akt phosphorylation.

    PubMed

    Salani, Barbara; Ravera, Silvia; Amaro, Adriana; Salis, Annalisa; Passalacqua, Mario; Millo, Enrico; Damonte, Gianluca; Marini, Cecilia; Pfeffer, Ulrich; Sambuceti, Gianmario; Cordera, Renzo; Maggi, Davide

    2015-05-19

    Pyruvate kinase M2 (PKM2) acts at the crossroad of growth and metabolism pathways in cells. PKM2 regulation by growth factors can redirect glycolytic intermediates into key biosynthetic pathway. Here we show that IGF1 can regulate glycolysis rate, stimulate PKM2 Ser/Thr phosphorylation and decrease cellular pyruvate kinase activity. Upon IGF1 treatment we found an increase of the dimeric form of PKM2 and the enrichment of PKM2 in the nucleus. This effect was associated to a reduction of pyruvate kinase enzymatic activity and was reversed using metformin, which decreases Akt phosphorylation. IGF1 induced an increased nuclear localization of PKM2 and STAT3, which correlated with an increased HIF1?, HK2, and GLUT1 expression and glucose entrapment. Metformin inhibited HK2, GLUT1, HIF-1? expression and glucose consumption. These findings suggest a role of IGFIR/Akt axis in regulating glycolysis by Ser/Thr PKM2 phosphorylation in cancer cells. PMID:25790097

  8. Phosphorylation and Glycosylation of Nucleoporins

    Microsoft Academic Search

    Mill W. Miller; Maria R. Caracciolo; William K. Berlin; John A. Hanover

    1999-01-01

    The nuclear pore complex mediates macromolecular transport between the nucleus and cytoplasm. Many nuclear pore components (nucleoporins) are modified by both phosphate and O-linked N-acetylglucosamine (O-GlcNAc). Among its many functions, protein phosphorylation plays essential roles in cell cycle progression. The role of O-GlcNAc addition is unknown. Here, levels of nucleoporin phosphorylation and glycosylation during cell cycle progression are examined. Whereas

  9. DNA-dependent phosphorylation of histone H2A.X during nucleosome assembly in Xenopus laevis oocytes: involvement of protein phosphorylation in nucleosome spacing.

    PubMed

    Kleinschmidt, J A; Steinbeisser, H

    1991-10-01

    ATP is required for physiological nucleosome alignment in chromatin reconstituted from high-speed nuclear supernatants of Xenopus laevis oocytes. Here we show that during in vitro nucleosome assembly the histone variant H2A.X becomes phosphorylated upon transfer onto DNA, a process which is also observed in vivo. Histone H2A.X phosphorylation increases in the early phase of the assembly reaction, reaching a steady state after approximately 16 min and is maintained with a half-life of the phosphate groups of approximately 2 h. After 6 h, the overall phosphorylation state of H2A.X is reduced, indicating that the phosphorylation-dephosphorylation ratio decreases considerably over time. Addition of alkaline phosphatase leads to a persistently lowered state of H2A.X phosphorylation, in contrast to other nuclear phosphoproteins which undergo rapid rephosphorylation. This suggests that H2A.X phosphorylation is a unique step in the histone-to-DNA transfer process. Selective inhibition of DNA-dependent phosphorylation of H2A.X and of other proteins causes a loss of the physiological 180 bp spacing. PMID:1915279

  10. Regulation of striatal tyrosine hydroxylase phosphorylation by acute and chronic haloperidol.

    PubMed

    Håkansson, Kerstin; Pozzi, Laura; Usiello, Alessandro; Haycock, John; Borrelli, Emiliana; Fisone, Gilberto

    2004-08-01

    The typical neuroleptic haloperidol increases the state of phosphorylation and activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines. Here we show that the increases in TH phosphorylation produced by haloperidol at Ser31 and Ser40, two sites critically involved in the regulation of enzymatic activity, are abolished in dopamine D2 receptor-null mice and mimicked by the selective dopamine D2 receptor antagonist, eticlopride. Moreover, the ability of haloperidol and eticlopride to stimulate phosphorylation at both seryl residues is prevented by treatment with SL327, a compound that blocks activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). We also show that chronic administration of haloperidol reduces the basal levels of phosphoSer31-TH and decreases the ability of the drug to stimulate Ser40 phosphorylation. These results provide a model accounting for the stimulation exerted by haloperidol on dopamine synthesis. According to this model, haloperidol increases TH activity via blockade of dopamine D2 receptors, disinhibition of dopaminergic projection neurons and ERK1/2-dependent phosphorylation of TH at Ser31 and Ser40. These studies also show that lower levels of phosphorylated TH are associated with chronic neuroleptic treatment and may be related to depressed dopaminergic transmission in nigrostriatal neurons. PMID:15305880

  11. Radical mechanisms of enzymatic catalysis.

    PubMed

    Frey, P A

    2001-01-01

    Two classes of enzymatic mechanisms that proceed by free radical chemistry initiated by the 5'-deoxyadenosyl radical are discussed. In the first class, the mechanism of the interconversion of L-lysine and L-beta-lysine catalyzed by lysine 2,3-aminomutase (LAM) involves four radicals, three of which have been spectroscopically characterized. The reversible formation of the 5'-deoxyadenosyl radical takes place by the chemical cleavage of S-adenosylmethionine (SAM) reacting with the [4Fe-4S]+ center in LAM. In other reactions of SAM with iron-sulfur proteins, SAM is irreversibly consumed to generate the 5'-deoxyadenosyl radical, which activates an enzyme by abstracting a hydrogen atom from an enzymatic glycyl residue to form a glycyl radical. The glycyl radical enzymes include pyruvate formate-lyase, anaerobic ribonucleotide reductase from Escherichia coli, and benzylsuccinate synthase. Biotin synthase and lipoate synthase are SAM-dependent [4Fe-4S] proteins that catalyze the insertion of sulfur into unactivated C-H bonds, which are cleaved by the 5'-deoxyadenosyl radical from SAM. In the second class of enzymatic mechanisms using free radicals, adenosylcobalamin-dependent reactions, the 5'-deoxyadenosyl radical arises from homolytic cleavage of the cobalt-carbon bond, and it initiates radical reactions by abstracting hydrogen atoms from substrates. Three examples are described of suicide inactivation through the formation of exceptionally stable free radicals at enzymatic active sites. PMID:11395404

  12. 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...

  13. Non-enzymatic oxidation of NADH by quinones

    NASA Astrophysics Data System (ADS)

    Scherbak, Nikolai; Strid, Åke; Eriksson, Leif A.

    2005-10-01

    Non-enzymatic oxidation of NADH by a large number of different quinones has been explored both theoretically and experimentally. It is concluded that the smaller benzo- and naphtho-quinones are capable of oxidising NADH in aqueous solution, whereas the larger anthraquinone is not. The mechanisms of stepwise electron and proton transfers are explored, and ruled out in favour of direct hydride transfer. For menadione (2-methyl-1,4-naphthoquinone), no reaction is observed experimentally; theoretically we find that there is a very close balance between the energetic cost of hydride removal from NADH and the energy gain of formation of the menadione semiquinone radical anion.

  14. Interphase phosphorylation of lamin A

    PubMed Central

    Kochin, Vitaly; Shimi, Takeshi; Torvaldson, Elin; Adam, Stephen A.; Goldman, Anne; Pack, Chan-Gi; Melo-Cardenas, Johanna; Imanishi, Susumu Y.; Goldman, Robert D.; Eriksson, John E.

    2014-01-01

    ABSTRACT Nuclear lamins form the major structural elements that comprise the nuclear lamina. Loss of nuclear structural integrity has been implicated as a key factor in the lamin A/C gene mutations that cause laminopathies, whereas the normal regulation of lamin A assembly and organization in interphase cells is still undefined. We assumed phosphorylation to be a major determinant, identifying 20 prime interphase phosphorylation sites, of which eight were high-turnover sites. We examined the roles of these latter sites by site-directed mutagenesis, followed by detailed microscopic analysis – including fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and nuclear extraction techniques. The results reveal three phosphorylation regions, each with dominant sites, together controlling lamin A structure and dynamics. Interestingly, two of these interphase sites are hyper-phosphorylated in mitotic cells and one of these sites is within the sequence that is missing in progerin of the Hutchinson-Gilford progeria syndrome. We present a model where different phosphorylation combinations yield markedly different effects on the assembly, subunit turnover and the mobility of lamin A between, and within, the lamina, the nucleoplasm and the cytoplasm of interphase cells. PMID:24741066

  15. Interphase phosphorylation of lamin A.

    PubMed

    Kochin, Vitaly; Shimi, Takeshi; Torvaldson, Elin; Adam, Stephen A; Goldman, Anne; Pack, Chan-Gi; Melo-Cardenas, Johanna; Imanishi, Susumu Y; Goldman, Robert D; Eriksson, John E

    2014-06-15

    Nuclear lamins form the major structural elements that comprise the nuclear lamina. Loss of nuclear structural integrity has been implicated as a key factor in the lamin A/C gene mutations that cause laminopathies, whereas the normal regulation of lamin A assembly and organization in interphase cells is still undefined. We assumed phosphorylation to be a major determinant, identifying 20 prime interphase phosphorylation sites, of which eight were high-turnover sites. We examined the roles of these latter sites by site-directed mutagenesis, followed by detailed microscopic analysis - including fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and nuclear extraction techniques. The results reveal three phosphorylation regions, each with dominant sites, together controlling lamin A structure and dynamics. Interestingly, two of these interphase sites are hyper-phosphorylated in mitotic cells and one of these sites is within the sequence that is missing in progerin of the Hutchinson-Gilford progeria syndrome. We present a model where different phosphorylation combinations yield markedly different effects on the assembly, subunit turnover and the mobility of lamin A between, and within, the lamina, the nucleoplasm and the cytoplasm of interphase cells. PMID:24741066

  16. The Enzymatic Oxidation of Graphene Oxide

    PubMed Central

    Kotchey, Gregg P.; Allen, Brett L.; Vedala, Harindra; Yanamala, Naveena; Kapralov, Alexander A.; Tyurina, Yulia Y.; Klein-Seetharaman, Judith; Kagan, Valerian E.; Star, Alexander

    2011-01-01

    Two-dimensional graphitic carbon is a new material with many emerging applications, and studying its chemical properties is an important goal. Here, we reported a new phenomenon – the enzymatic oxidation of a single layer of graphitic carbon by horseradish peroxidase (HRP). In the presence of low concentrations of hydrogen peroxide (~40 µM), HRP catalyzed the oxidation of graphene oxide, which resulted in the formation of holes on its basal plane. During the same period of analysis, HRP failed to oxidize chemically reduced graphene oxide (RGO). The enzymatic oxidation was characterized by Raman, UV-Vis, EPR and FT-IR spectroscopy, TEM, AFM, SDS-PAGE, and GC-MS. Computational docking studies indicated that HRP was preferentially bound to the basal plane rather than the edge for both graphene oxide and RGO. Due to the more dynamic nature of HRP on graphene oxide, the heme active site of HRP was in closer proximity to graphene oxide compared to RGO, thereby facilitating the oxidation of the basal plane of graphene oxide. We also studied the electronic properties of the reduced intermediate product, holey reduced graphene oxide (hRGO), using field-effect transistor (FET) measurements. While RGO exhibited a V-shaped transfer characteristic similar to a single layer of graphene that was attributed to its zero band gap, hRGO demonstrated a p-type semiconducting behavior with a positive shift in the Dirac points. This p-type behavior rendered hRGO, which can be conceptualized as interconnected graphene nanoribbons, as a potentially attractive material for FET sensors. PMID:21344859

  17. Integrated enzymatic reactions and analysis

    Microsoft Academic Search

    Ernst Wehtje; Patrick Adlercreutz; Bo Mattiasson

    1992-01-01

    Summary  Enzymatic reactions were performed in a modified auto-injector unit of a Shimadzu HPLC system. The reactions were analyzed\\u000a by automated injections directly into the HPLC separation system. Two reactions were studied, and the enzymes mandelonitrile\\u000a lyase and ?-chymotrypsin were immobilized by adsorption onto a solid support, e.g., Celite and Chromosorb. The reactions were\\u000a performed in various organic solvents e.g., diisopropyl

  18. Human and Quail Aromatase Activity Is Rapidly and Reversibly Inhibited by Phosphorylating Conditions

    PubMed Central

    Harada, Nobuhiro; Balthazart, Jacques; Cornil, Charlotte A.

    2011-01-01

    Besides their slow genomic actions, estrogens also induce rapid physiological responses. To be functionally relevant, these effects must be associated with rapid changes in local concentrations of estrogens. Rapid changes in aromatase activity (AA) controlled by calcium-dependent phosphorylations of the enzyme can alter in a rapid manner local estrogen concentrations, but so far this mechanism was identified only in the avian (quail) brain. We show here that AA is also rapidly down-regulated by phosphorylating conditions in quail ovary homogenates and in various cell lines transfected with human aromatase (HEK 293, Neuro2A, and C6). Enzymatic activity was also rapidly inhibited after depolarization of aromatase-expressing HEK 293 cells with 100 mm KCl, and activity was fully restored when cells returned to control conditions. Western blot analysis demonstrated that the reduction of enzymatic activity is not due to protein degradation. We next investigated by site-directed mutagenesis the potential implication in the control of AA of specific aromatase residues identified by bioinformatic analysis. Mutation of the amino acids S118, S247, S267, T462, T493, or S497 to alanine, alone or in combination, did not block the rapid inhibition of enzymatic activity induced by phosphorylating conditions, but basal AA was markedly decreased in the S118A mutant. Altogether, these results demonstrate that the rapid inhibition of AA is a widespread and fully reversible process and that phosphorylation of specific residues modulate AA. These processes provide a new general mechanism by which local estrogen concentration can be rapidly altered in the brain and other tissues. PMID:21914772

  19. Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants

    PubMed Central

    Bekešová, Slávka; Komis, George; K?enek, Pavel; Vyplelová, Petra; Ove?ka, Miroslav; Luptov?iak, Ivan; Illés, Peter; Kucha?ová, Anna; Šamaj, Jozef

    2015-01-01

    The aim of the present study is to rationalize acrylamide pendant Phos-Tag™ in-gel discrimination of phosphorylated and non-phosphorylated plant protein species with standard immunoblot analysis, and optimize sample preparation, efficient electrophoretic separation and transfer. We tested variants of the method including extraction buffers suitable for preservation of phosphorylated protein species in crude extracts from plants and we addressed the importance of the cation (Mn2+ or Zn2+) used in the gel recipe for efficient transfer to PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of Medicago sativa stress-induced mitogen activated protein kinase (SIMK) in stress-treated wild type plants and transgenic SIMKK RNAi line. We further show the hyperosmotically-induced phosphorylation of the previously uncharacterized HvMPK4 of barley. The method is validated using inducible phosphorylation of barley and wheat ?-tubulin and of Arabidopsis MPK6. Acrylamide pendant Phos-Tag™offers a flexible tool for studying protein phosphorylation in crops and Arabidopsis circumventing radioactive labeling and the use of phosphorylation specific antibodies.

  20. Monitoring protein phosphorylation by acrylamide pendant Phos-Tag™ in various plants.

    PubMed

    Bekešová, Slávka; Komis, George; K?enek, Pavel; Vyplelová, Petra; Ove?ka, Miroslav; Luptov?iak, Ivan; Illés, Peter; Kucha?ová, Anna; Šamaj, Jozef

    2015-01-01

    The aim of the present study is to rationalize acrylamide pendant Phos-Tag™ in-gel discrimination of phosphorylated and non-phosphorylated plant protein species with standard immunoblot analysis, and optimize sample preparation, efficient electrophoretic separation and transfer. We tested variants of the method including extraction buffers suitable for preservation of phosphorylated protein species in crude extracts from plants and we addressed the importance of the cation (Mn(2+) or Zn(2+)) used in the gel recipe for efficient transfer to PVDF membranes for further immunoblot analysis. We demonstrate the monitoring of Medicago sativa stress-induced mitogen activated protein kinase (SIMK) in stress-treated wild type plants and transgenic SIMKK RNAi line. We further show the hyperosmotically-induced phosphorylation of the previously uncharacterized HvMPK4 of barley. The method is validated using inducible phosphorylation of barley and wheat ?-tubulin and of Arabidopsis MPK6. Acrylamide pendant Phos-Tag™offers a flexible tool for studying protein phosphorylation in crops and Arabidopsis circumventing radioactive labeling and the use of phosphorylation specific antibodies. PMID:26029234

  1. SYMPOSIUM ON PLANT PROTEIN PHOSPHORYLATION

    SciTech Connect

    JOHN C WALKER

    2011-11-01

    Protein phosphorylation and dephosphorylation play key roles in many aspects of plant biology, including control of cell division, pathways of carbon and nitrogen metabolism, pattern formation, hormonal responses, and abiotic and biotic responses to environmental signals. A Symposium on Plant Protein Phosphorylation was hosted on the Columbia campus of the University of Missouri from May 26-28, 2010. The symposium provided an interdisciplinary venue at which scholars studying protein modification, as it relates to a broad range of biological questions and using a variety of plant species, presented their research. It also provided a forum where current international challenges in studies related to protein phosphorylation could be examined. The symposium also stimulated research collaborations through interactions and networking among those in the research community and engaged students and early career investigators in studying issues in plant biology from an interdisciplinary perspective. The proposed symposium, which drew 165 researchers from 13 countries and 21 States, facilitated a rapid dissemination of acquired knowledge and technical expertise regarding protein phosphorylation in plants to a broad range of plant biologists worldwide.

  2. Large-scale determination of absolute phosphorylation stoichiometries in human cells by motif-targeting quantitative proteomics.

    PubMed

    Tsai, Chia-Feng; Wang, Yi-Ting; Yen, Hsin-Yung; Tsou, Chih-Chiang; Ku, Wei-Chi; Lin, Pei-Yi; Chen, Hsuan-Yu; Nesvizhskii, Alexey I; Ishihama, Yasushi; Chen, Yu-Ju

    2015-01-01

    Our ability to model the dynamics of signal transduction networks will depend on accurate methods to quantify levels of protein phosphorylation on a global scale. Here we describe a motif-targeting quantitation method for phosphorylation stoichiometry typing. Proteome-wide phosphorylation stoichiometry can be obtained by a simple phosphoproteomic workflow integrating dephosphorylation and isotope tagging with enzymatic kinase reaction. Proof-of-concept experiments using CK2-, MAPK- and EGFR-targeting assays in lung cancer cells demonstrate the advantage of kinase-targeted complexity reduction, resulting in deeper phosphoproteome quantification. We measure the phosphorylation stoichiometry of >1,000 phosphorylation sites including 366 low-abundance tyrosine phosphorylation sites, with high reproducibility and using small sample sizes. Comparing drug-resistant and sensitive lung cancer cells, we reveal that post-translational phosphorylation changes are significantly more dramatic than those at the protein and messenger RNA levels, and suggest potential drug targets within the kinase-substrate network associated with acquired drug resistance. PMID:25814448

  3. FOR THE RECORD Lipopolysaccharide phosphorylating enzymes encoded

    E-print Network

    Srinivasan, N.

    conserved components, namely, lipid A, core (comprised of phosphoryl derivatives of sugar residues) and the O-antigen. The phosphoryl substituents of the core serve to stabilize the outer membrane by means

  4. Phosphorylation of rat brain choline acetyltransferase and its relationship to enzyme activity.

    PubMed

    Schmidt, B M; Rylett, R J

    1993-11-01

    Choline acetyltransferase catalyzes the formation of acetylcholine from choline and acetyl-CoA in cholinergic neurons. The present study examined conditions for modulation of kinase-mediated phosphorylation of this enzyme. By using a monospecific polyclonal rabbit anti-human choline acetyltransferase antibody to immunoprecipitate cytosolic and membrane-associated subcellular pools of enzyme from rat hippocampal synaptosomes, we determined that only the cytosolic fraction of the enzyme (67,000 +/- 730 daltons) was phosphorylated under basal, unstimulated conditions. The quantity of this endogenous phosphoprotein was dependent, in part, upon the level of intracellular calcium, with 32Pi incorporation into the enzyme in nerve terminals incubated in nominally calcium-free medium only 43 +/- 7% of control. The corresponding enzymatic activity of cytosolic choline acetyltransferase did not appear to be altered by lowered cytosolic calcium, whereas membrane-associated choline acetyltransferase activity was decreased to 58 +/- 11% of control. Depolarization of synaptosomes with 50 microM veratridine neither altered the extent of phosphorylation or specific activity of cytosolic choline acetyltransferase, nor induced detectable phosphorylation of membrane-associated choline acetyltransferase, although the specific activity of the membrane-associated enzyme was increased to 132 +/- 5% of control. In summary, phosphorylation of choline acetyltransferase does not appear to regulate cholinergic neurotransmission by a direct action on catalytic activity of the enzyme. PMID:8228993

  5. Mammalian liver cytochrome c is tyrosine-48 phosphorylated in vivo, inhibiting mitochondrial respiration

    PubMed Central

    Yu, Hong; Lee, Icksoo; Salomon, Arthur R.; Yu, Kebing; Hüttemann, Maik

    2009-01-01

    Cytochrome c (Cyt c) is part of the mitochondrial electron transport chain (ETC), accepting electrons from bc1 complex and transferring them to cytochrome c oxidase (CcO). The ETC generates the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. In addition, the release of Cyt c from the mitochondria often commits a cell to undergo apoptosis. Considering its central role in life (respiration) and death (apoptosis) decisions one would expect tight regulation of Cyt c function. Reversible phosphorylation is a main cellular regulatory mechanism, but the effect of cell signaling targeting the mitochondrial oxidative phosphorylation system is not well understood, and only a small number of proteins that can be phosphorylated have been identified to date. We have recently shown that Cyt c isolated from cow heart tissue is phosphorylated on tyrosine 97 in vivo, which leads to inhibition of respiration in the reaction with CcO. In this study we isolated Cyt c from a different organ, cow liver, under conditions preserving the physiological phosphorylation state. Western analysis with a phospho-tyrosine specific antibody suggested that liver Cyt c is phosphorylated. Surprisingly, the phosphorylation site was unambiguously assigned to Tyr-48 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS), and not to the previously identified phospho-Tyr-97 in cow heart. As is true of Tyr-97, Tyr-48 is conserved in eukaryotes. As one possible consequence of Tyr-48 phosphorylation we analyzed the in vitro reaction kinetics with isolated cow liver CcO revealing striking differences. Maximal turnover of Tyr-48 phosphorylated Cyt c was 3.7 s?1 whereas dephosphorylation resulted in a 2.2 fold increase in activity to 8.2 s?1. Effects of Tyr-48 phosphorylation based on the Cyt c crystal structure are discussed. PMID:18471988

  6. Enzymatic glycosylation of multivalent scaffolds.

    PubMed

    Bojarová, Pavla; Rosencrantz, Ruben R; Elling, Lothar; K?en, Vladimír

    2013-06-01

    The design of glycoclusters, glycodendrimers, glycopolymers and other complex glycostructures that mimic the multivalent carbohydrate display on the cell surface is of immense interest for diagnosis and therapy. This review presents a detailed insight into the exciting possibilities of multiple glycosylation using enzymes, particularly glycosyltransferases (EC 2.4). A representative choice of available scaffolds for the enzyme action is practically infinite and comprises synthetic polymers, carbosilane dendrimers, multiantennary glycans or hyperbranched conjugates. The introduced glyco-patterns range from common sialyl Lewis(x) and sialyl lacto-chains to chemically functionalized carbohydrate units for detection purposes. The possibilities of in vitro enzymatic production of N- and O-glycans and other natural polymers are also discussed. In harmony with their natural tasks, glycosyltransferases may in vitro complete the imperfect glycosylation pattern of proteins, recombinantly produced in pro- and eukaryotic hosts. What is more, the required enzymatic battery may be directly co-expressed with the protein, in order to elegantly accomplish the production of eukaryotic glycans. Ingenious metabolic labeling enables facile imaging of glycostructures. The boom of glycoarray technology opens vast possibilities in high-throughput screening for novel enzymes and substrate specificities as well as in the synthesis. Though there is still a long way until the Nature's ideal of multivalent glycans is achievable in the laboratory, the sketched pathways to multivalent glycostructures open tremendous possibilities for the future glycobiological research. PMID:23348496

  7. Enzymatic synthesis of DNA on glycerol nucleic acid templates without stable duplex formation between

    E-print Network

    Heller, Eric

    Enzymatic synthesis of DNA on glycerol nucleic acid templates without stable duplex formation for review May 8, 2007) Glycerol nucleic acid (GNA) is an interesting alternative base- pairing system based is not required for template-dependent polymerization. information transfer polymerase Nucleic acid analogs

  8. A hydrocarbon ruler measures palmitate in the enzymatic acylation of endotoxin

    E-print Network

    Bishop, Russell

    A hydrocarbon ruler measures palmitate in the enzymatic acylation of endotoxin Victoria E Ahn1 that helps pathogenic bacteria to evade the host immune response by transferring a palmitate chain from the hydrocarbon ruler mechanism for palmitate recognition. A striking structural similarity between Pag

  9. Locations and immunoreactivities of phosphorylation sites on bovine and porcine tau proteins and a PHF-tau fragment.

    PubMed

    Poulter, L; Barratt, D; Scott, C W; Caputo, C B

    1993-05-01

    Tau protein is a phosphorylated neuronal microtubule-associated protein. Tau protein is also present in the major pathological lesions of Alzheimer's disease in an insoluble hyperphosphorylated state as paired helical filaments (PHFs). We have investigated the phosphorylation state of control taus and a fragment of PHF-tau. Tau samples were digested with protease, separated by reversed-phase high-performance liquid chromatography, and analyzed by mass spectrometry and Edman microsequencing. The serine homologous with S404 of human tau 441 was phosphorylated on bovine and porcine tau and up to two phosphates were present on a peptide of amino acids 182-240 of bovine tau (193-251 of human tau 441). The serine within the KSPV motif was not phosphorylated on bovine or porcine tau. PHF-tau fragments, isolated from pronase-treated PHFs encompassed a 93-amino acid region within the microtubule binding domain. Enzymatic digestion and mass spectrometric analysis showed no phosphate was present and a second carboxyl terminus was identified at E380. Antibodies T3P and SMI34, which recognize PHF-tau and peptides phosphorylated at the sequence KSPV, both reacted with bovine and porcine tau even though the KSPV sequence was not phosphorylated. These data indicate that the 93-amino acid sequence of F5.5 tau from PHFs is not phosphorylated, and the serine equivalent to S404 of human tau is phosphorylated in bovine and porcine tau. Antibodies T3P and SMI34 react with phosphorylated epitopes that are not unique to PHF-tau and that are not necessarily at the KSPV site. PMID:8486651

  10. beta. -Sulfopyruvate: chemical and enzymatic syntheses and enzymatic assay

    SciTech Connect

    Weinstein, C.L.; Griffith, O.W.

    1986-01-01

    BETA-Sulfopyruvic acid (2-carboxy-2-oxoethanesulfonic acid) is prepared in greater than 90% yield by reaction of bromopyruvic acid with sodium sulfite. ..beta..-(/sup 35/S)Sulfopyruvate is prepared by transamination between (/sup 35/)cysteinesulfonate (cysteate) and ..cap alpha..-ketoglutarate using mitochondrial aspartate aminotransferase isolated from rat liver. Following either chemical or enzymatic synthesis the crude reaction product is conveniently purified by chromatography on Dowex 1; ..beta..-sulfopyruvate is isolated as the stable, water-soluble dilithium salt. ..beta..-Sulfopyruvate is shown to be an alternative substrate of mitochondrial malate dehydrogenase; in the presence of 0.25 mM NADH, ..beta..-sulfopyruvate is reduced with an apparent K/sub m/ of 6.3 mM and a V/sub max/ equal to about 40% of that observed with oxaloacetate. This finding forms the basis of a convenient spectrophotometric assay of ..beta..-sulfopyruvate.

  11. Ethanol from biomass by enzymatic hydrolysis

    Microsoft Academic Search

    1988-01-01

    Enzymes are biological catalysts that generally are designed to do one job well, but to do one job only. Therefore, the enzymes that catalyze the hydrolysis of cellulose to sugar do not break down the sugars. Thus, enzymatic processes are capable of yields approaching 100%. Enzymatic hydrolysis processes have been under development for only 10 years. Although improvements have been

  12. Phosphorylation and metabolism of sucrose and its five linkage-isomeric alpha-D-glucosyl-D-fructoses by Klebsiella pneumoniae.

    PubMed

    Thompson, J; Robrish, S A; Pikis, A; Brust, A; Lichtenthaler, F W

    2001-03-22

    Not only sucrose but the five isomeric alpha-D-glucosyl-D-fructoses trehalulose, turanose, maltulose, leucrose, and palatinose are utilized by Klebsiella pneumoniae as energy sources for growth, thereby undergoing phosphorylation by a phosphoenolpyruvate-dependent phosphotransferase system uniformly at 0-6 of the glucosyl moiety. Similarly, maltose, isomaltose, and maltitol, when exposed to these conditions, are phosphorylated regiospecifically at O-6 of their non-reducing glucose portion. The structures of these novel compounds have been established unequivocally by enzymatic analysis, acid hydrolysis, FAB negative-ion spectrometry, and 1H and 13C NMR spectroscopy. In cells of K. pneumoniae, hydrolysis of sucrose 6-phosphate is catalyzed by sucrose 6-phosphate hydrolase from Family 32 of the glycosylhydrolase superfamily. The five 6'-O-phosphorylated alpha-D-glucosyl-fructoses are hydrolyzed by an inducible (approximately 49-50 Kda) phospho-alpha-glucosidase from Family 4 of the glycosylhydrolase superfamily. PMID:11322729

  13. Phosphorylation of DNA topoisomerase II by casein kinase II: modulation of eukaryotic topoisomerase II activity in vitro.

    PubMed Central

    Ackerman, P; Glover, C V; Osheroff, N

    1985-01-01

    The phosphorylation of Drosophila melanogaster DNA topoisomerase II by purified casein kinase II was characterized in vitro. Under the conditions used, the kinase incorporated a maximum of 2-3 molecules of phosphate per homodimer of topoisomerase II. No autophosphorylation of the topoisomerase was observed. The only amino acid residue modified by casein kinase II was serine. Apparent Km and Vmax values for the phosphorylation reaction were 0.4 microM topoisomerase II and 3.3 mumol of phosphate incorporated per min per mg of kinase, respectively. Phosphorylation stimulated the DNA relaxation activity of topoisomerase II by 3-fold over that of the dephosphorylated enzyme, and the effects of modification could be reversed by treatment with alkaline phosphatase. Therefore, this study demonstrates that post-translational enzymatic modifications can be used to modulate the interaction between topoisomerase II and DNA. Images PMID:2987912

  14. Enzymatic activities linked to cardiac energy metabolism of Trypanosoma evansi-infected rats and their possible functional correlations to disease pathogenesis.

    PubMed

    Baldissera, Matheus D; Rech, Virginia C; Grings, Mateus; Gressler, Lucas T; Vaucher, Rodrigo A; Schwertz, Claiton I; Mendes, Ricardo E; Leipnitz, Guilhian; Stefani, Lenita M; Monteiro, Silvia G; DA Silva, Aleksandro S

    2015-08-01

    The aim of this study was to investigate the activities of important enzymes involved in the phosphoryl transfer network (adenylate kinase and creatine kinase (CK)), lactate dehydrogenase (LDH), respiratory chain complexes and biomarkers of cardiac function in rat experimentally infected by Trypanosoma evansi. Rat heart samples were evaluated at 5 and 15 days post-infection (PI). At 5 day PI, there was an increase in LDH and CK activities, and a decrease in respiratory chain complexes II, IV and succinate dehydrogenase activities. In addition, on day 15 PI, a decrease in the respiratory chain complex IV activity was observed. Biomarkers of cardiac function were higher in infected animals on days 5 and 15 PI. Considering the importance of the energy metabolism for heart function, it is possible that the changes in the enzymatic activities involved in the cardiac phosphotransfer network and the decrease in respiratory chain might be involved partially in the role of biomarkers of cardiac function of T. evansi-infected rats. PMID:25758981

  15. Prebiotic phosphorylation of thymidine at 65 C in simulated desert conditions.

    NASA Technical Reports Server (NTRS)

    Bishop, M. J.; Lohrmann, R.; Orgel, L. E.

    1972-01-01

    The phosphorylation of thymidine is described for a variety of conditions at 65 C to demonstrate that the reaction could readily take place in deserts at the present time. This might be used as an indication that urea-phosphate mixtures could have been important as phosphorylating agents on the primitive earth. Reaction products were identified by comparing their chromatographic and electrophoretic mobilities with those of authentic materials and by enzymatic degradation. The results show that good yields of nucleotides are obtained when nucleosides are heated with urea-phospate mixtures at 65 C. Reactions proceed more rapidly at moderate humidities than in a stream of dry nitrogen. Occasional wetting results in even faster and more extensive reactions. There was no reaction for a mixture of urea and trimetaphosphate.

  16. Salt stress-induced protein phosphorylation

    SciTech Connect

    Godoy, J.A.; Torres-Schumann, S.; Llobell, A.; Pintor-Toro, J.A.

    1989-04-01

    Protein phosphorylation induced by salt stress in tomato germinating seeds were investigated by two-dimensional polyacrilamide gel electrophoresis of proteins labeled in vivo with ({sup 32}P)-Phosphate. NaCl induced the phosphorylation of a 14 Kd polypeptide. Pulse-chase experiments revealed that the phosphorylated molecules of this polypeptide are only stable while the stress is present. Phosphorylated 14 Kd polypeptides could be detected in radicles of salt-shocked seedlings after 6 hours stress period. 14 Kd polypeptide phosphorylation was also observed in seeds germinating in the presence of abscisic acid (ABA). The amount of phosphorylated 14 Kd polypeptide was significantly increased in seeds treated simultaneously with NaCl and ABA.

  17. Phosphorylated Tau Can Promote Tubulin Assembly

    Microsoft Academic Search

    Huang-Chun Tseng; Quan Lu; Eric Henderson; Donald J. Graves

    1999-01-01

    Phosphorylation can affect the function of microtubule-associated protein tau. Here, the human brain tau with 441 amino acids was phosphorylated by cyclic-AMP-dependent protein kinase (PKA) or glycogen synthase kinase-3beta . PKA-phosphorylated tau (2.7 mol phosphates\\/mol) does not promote tubulin assembly as judged by spectrophotometric and atomic force microscopy measurements, unless trimethylamine N-oxide (TMAO), a natural occurring osmolyte, is included in

  18. Protein kinase C phosphorylation of PLC?1 regulates its cellular localization.

    PubMed

    Aisiku, Omozuanvbo; Dowal, Louisa; Scarlata, Suzanne

    2011-05-15

    Activation of phospholipase C? (PLC?) by G proteins leads to a chain of events that result in an increase in intracellular calcium and activation of protein kinase C (PKC). It has been found that PKC phosphorylates PLC?1 on S887 in vitro without affecting its enzymatic activity or its ability to be activated by G?(q) proteins. To understand whether S887 phosphorylation affects the enzyme's activity in cells, we constructed two mutants that mimic the wild type and PKC-phosphorylated enzymes (S887A and S887D). We find that these constructs bind similarly to G?(q) in vitro. When expressed in HEK293 cells, both mutants associate identically to G?(q) in both the basal and stimulated states. Both mutants diffuse with similar rates and also interact identically with another known binding partner, translin-associated factor X (TRAX), which associates with PLC?1 in the cytosol and nucleus. However, the two mutants localize differently in the cell. We find that S887A has a much higher nuclear localization than its S887D counterpart both in HEK293 cells and PC12 cells. Our studies suggest that PKC phosphorylation regulates the level of PLC?1 cytosolic and nuclear activity by regulating its cellular compartmentalization. PMID:21338571

  19. Regulation of Polar Peptidoglycan Biosynthesis by Wag31 Phosphorylation in Mycobacteria

    PubMed Central

    2010-01-01

    Background Sensing and responding to environmental changes is a central aspect of cell division regulation. Mycobacterium tuberculosis contains eleven Ser/Thr kinases, two of which, PknA and PknB, are key signaling molecules that regulate cell division/morphology. One substrate of these kinases is Wag31, and we previously showed that partial depletion of Wag31 caused morphological changes indicative of cell wall defects, and that the phosphorylation state of Wag31 affected cell growth in mycobacteria. In the present study, we further characterized the role of the Wag31 phosphorylation in polar peptidoglycan biosynthesis. Results We demonstrate that the differential growth among cells expressing different wag31 alleles (wild-type, phosphoablative, or phosphomimetic) is caused by, at least in part, dissimilar nascent peptidoglycan biosynthesis. The phosphorylation state of Wag31 is found to be important for protein-protein interactions between the Wag31 molecules, and thus, for its polar localization. Consistent with these results, cells expressing a phosphomimetic wag31 allele have a higher enzymatic activity in the peptidoglycan biosynthetic pathway. Conclusions The Wag31Mtb phosphorylation is a novel molecular mechanism by which Wag31Mtb regulates peptidoglycan synthesis and thus, optimal growth in mycobacteria. PMID:21190553

  20. Hydrogen-deuterium exchange in imidazole as a tool for studying histidine phosphorylation.

    PubMed

    Cebo, Ma?gorzata; Kielmas, Martyna; Adamczyk, Justyna; Cebrat, Marek; Szewczuk, Zbigniew; Stefanowicz, Piotr

    2014-12-01

    Isotope exchange at the histidine C2 atom of imidazole in D2O solution is well known to occur at a significantly slower rate than the exchange of amide protons. Analysis of the kinetics of this isotope-exchange reaction is proposed herein as a method of detecting histidine phosphorylation. This modification of His-containing peptides is challenging to pinpoint because of its instability under acidic conditions as well as during CID-MS analysis. In this work, we investigated the effect of phosphorylation of the histidine side chain in peptides on deuterium-hydrogen exchange (DHX) in the imidazole. The results demonstrate that phosphorylation dramatically slows the rate of the DHX reaction. This phenomenon can be applied to detect phosphorylation of peptides at the histidine residue (e.g., in enzymatic digests). We also found that the influence of the peptide sequence on the exchange kinetics is relatively small. A CID fragmentation experiment revealed that there was no detectable hydrogen scrambling in peptides deuterated at C2 of the imidazole ring. Therefore, MS/MS can be used to directly identify the locations of deuterium ions incorporated into peptides containing multiple histidine moieties. PMID:25354888

  1. Recent Advances in Carbon Nanotube-Based Enzymatic Fuel Cells

    PubMed Central

    Cosnier, Serge; Holzinger, Michael; Le Goff, Alan

    2014-01-01

    This review summarizes recent trends in the field of enzymatic fuel cells. Thanks to the high specificity of enzymes, biofuel cells can generate electrical energy by oxidation of a targeted fuel (sugars, alcohols, or hydrogen) at the anode and reduction of oxidants (O2, H2O2) at the cathode in complex media. The combination of carbon nanotubes (CNT), enzymes and redox mediators was widely exploited to develop biofuel cells since the electrons involved in the bio-electrocatalytic processes can be efficiently transferred from or to an external circuit. Original approaches to construct electron transfer based CNT-bioelectrodes and impressive biofuel cell performances are reported as well as biomedical applications. PMID:25386555

  2. Chaperon-like Activation of Serum-Inducible Tryptophanyl-tRNA Synthetase Phosphorylation through Refolding as a Tool for Analysis of Clinical Samples1

    PubMed Central

    Paley, Elena L

    2011-01-01

    Tryptophanyl-tRNA synthetase (TrpRS) expression alters in colorectal (CRC), pancreatic (PC), and cervical (CC) cancers. Here, phosphorylation of unfolded TrpRS and its fragments is stimulated by human cancer sera (CS; n = 13) and serum of rabbit tumor induced by Rous sarcoma virus, unaffected by donor sera (NS; 11/15) and abolished by alkaline phosphatase. At 20 years of follow-up, serum-inducible TrpRS phosphorylation found years before healthy donors (3/15) diagnosed with PC, CRC, or leukemia. I have examined a specificity of serum-inducible TrpRS phosphorylation and found, surprisingly, that serine phosphorylation of unfolded TrpRS is stimulated by anti-TrpRS rabbit antisera but is unaffected by rabbit nonimmune sera and antisera to other antigens. Anti-TrpRS immunoglobulin G (IgG) inhibits phosphorylation of full-length TrpRS and stimulates phosphorylation of its 20-kDa fragment. Phosphorylation of this fragment is stimulated also by CS but not NS. 2-Mercaptoethanol and cyclic AMP exerted synergistic inhibitory effect on TrpRS phosphorylation. Anti-TrpRS sera and casein act as chaperones increasing TrpRS phosphorylation through refolding. Histone-specific protein kinase activity in CS (n = 44) and anti-TrpRS sera was lower than that in NS (n = 11), rabbit nonimmune sera and antisera to other antigens. TrpRS inhibitors, tryptamine, and tryptophanol stimulate in vivo accumulation of enzymatically inactive, nonphosphorylated, aggregated and anti-TrpRS IgG refoldable TrpRS. Phosphorylation of postsurgical tissues (n = 18) reveals TrpRS in ovarian cancer (OVC) and CC but not in normal placenta and liver. In OVC, TrpRS phosphorylation increase correlates with elevated tryptophan-dependent ATP-inorganic pyrophosphate exchange. Although not inducing cancer, TrpRS triggers signaling concomitant with cancer. PMID:22191002

  3. ATP synthesis by decarboxylation phosphorylation.

    PubMed

    Dimroth, Peter; von Ballmoos, Christoph

    2008-01-01

    Adenosine triphosphate (ATP) is used as a general energy source by all living cells. The free energy released by hydrolyzing its terminal phosphoric acid anhydride bond to yield ADP and phosphate is utilized to drive various energy-consuming reactions. The ubiquitous F(1)F(0) ATP synthase produces the majority of ATP by converting the energy stored in a transmembrane electrochemical gradient of H(+) or Na(+) into mechanical rotation. While the mechanism of ATP synthesis by the ATP synthase itself is universal, diverse biological reactions are used by different cells to energize the membrane. Oxidative phosphorylation in mitochondria or aerobic bacteria and photophosphorylation in plants are well-known processes. Less familiar are fermentation reactions performed by anaerobic bacteria, wherein the free energy of the decarboxylation of certain metabolites is converted into an electrochemical gradient of Na(+) ions across the membrane (decarboxylation phosphorylation). This chapter will focus on the latter mechanism, presenting an updated survey on the Na(+)-translocating decarboxylases from various organisms. In the second part, we provide a detailed description of the F(1)F(0) ATP synthases with special emphasis on the Na(+)-translocating variant of these enzymes. PMID:18049805

  4. Rapid ELISA-Based Measurement of Protein Phosphorylation Using RayBio Phosphorylation ELISA Kits

    E-print Network

    Cai, Long

    Rapid ELISA-Based Measurement of Protein Phosphorylation Using RayBio® Phosphorylation ELISA Kits 510600 China. *To whom correspondence should be addressed. Abstract RayBio® Phosphorylation ELISA is a rapid, convenient and sensitive sandwich ELISA for the in vitro measurement of key signaling pathway

  5. Extracellular Phosphorylation and Phosphorylated Proteins: Not Just Curiosities But Physiologically Important

    NSDL National Science Digital Library

    Garif Yalak (ETH Zurich; Department of Health Sciences and Technology REV)

    2012-12-18

    With 3 figures, 1 table, and 221 references plus 2 supplementary figures and 1 supplementary table, this Review describes the evidence supporting physiological and pathological functions of phosphorylated extracellular proteins. In addition, evidence for extracellular phosphorylation in various contexts and tissues is also presented. To facilitate the study of the extracellular phosphoproteome, annotation of the phosphorylated proteins found in proteomic databases is supplied.

  6. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome

    Microsoft Academic Search

    Matthias Mann; Shao-En Ong; Mads Grønborg; Hanno Steen; Ole N. Jensen; Akhilesh Pandey

    2002-01-01

    In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling networks also requires quantitation of these phosphorylation events. In this article, we outline several methods for enrichment of phosphorylated proteins and

  7. New Prospects for an Old Enzyme:  Mammalian Cytochrome c Is Tyrosine-Phosphorylated in Vivo †

    Microsoft Academic Search

    Icksoo Lee; Arthur R. Salomon; Kebing Yu; Jeffrey W. Doan; Lawrence I. Grossman; Maik Hüttemann

    2006-01-01

    Mammalian cytochrome c (Cyt c) has two primary functions: transfer of electrons from the bc1 complex to cytochrome c oxidase (COX) as part of the mitochondrial electron transport chain (ETC), and participation in type II apoptosis. Several studies have indicated that components of the ETC can be phosphorylated, and we have recently shown that the Cyt c electron acceptor COX

  8. IMPORTANCE OF ENZYMATIC BIOTRANSFORMATION IN IMMUNOTOXICOLOGY

    EPA Science Inventory

    Many immunotoxic compounds, such as benzene and other organic solvents, pesticides, mycotoxins and polycyclic aromatic hydrocarbons, can alter immune function only after undergoing enzyme-mediated reactions within various tissues. In the review that follows, the role of enzymatic...

  9. Extracellular enzymatic activity of Malassezia spp. isolates

    Microsoft Academic Search

    Francesca Mancianti; Antonello Rum; Simona Nardoni; Michele Corazza

    2001-01-01

    Extracellular enzymatic activity of different species of Malassezia spp was evaluated. Thirty-three isolates of animal origin (dogs and cats) and stock culture samples were studied. Twenty\\u000a isolates of M. pachydermatis, 8 of M. furfur, 2 of M. sympodialis and M. globosa and one of M. restricta, M. obtusa and M. slooffiae were examined. The enzymatic activity was investigatedusing Api Zym

  10. Extracellular enzymatic activity in 11 Cryptococcus species

    Microsoft Academic Search

    P. García-Martos; P. Marín; J. M. Hernández-Molina; L. García-Agudo; S. Aoufi; J. Mira

    2001-01-01

    The extracellular enzymatic activity of 36 strains of yeast belonging to 11 species of the genus Cryptococcus, has been investigated, using the API-ZYM (BioMérieux, France) commercial system, with the objective of determining the differences\\u000a in the enzymatic profiles of the various species. The strains studied were : 9 of C. neoformans, 7 of C. albidus, 6 of C. laurentii, 5

  11. Enzymatic hydrolysis of low substituted carboxymethyl cellulose

    E-print Network

    Chanona Dominquez, Guadalupe

    1984-01-01

    ENZYMATIC HYDROLYSIS OF LOW SUBSTITUTED CARBOXYMETHYL CELLULOSE A Thesis by GUADALUPE CHANONA DOMINGUEZ Submitted to the Graduate College of Texas A&M University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE... December 1984 Major Subject: Agricultural Engineering ENZYMATIC HYDROLYSIS OF LOW SUBSTITUTED CARBOXYMETHYL CELLULOSE A Thesis by GUADALUPE CHANONA DOMINGUEZ Approved as to style and content by: Cady R. Engler airman) Ed. J. Soltes (Member...

  12. Protein-tyrosine phosphorylation in the Archaea.

    PubMed Central

    Smith, S C; Kennelly, P J; Potts, M

    1997-01-01

    Sulfolobus sulfataricus ATCC 35091, Haloferax volcanii, and Methanosarcina thermophila TM-1, representing the Euryarchaeota and Crenarchaeota subdomains of the Archaea, contain proteins which are phosphorylated on tyrosine. These data raise fundamental questions as to the origin and evolution of tyrosine phosphorylation, a protein modification that is of pivotal importance in the regulation of the physiology of eukaryotic cells. PMID:9079930

  13. Nuclear genetic defects of oxidative phosphorylation

    Microsoft Academic Search

    Eric A. Shoubridge

    2001-01-01

    ATP generated by oxidative phosphorylation is necessary for the normal function of most cells in the body. Partial deficiencies in this system are an important cause of a large and diverse group of multisystem disorders. As both the nuclear and mitochondrial genomes encode structural components of the enzyme complexes of the oxidative phosphorylation system, the disorders can be transmitted either

  14. Phosphorylation of extracellular carbohydrates by intact cells. Chicken hepatocytes specifically adhere to and phosphorylate immobilized N-acetylglucosamine.

    PubMed

    Brandley, B K; Schnaar, R L

    1985-10-15

    Cell-cell adhesion is a multi-step process which may be initiated by binding of cell surface carbohydrates to complementary carbohydrate receptors on apposing cell surfaces. We have modeled such interactions using polyacrylamide gels covalently derivatized with glycosides, to which intact cells specifically adhere; chicken hepatocytes adhere to gels derivatized with N-acetylglucosamine (GlcNAc). Initially adhesion is blocked (or reversed) by soluble GlcNAc, but becomes sugar-resistant rapidly at 37 degrees C, perhaps due to cellular modification of the carbohydrate-derivatized surface (Guarnaccia, S. P., Kuhlenschmidt, M. S., Slife, C. W., and Schnaar, R. L. (1982) J. Biol. Chem. 257, 14293-14299). We report here that, subsequent to recognition and adhesion, intact chicken hepatocytes transfer phosphate covalently to GlcNAc-derivatized gels. Metabolically radiolabeled cells (32Pi) were incubated on polyacrylamide gels derivatized with various aminohexyl glycosides. Noncovalently bound material was then removed from the gels by extensive washing in detergents and salt solutions. Subsequent radiochemical analysis revealed that phosphate was transferred selectively to GlcNAc-derivatized gels (up to 20-fold more than to glucose-, galactose-, or mannose-derivatized gels). Soluble GlcNAc (but not other sugars) or low temperature inhibited phosphate transfer. The phosphorylation was mediated by intact cells; cell lysate was itself incapable of specific phosphate transfer and attenuated specific transfer when added to intact cells. When GlcNAc was immobilized using a cleavable (disulfide-containing) linker arm the transferred phosphate radiolabel could be solubilized by disulfide reduction and recovered for further analysis. The released phosphorylated product migrated as a single low molecular weight species upon gel permeation chromatography, paper electrophoresis, and cellulose thin layer chromatography. Acid hydrolysis of the phosphorylated product generated a compound with the mobility of GlcNAc-6-P in five different separation systems. Treatment with alkaline phosphatase converted the radiolabel to a compound with the properties of inorganic phosphate. These data indicate that; subsequent to carbohydrate recognition and adhesion, intact hepatocytes generate phosphomonoesters of recognized carbohydrates outside of their plasma membranes. PMID:4044599

  15. Evidence that autophosphorylation of solubilized receptors for epidermal growth factor is mediated by intermolecular cross-phosphorylation

    SciTech Connect

    Honegger, A.M.; Kris, R.M.; Schlessinger, J. (Rorer Biotechnology, Inc., King of Prussia, PA (USA)); Ullrich, A. (Genetech, Inc., San Francisco, CA (USA))

    1989-02-01

    Structurally distinguishable mutants of human epidermal growth factor receptor (EGFR) were used to investigate the mechanism of EGFR autophosphorylation. Mutant receptors generated by site-directed mutagenesis were expressed in transfected NIH 3T3 cells lacking endogenous receptors. After coincubation of cell lysates in the presence or absence of EGF, receptor immunoprecipitates were incubated with ({gamma}-{sup 32}P)ATP. A kinase-negative mutant EGFR (K721A), in which Lys-721 in the ATP binding site was replaced by an alanine residue, was shown to be phosphorylated in an EGF-dependent manner by an enzymatically active EGFR deletion mutant lacking two autophosphorylation sites. A mutant EGFR lacking the EGF-binding domain as well as the phosphorylation sites also phosphorylated the kinase-negative mutant. In both cases the kinase-negative mutant K721A was phosphorylated on sites virtually identical to the sites that are autophosphorylated by wild-type recombinant or native human EGFRs. With four different site-specific anti-EGFR antibodies, it was shown that deletion mutants devoid of epitopes recognized by the antibodies were coimmunoprecipitated together with wild-type or mutant receptors recognized by the antibodies. This indicates that EGFR oligomers were preserved during immunoprecipitation. On the basis of these results, the authors propose that autophosphorylation of solubilized EGFR is mediated by intermolecular cross-phosphorylation, probably facilitated by receptor oligomerization.

  16. Conformational motions regulate phosphoryl transfer in related protein tyrosine phosphatases.

    PubMed

    Whittier, Sean K; Hengge, Alvan C; Loria, J Patrick

    2013-08-23

    Many studies have implicated a role for conformational motions during the catalytic cycle, acting to optimize the binding pocket or facilitate product release, but a more intimate role in the chemical reaction has not been described. We address this by monitoring active-site loop motion in two protein tyrosine phosphatases (PTPs) using nuclear magnetic resonance spectroscopy. The PTPs, YopH and PTP1B, have very different catalytic rates; however, we find in both that the active-site loop closes to its catalytically competent position at rates that mirror the phosphotyrosine cleavage kinetics. This loop contains the catalytic acid, suggesting that loop closure occurs concomitantly with the protonation of the leaving group tyrosine and explains the different kinetics of two otherwise chemically and mechanistically indistinguishable enzymes. PMID:23970698

  17. Pseudomonas aeruginosa Exotoxin Y Is a Promiscuous Cyclase That Increases Endothelial Tau Phosphorylation and Permeability*

    PubMed Central

    Ochoa, Cristhiaan D.; Alexeyev, Mikhail; Pastukh, Viktoriya; Balczon, Ron; Stevens, Troy

    2012-01-01

    Exotoxin Y (ExoY) is a type III secretion system effector found in ? 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY causes inter-endothelial gaps and vascular leak, the mechanisms by which this occurs are poorly understood. Using both a bacteria-delivered and a codon-optimized conditionally expressed ExoY, we report that this toxin is a dual soluble adenylyl and guanylyl cyclase that results in intracellular cAMP and cGMP accumulation. The enzymatic activity of ExoY caused phosphorylation of endothelial Tau serine 214, accumulation of insoluble Tau, inter-endothelial cell gap formation, and increased macromolecular permeability. To discern whether the cAMP or cGMP signal was responsible for Tau phosphorylation and barrier disruption, pulmonary microvascular endothelial cells were engineered for the conditional expression of either wild-type guanylyl cyclase, which synthesizes cGMP, or a mutated guanylyl cyclase, which synthesizes cAMP. Sodium nitroprusside stimulation of the cGMP-generating cyclase resulted in transient Tau serine 214 phosphorylation and gap formation, whereas stimulation of the cAMP-generating cyclase induced a robust increase in Tau serine 214 phosphorylation, gap formation, and macromolecular permeability. These results indicate that the cAMP signal is the dominant stimulus for Tau phosphorylation. Hence, ExoY is a promiscuous cyclase and edema factor that uses cAMP and, to some extent, cGMP to induce the hyperphosphorylation and insolubility of endothelial Tau. Because hyperphosphorylated and insoluble Tau are hallmarks in neurodegenerative tauopathies such as Alzheimer disease, acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases. PMID:22637478

  18. Pseudomonas aeruginosa exotoxin Y is a promiscuous cyclase that increases endothelial tau phosphorylation and permeability.

    PubMed

    Ochoa, Cristhiaan D; Alexeyev, Mikhail; Pastukh, Viktoriya; Balczon, Ron; Stevens, Troy

    2012-07-20

    Exotoxin Y (ExoY) is a type III secretion system effector found in ~ 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY causes inter-endothelial gaps and vascular leak, the mechanisms by which this occurs are poorly understood. Using both a bacteria-delivered and a codon-optimized conditionally expressed ExoY, we report that this toxin is a dual soluble adenylyl and guanylyl cyclase that results in intracellular cAMP and cGMP accumulation. The enzymatic activity of ExoY caused phosphorylation of endothelial Tau serine 214, accumulation of insoluble Tau, inter-endothelial cell gap formation, and increased macromolecular permeability. To discern whether the cAMP or cGMP signal was responsible for Tau phosphorylation and barrier disruption, pulmonary microvascular endothelial cells were engineered for the conditional expression of either wild-type guanylyl cyclase, which synthesizes cGMP, or a mutated guanylyl cyclase, which synthesizes cAMP. Sodium nitroprusside stimulation of the cGMP-generating cyclase resulted in transient Tau serine 214 phosphorylation and gap formation, whereas stimulation of the cAMP-generating cyclase induced a robust increase in Tau serine 214 phosphorylation, gap formation, and macromolecular permeability. These results indicate that the cAMP signal is the dominant stimulus for Tau phosphorylation. Hence, ExoY is a promiscuous cyclase and edema factor that uses cAMP and, to some extent, cGMP to induce the hyperphosphorylation and insolubility of endothelial Tau. Because hyperphosphorylated and insoluble Tau are hallmarks in neurodegenerative tauopathies such as Alzheimer disease, acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases. PMID:22637478

  19. Oxidized phospholipids mediate occludin expression and phosphorylation in vascular endothelial cells.

    PubMed

    DeMaio, Lucas; Rouhanizadeh, Mahsa; Reddy, Srinivasa; Sevanian, Alex; Hwang, Juliana; Hsiai, Tzung K

    2006-02-01

    Oxidized l-alpha-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), a component of minimally modified LDL, induces production of proinflammatory cytokines and development of atherosclerotic lesions. We tested the hypothesis that OxPAPC alters expression, phosphorylation, and localization of tight junction (TJ) proteins, particularly occludin, a transmembrane TJ protein. OxPAPC reduced total occludin protein and increased occludin phosphorylation dose dependently (10-50 microg/ml) and time dependently in bovine aortic endothelial cells. OxPAPC decreased occludin mRNA and reduced the immunoreactivity of zonula occludens-1 at the cell-cell contacts. Furthermore, OxPAPC increased the diffusive flux of 10-kDa dextran in a dose-dependent manner. O2-* production by bovine aortic endothelial cells increased nearly twofold after exposure to OxPAPC. Also, enzymatic generation of O2-* by xanthine oxidase-lumazine and H2O2 by glucose oxidase-glucose increased occludin phosphorylation, implicating reactive oxygen species as modulators of the OxPAPC effects on occludin phosphorylation. Superoxide dismutase and/or catalase blocked the effects of OxPAPC on occludin protein content and phosphorylation, occludin mRNA, zonula occludens-1 immunoreactivity, and diffusive flux of 10-kDa dextran. These findings suggest that changes in TJ proteins are potential mechanisms by which OxPAPC compromises the barrier properties of the vascular endothelium. OxPAPC-induced disruption of TJs, which likely facilitates transmigration of LDL and inflammatory cells into the subendothelial layers, may be mediated by reactive oxygen species. PMID:16172163

  20. In vivo analysis of Yorkie phosphorylation sites.

    PubMed

    Oh, H; Irvine, K D

    2009-04-30

    The co-activator Yorkie (Yki) mediates transcriptional regulation effected by the Drosophila Fat-Warts (Wts)-Hippo (Hpo) pathways. Yki is inhibited by Wts-mediated phosphorylation, and a Wts phosphorylation site at Ser168 has been identified. Here we identify two additional Wts phosphorylation sites on Yki, and examine the respective contribution of all three sites to Yki nuclear localization and activity. Our results show that although Ser168 is the most critical site, all three phosphorylation sites influence Yki localization and activity in vivo, and can be sites of regulation by Wts. Thus, investigations of the role of Yki and its mammalian homolog Yes-associated protein (YAP) in development and oncogenesis should include evaluations of additional sites. The WW domains of Yki are not required for its phosphorylation, but instead are positively required for its activity. We also identify two potential sites of phosphorylation by an unknown kinase, which could influence phosphorylation of Ser168 by Wts, suggesting that there are additional mechanisms for regulating Yki/YAP activity. PMID:19330023

  1. Tyrosine Phosphorylation as a Conformational Switch

    PubMed Central

    Deshmukh, Lalit; Meller, Nahum; Alder, Nathan; Byzova, Tatiana; Vinogradova, Olga

    2011-01-01

    Reversible protein phosphorylation is vital for many fundamental cellular processes. The actual impact of adding and removing phosphate group(s) is 3-fold: changes in the local/global geometry, alterations in the electrostatic potential and, as the result of both, modified protein-target interactions. Here we present a comprehensive structural investigation of the effects of phosphorylation on the conformational as well as functional states of a crucial cell surface receptor, ?IIb?3 integrin. We have analyzed phosphorylated (Tyr747 and Tyr759) ?3 integrin cytoplasmic tail (CT) primarily by NMR, and our data demonstrate that under both aqueous and membrane-mimetic conditions, phosphorylation causes substantial conformational rearrangements. These changes originate from novel ionic interactions and revised phospholipid binding. Under aqueous conditions, the critical Tyr747 phosphorylation prevents ?3CT from binding to its heterodimer partner ?IIbCT, thus likely maintaining an activated state of the receptor. This conclusion was tested in vivo and confirmed by integrin-dependent endothelial cells adhesion assay. Under membrane-mimetic conditions, phosphorylation results in a modified membrane embedding characterized by significant changes in the secondary structure pattern and the overall fold of ?3CT. Collectively these data provide unique molecular insights into multiple regulatory roles of phosphorylation. PMID:21956114

  2. Histone H1 phosphorylation in breast cancer.

    PubMed

    Harshman, Sean W; Hoover, Michael E; Huang, Chengsi; Branson, Owen E; Chaney, Sarah B; Cheney, Carolyn M; Rosol, Thomas J; Shapiro, Charles L; Wysocki, Vicki H; Huebner, Kay; Freitas, Michael A

    2014-05-01

    Breast cancer is the second leading cause of cancer-related deaths in women. The need for new clinical biomarkers in breast cancer is necessary to further predict prognosis and therapeutic response. In this article, the LC-MS histone H1 phosphorylation profiles were established for three distinct breast cancer cell lines. The results show that the extent of H1 phosphorylation can distinguish between the different cell lines. The histone H1 from the metastatic cell line, MDA-MB-231, was subjected to chemical derivitization and LC-MS/MS analysis. The results suggest that the phosphorylation at threonine 146 is found on both histone H1.2 and histone H1.4. Cell lines were then treated with an extracellular stimulus, estradiol or kinase inhibitor LY294002, to monitor changes in histone H1 phosphorylation. The data show that histone H1 phosphorylation can increase and decrease in response to extracellular stimuli. Finally, primary breast tissues were stained for the histone H1 phosphorylation at threonine 146. Variable staining patterns across tumor grades and subtypes were observed with pT146 labeling correlating with tumor grade. These results establish the potential for histone H1 phosphorylation at threonine 146 as a clinical biomarker in breast cancer. PMID:24601643

  3. Histone H1 Phosphorylation in Breast Cancer

    PubMed Central

    2015-01-01

    Breast cancer is the second leading cause of cancer-related deaths in women. The need for new clinical biomarkers in breast cancer is necessary to further predict prognosis and therapeutic response. In this article, the LC-MS histone H1 phosphorylation profiles were established for three distinct breast cancer cell lines. The results show that the extent of H1 phosphorylation can distinguish between the different cell lines. The histone H1 from the metastatic cell line, MDA-MB-231, was subjected to chemical derivitization and LC-MS/MS analysis. The results suggest that the phosphorylation at threonine 146 is found on both histone H1.2 and histone H1.4. Cell lines were then treated with an extracellular stimulus, estradiol or kinase inhibitor LY294002, to monitor changes in histone H1 phosphorylation. The data show that histone H1 phosphorylation can increase and decrease in response to extracellular stimuli. Finally, primary breast tissues were stained for the histone H1 phosphorylation at threonine 146. Variable staining patterns across tumor grades and subtypes were observed with pT146 labeling correlating with tumor grade. These results establish the potential for histone H1 phosphorylation at threonine 146 as a clinical biomarker in breast cancer. PMID:24601643

  4. Phosphorylation state-dependent interaction between AKAP7?/? and phospholamban increases phospholamban phosphorylation.

    PubMed

    Rigatti, Marc; Le, Andrew V; Gerber, Claire; Moraru, Ion I; Dodge-Kafka, Kimberly L

    2015-09-01

    Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca(2+) cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca(2+) re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7?/? [2,3]. Here, we further these findings by showing that phosphorylation of PLB requires interaction with AKAP7?/? and that this interaction occurs only when PLB is unphosphorylated. Additionally, we find that two mutants of PLB (R9C and ?14), which are associated with dilated cardiomyopathy in humans, prevent association with AKAP7?/? and display reduced phosphorylation in vitro. This finding implicates the AKAP7?/?-PLB interaction in the pathology of the disease phenotype. Further exploration of the AKAP7?/?-PLB association demonstrated a phosphorylation state-dependence of the interaction. Computational modeling revealed that this mode of interaction allows for small amounts of AKAP and PKA (100-200nM) to regulate the phosphorylation of large quantities of PLB (50?M). Our results confirm that AKAP7?/? binding to PLB is important for phosphorylation of PLB, and describe a novel phosphorylation state-dependent binding mechanism that explains how phosphorylation of highly abundant PKA substrates can be regulated by AKAPs present at ~100-200 fold lower concentrations. PMID:26027516

  5. Enzymatic mechanism of copper-containing nitrite reductase.

    PubMed

    Li, Yan; Hodak, Miroslav; Bernholc, J

    2015-02-10

    Copper-containing nitrite reductases (CuNiRs) catalyze the reduction of nitrite to nitric oxide, a key step in the denitrification process that maintains balance between organic and inorganic nitrogen. Despite their importance, their functioning is not well understood. In this work, we carry out first-principles calculations and show that the available structural data are consistent only with a single mechanism. For this mechanism, we determine the activation energies, transition states, and minimum energy pathways of CuNiR. The calculations lead to an updated enzymatic mechanism and resolve several controversial issues. In particular, our work identifies the origins of the two protons necessary for the enzymatic function and shows that the transformation from the initial O-coordination of substrate to the final N-coordination of product is achieved by electron transfer from T1 copper to T2 copper, rather than by the previously reported side-on coordination of a NO intermediate, which only takes place in the reduced enzyme. We also examine the role of structural change in the critical residue Asp(98), reported in one experimental study, and find that while the structural change affects the energetics of substrate attachment and product release at the T2 copper reaction center, it does not significantly affect the activation energy and reaction pathways of the nitrite reduction process. PMID:25594136

  6. Use of enzymatic cleaners on US Navy ships. Research report

    SciTech Connect

    Venkatachalam, R.S.

    1996-03-01

    The Naval Surface Warfare Center, Carderock Division, conducted a study to determine the feasibility of using enzymatic and bacterial products in cleaning applications aboard U.S. Navy ships. A review of the most recent technical literature and a survey of potential suppliers were conducted. In addition, shipboard systems, subsystems and housekeeping processes were evaluated to identify suitable applications for enzymatic and bacterial cleaners. The study identified numerous commercial products that, based on manufacturers` claims, would be effective and safe for use aboard ship to clean walls, floors, galley work surfaces, engine and machine parts, drains, pipes, grease traps, collection, holding and transfer (CHT) tanks, ballast tanks and bilge areas. However, the study also revealed the absence of standardized test protocols essential for validation of manufacturers` claims, and recommended the cooperative development of such protocols by representatives from the commercial sector, Government and academia. The need to obtain meaningful cost information based on actual use scenarios and to investigate any permitting issues associated with the discharge of related wastes to pierside facilities was also identified.

  7. AIF deficiency compromises oxidative phosphorylation

    PubMed Central

    Vahsen, Nicola; Candé, Céline; Brière, Jean-Jacques; Bénit, Paule; Joza, Nicholas; Larochette, Nathanael; Mastroberardino, Pier Giorgio; Pequignot, Marie O; Casares, Noelia; Lazar, Vladimir; Feraud, Olivier; Debili, Najet; Wissing, Silke; Engelhardt, Silvia; Madeo, Frank; Piacentini, Mauro; Penninger, Josef M; Schägger, Hermann; Rustin, Pierre; Kroemer, Guido

    2004-01-01

    Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF-deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death. PMID:15526035

  8. Enzymatic aerobic ring rearrangement of optically active furylcarbinols.

    PubMed

    Thiel, Daniel; Dokni?, Diana; Deska, Jan

    2014-01-01

    Biogenic furans are currently discussed as highly attractive alternative feedstock in a post-fossil society; thus, also the creation of sustainable furan valorization pathways appears of great importance. Here an artificial Achmatowicz monooxygenase activity for the aerobic ring expansion of furans is achieved by the combination of commercial glucose oxidase as oxygen-activating biocatalyst and wild-type chloroperoxidase as oxygen-transfer mediator, providing a biological ready-to-use solution for this truly synthetic furan rearrangement. In concert with enzymatic transformations for the enantioselective preparation of optically active furylcarbinols, purely biocatalytic reaction cascades for the stereocontrolled construction of complex pyranones are obtained, exhibiting high functional group tolerance even to oxidation-sensitive moieties. PMID:25335580

  9. Phosphorylation of MAP4 affects microtubule properties

    E-print Network

    Bulinski, Chloë

    , Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachiohji, Tokyo 192-0397 To whom correspondence). Consistent with this hypothesis, MAP4's level of phosphorylation has been shown to increase at the G2/M

  10. Protein Kinase C Phosphorylation of PLC?1 Regulates its Cellular Localization

    PubMed Central

    Aisiku, Omozuanvbo; Dowal, Louisa; Scarlata, Suzanne

    2011-01-01

    Activation of phospholipase C? (PLC?) by G proteins leads to a chain of events that result in an increase in intracellular calcium and activation of protein kinase C (PKC). It has been found that PKC phosphorylates PLC?1 on S887 in vitro without affecting its enzymatic activity or its ability to be activated by G?(q) proteins. To understand whether S887 phosphorylation affects the enzyme's activity in cells, we constructed two mutants that mimic the wild type and PKC-phosphoryated enzymes (S887A and S887D). We find that these constructs bind similarly to G?(q) in vitro. When expressed in HEK293 cells, both mutants associate identically to G?(q) in both the basal and stimulated states. Both mutants diffuse with similar rates and also interact identically with another known binding partner, Translin-Associated factor X (TRAX), which associates with PLC?1 in the cytosol and nucleus. However, the two mutants localize differently in the cell. We find that S887A has a much higher nuclear localization than its S887D counterpart both in HEK293 cells and PC12 cells. Our studies suggest that PKC phosphorylation regulates the level of PLC?1 cytosolic and nuclear activity by regulating its cellular compartmentalization. PMID:21338571

  11. Ascorbate stimulates endothelial nitric oxide synthase enzyme activity by rapid modulation of its phosphorylation status.

    PubMed

    Ladurner, Angela; Schmitt, Christoph A; Schachner, Daniel; Atanasov, Atanas G; Werner, Ernst R; Dirsch, Verena M; Heiss, Elke H

    2012-05-15

    Long-term exposure to ascorbate is known to enhance endothelial nitric oxide synthase (eNOS) activity by stabilizing the eNOS cofactor tetrahydrobiopterin (BH4). We investigated acute effects of ascorbate on eNOS function in primary (HUVEC) and immortalized human endothelial cells (EA.hy926), aiming to provide a molecular explanation for the rapid vasodilatation seen in vivo upon administration of ascorbate. Enzymatic activity of eNOS and intracellular BH4 levels were assessed by means of an arginine-citrulline conversion assay and HPLC analysis, respectively. Over a period of 4h, ascorbate steadily increased eNOS activity, although endothelial BH4 levels remained unchanged compared to untreated control cells. Immunoblot analyses revealed that as early as 5 min after treatment ascorbate dose-dependently increased phosphorylation at eNOS-Ser1177 and concomitantly decreased phosphorylation at eNOS-Thr495, a phosphorylation pattern indicative of increased eNOS activity. By employing pharmacological inhibitors, siRNA-mediated knockdown approaches, and overexpression of the catalytic subunit of protein phosphatase 2A (PP2A), we show that this effect was at least partly owing to reduction of PP2A activity and subsequent activation of AMP-activated kinase. In this report, we unravel a novel mechanism for how ascorbate rapidly activates eNOS independent of its effects on BH4 stabilization. PMID:22542797

  12. Ascorbate stimulates endothelial nitric oxide synthase enzyme activity by rapid modulation of its phosphorylation status

    PubMed Central

    Ladurner, Angela; Schmitt, Christoph A.; Schachner, Daniel; Atanasov, Atanas G.; Werner, Ernst R.; Dirsch, Verena M.; Heiss, Elke H.

    2012-01-01

    Long-term exposure to ascorbate is known to enhance endothelial nitric oxide synthase (eNOS) activity by stabilizing the eNOS cofactor tetrahydrobiopterin (BH4). We investigated acute effects of ascorbate on eNOS function in primary (HUVEC) and immortalized human endothelial cells (EA.hy926), aiming to provide a molecular explanation for the rapid vasodilatation seen in vivo upon administration of ascorbate. Enzymatic activity of eNOS and intracellular BH4 levels were assessed by means of an arginine–citrulline conversion assay and HPLC analysis, respectively. Over a period of 4 h, ascorbate steadily increased eNOS activity, although endothelial BH4 levels remained unchanged compared to untreated control cells. Immunoblot analyses revealed that as early as 5 min after treatment ascorbate dose-dependently increased phosphorylation at eNOS-Ser1177 and concomitantly decreased phosphorylation at eNOS-Thr495, a phosphorylation pattern indicative of increased eNOS activity. By employing pharmacological inhibitors, siRNA-mediated knockdown approaches, and overexpression of the catalytic subunit of protein phosphatase 2A (PP2A), we show that this effect was at least partly owing to reduction of PP2A activity and subsequent activation of AMP-activated kinase. In this report, we unravel a novel mechanism for how ascorbate rapidly activates eNOS independent of its effects on BH4 stabilization. PMID:22542797

  13. Enzymatic hydrogen production by light-sensitized anodized tubular TiO 2 photoanode

    Microsoft Academic Search

    Sanghyun Bae; Eunjung Shim; Jaekyung Yoon; Hyunku Joo

    2008-01-01

    Preliminary experiments with a slurry system of enzyme and powdery photocatalyst mixed in one compartment suggested that the electron transfer from light-sensitized photocatalyst to enzyme is the rate-determining step. Hence, in this study an anodized tubular TiO2 electrode (ATTE) on a titanium substrate was examined as a photoanode in an anodic cell for enzymatic hydrogen production in a cathodic cell.

  14. Phosphorylation of human skeletal muscle myosin

    SciTech Connect

    Houston, M.E.; Lingley, M.D.; Stuart, D.S.; Hoffman-Goetz, L.

    1986-03-01

    Phosphorylation of the P-light chains (phosphorylatable light chains) in human skeletal muscle myosin was studied in vitro and in vivo under resting an d contracted conditions. biopsy samples from rested vastus lateralis muscle of male and female subjects were incubated in oxygenated physiological solution at 30/sup 0/C. Samples frozen following a quiescent period showed the presence of only unphosphorylated P-light chains designated LC2f (light chain two of fast myosin) CL2s and LC2s'(light chains two of slow myosin). Treatment with caffeine (10 mM) or direct electrical stimulation resulted in the appearance of three additional bands which were identified as the phosphorylated forms of the P-light chains i.e. LC2f-P, LC2s-P and LC2s'-P. The presence of phosphate was confirmed by prior incubation with (/sup 30/P) orthophosphate. Muscle samples rapidly frozen from resting vastus lateralis muscle revealed the presence of unphosphorylated and phosphorylated P-light chains in approximately equal ratios. Muscle samples rapidly frozen following a maximal 10 second isometric contraction showed virtually only phosphorylated fast and slow P-light chains. These results reveal that the P-light chains in human fast and slow myosin may be rapidly phosphorylated, but the basal level of phosphorylation in rested human muscle considerably exceeds that observed in animal muscles studied in vitro or in situ.

  15. Regulation of serine (Ser)-31 and Ser40 tyrosine hydroxylase phosphorylation during morphine withdrawal in the hypothalamic paraventricular nucleus and nucleus tractus solitarius-A2 cell group: role of ERK1/2.

    PubMed

    Núñez, Cristina; Laorden, M Luisa; Milanés, M Victoria

    2007-12-01

    Our previous studies have shown that naloxone-induced morphine withdrawal increases the hypothalamic-pituitary-adrenocortical (HPA) axis activity, which is dependent on a hyperactivity of noradrenergic pathways [nucleus tractus solitarius (NTS) A(2)] innervating the hypothalamic paraventricular nucleus (PVN). Short-term regulation of catecholamine biosynthesis occurs through phosphorylation of tyrosine hydroxylase (TH), which enhances enzymatic activity. In the present study, the effect of morphine withdrawal on site-specific TH phosphorylation in the PVN and NTS-A(2) was determined by quantitative blot immunolabeling and immunohistochemistry using phosphorylation state-specific antibodies. We show that naloxone-induced morphine withdrawal phosphorylates TH at Serine (Ser)-31 but not Ser40 in PVN and NTS-A(2), which is associated with both an increase in total TH immunoreactivity in NTS-A(2) and an enhanced TH activity in the PVN. In addition, we demonstrated that TH neurons phosphorylated at Ser31 coexpress c-Fos in NTS-A(2). We then tested whether pharmacological inhibition of ERK activation by ERK kinase contributes to morphine withdrawal-induced phosphorylation of TH at Ser31. We show that the ability of morphine withdrawal to stimulate phosphorylation at this seryl residue is reduced by SL327, an inhibitor of ERK(1/2) activation. These results suggest that morphine withdrawal increases noradrenaline turnover in the PVN, at least in part, via ERK(1/2)-dependent phosphorylation of TH at Ser31. PMID:17823252

  16. Vinexinß, an atypical "sensor" of retinoic acid receptor gamma signaling: union and sequestration, separation, and phosphorylation.

    PubMed

    Lalevée, Sébastien; Bour, Gaétan; Quinternet, Marc; Samarut, Eric; Kessler, Pascal; Vitorino, Marc; Bruck, Nathalie; Delsuc, Marc-André; Vonesch, Jean-Luc; Kieffer, Bruno; Rochette-Egly, Cécile

    2010-11-01

    The transcriptional activity of nuclear retinoic acid receptors (RARs) relies on the association/dissociation of coregulators at the ligand-binding domain. However, we determined that the N-terminal domain (NTD) also plays a role through its phosphorylation, and we isolated vinexin?, a cytoskeleton protein with three SH3 domains, as a new partner of the RAR? NTD. Here we deciphered the mechanism of the interaction and its role in RAR?-mediated transcription. By combining molecular and biophysical (surface plasmon resonance, NMR, and fluorescence resonance energy transfer) approaches, we demonstrated that the third SH3 domain of vinexin? interacts with a proline-rich domain (PRD) located in RAR? NTD and that phosphorylation at a serine located in the PRD abrogates the interaction. The affinity of the interaction was also evaluated. In vivo, vinexin? represses RAR?-mediated transcription and we dissected the underlying mechanism in chromatin immunoprecipitation experiments performed with F9 cells expressing RAR? wild type or mutated at the phosphorylation site. In the absence of retinoic acid (RA), vinexin? does not occupy RAR? target gene promoters and sequesters nonphosphorylated RAR? out of promoters. In response to RA, RAR? becomes phosphorylated and dissociates from vinexin?. This separation allows RAR? to occupy promoters. This is the first report of an RAR corepressor association/dissociation out of promoters and regulated by phosphorylation. PMID:20634350

  17. Novel Phosphorylations of IKK?/NEMO.

    PubMed

    Lee, Sun Hwa; Toth, Zsolt; Wong, Lai-Yee; Brulois, Kevin; Nguyen, Jim; Lee, June-Yong; Zandi, Ebrahim; Jung, Jae U

    2012-01-01

    Central to NF-?B signaling pathways is IKK?/NEMO, a regulatory subunit of the cytoplasmic I?B kinase (IKK) complex, which undergoes various posttranslational modifications, specifically phosphorylation, to regulate its function. Furthermore, Kaposi's sarcoma-associated herpesvirus (KSHV) FADD-like interleukin-1? (IL-1?) converting enzyme (FLICE) inhibitory protein (vFLIP) activates the NF-?B signaling pathway by directly interacting with IKK?/NEMO. However, the exact functions of IKK?/NEMO phosphorylation and its KvFLIP interaction in NF-?B activation remain elusive. Here, we report two novel phosphorylation sites of IKK?/NEMO and their negative effect on the IKK?/NEMO-mediated NF-?B signaling pathway. First, the Src family protein tyrosine kinases (SF-PTKs), including Src, Fyn, Lyn, and Fgr, interact with and phosphorylate tyrosine residue 374 (Y374) of IKK?/NEMO. Mutation of the Y374 residue to phenylalanine (Y374F) specifically abolished SF-PTK-mediated tyrosine phosphorylation, leading to increased tumor necrosis factor alpha (TNF-?)-induced NF-?B activity. Moreover, our mass spectrometry analysis found that the serine 377 residue (S377) of IKK?/NEMO underwent robust phosphorylation upon KvFLIP expression. Replacement of the IKK?/NEMO S377 residue by alanine (S377A) or glutamic acid (S377E) resulted in a significant increase or decrease of NF-?B activity and TNF-?-mediated IL-6 cytokine production, respectively. Our study thus demonstrates that the Y374 or S377 residue located at the C-terminal proline-rich domain of human IKK?/NEMO undergoes phosphorylation upon TNF-? treatment or KvFLIP expression, respectively, resulting in the suppression of IKK?/NEMO activity to induce NF-?B activation. This study suggests the potential phosphorylation-mediated feedback negative regulation of IKK?/NEMO activity in the NF-?B signaling pathway. IMPORTANCE Since unchecked regulation of NF-?B has been linked to uncontrolled proliferation and cell death, the downregulation of the NF-?B signaling pathway is as important as its activation. Specifically, the phosphorylation-mediated modification of IKK?/NEMO is a critical regulatory mechanism of NF-?B activity. Here, we report two novel phosphorylations of IKK?/NEMO and their negative effects on the NF-?B signaling pathway. First, the Src family protein tyrosine kinase interacts with and phosphorylates tyrosine residue 374 of IKK?/NEMO, suppressing tumor necrosis factor alpha (TNF-?)-induced NF-?B activity. Additionally, Kaposi's sarcoma-associated herpesvirus (KSHV) FADD-like interleukin-1? (IL-1?) converting enzyme (FLICE) inhibitory protein (KvFLIP) expression induces a robust phosphorylation of the serine 377 residue of IKK?/NEMO, resulting in a significant decrease of NF-?B activity. Our study thus demonstrates that the Y374 or S377 residue of IKK?/NEMO undergoes phosphorylation upon TNF-? treatment or KvFLIP expression, respectively, resulting in the suppression of IKK?/NEMO activity to induce NF-?B activation. This also suggests the potential phosphorylation-mediated feedback negative regulation of IKK?/NEMO activity in the NF-?B signaling pathway. PMID:23131831

  18. Ultrasonic acceleration of enzymatic processing of cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymatic bio-processing of cotton generates significantly less hazardous wastewater effluents, which are readily biodegradable, but it also has several critical shortcomings that impede its acceptance by industries: expensive processing costs and slow reaction rates. It has been found that the intr...

  19. Ethanol from biomass by enzymatic hydrolysis

    SciTech Connect

    Wright, J.D.

    1988-08-01

    Enzymes are biological catalysts that generally are designed to do one job well, but to do one job only. Therefore, the enzymes that catalyze the hydrolysis of cellulose to sugar do not break down the sugars. Thus, enzymatic processes are capable of yields approaching 100%. Enzymatic hydrolysis processes have been under development for only 10 years. Although improvements have been made in enzymatic technology, more are both possible and necessary. The important research issues include understanding the processes necessary to render the crystalline cellulose easily digestible, understanding and improving the basic mechanisms in the hydrolysis step, and developing better and less expensive enzymes. The hemicellulose fraction (25%) is primarily composed of xylan, which is simple to convert to the simple sugar xylose, but the xylose is difficult to ferment to ethanol. There were no practical systems for xylose fermentation 10 years ago. Today, methods have been identified using new yeasts, fungi, bacteria, and processes combining enzymes and yeasts. Although none of the fermentations is yet ready for commercial use, considerable progress has been made. The following sections describe current research efforts in each of the major areas (cellulose hydrolysis, xylose fermentation, and lignin conversion), with an emphasis on enzymatic hydrolysis using fungal enzymes.

  20. Accelerated Articles Enzymatically Amplified Surface Plasmon

    E-print Network

    directly on the surface concentration of DNA-RNA het- eroduplexes. DNA microarrays provide a powerful and RNA Microarrays for the Ultrasensitive Detection of Nucleic Acids Terry T. Goodrich, Hye Jin Lee A novel surface enzymatic amplification method that utilizes RNA microarrays in conjunction

  1. Enhanced enzymatic hydrolysis of cellulose in microgels.

    PubMed

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

    2015-06-16

    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

  2. pH & Rate of Enzymatic Reactions.

    ERIC Educational Resources Information Center

    Clariana, Roy B.

    1991-01-01

    A quantitative and inexpensive way to measure the rate of enzymatic reaction is provided. The effects of different pH levels on the reaction rate of an enzyme from yeast are investigated and the results graphed. Background information, a list of needed materials, directions for preparing solutions, procedure, and results and discussion are…

  3. Enzymatic biosynthesis of ricinoleic acid estolides

    Microsoft Academic Search

    A. Bódalo-Santoyo; J. Bastida-Rodríguez; M. F. Máximo-Martín; M. C. Montiel-Morte; M. D. Murcia-Almagro

    2005-01-01

    Candida rugosa lipase has been shown to have sufficient activity to catalyse the enzymatic synthesis of ricinoleic acid estolides in a batch reactor. The water requirements of the reactor change during the reaction: at the beginning of the process a minimum amount of water is necessary but, later, the reaction mixture must be dried out to obtain an estolide with

  4. ON THE SIMULATION OF ENZYMATIC DIGEST PATTERNS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A simulation methodology for predicting the time-course of enzymatic digestions is described. The model is based solely on the enzyme’s sub-site architecture and concomitant binding energies. This allows sub-site binding energies to be used to predict the evolution of the relative amounts of differ...

  5. Heavy metal pollution and soil enzymatic activity

    Microsoft Academic Search

    Germund Tyler

    1974-01-01

    The activity of hydrolytic soil enzymes was studied on spruce mor, polluted with Cu and Zn from a brass foundry in Sweden. Approximately straight regression lines were obtained between enzymatic activity or respiration rate and log Cu+Zn concentration, with highly significant negative regression coefficients for urease and acid phosphatase activity as well as respiration rate, whereas ß-glucosidase activity was not

  6. Calcineurin B homologous protein 3 negatively regulates cardiomyocyte hypertrophy via inhibition of glycogen synthase kinase 3 phosphorylation.

    PubMed

    Kobayashi, Soushi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

    2015-07-01

    Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca(2+)-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3?/?) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3?/? without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3?/?, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3? and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3? interacted with CHP3. However, a Ca(2+)-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3? and had the same inhibitory effect on GSK3?/? phosphorylation, suggesting that the action of CHP3 was independent of Ca(2+). These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3?/? phosphorylation and subsequent enzymatic activation of GSK3?/?. PMID:25935310

  7. Mutational analysis of Lck in CD45-negative T cells: dominant role of tyrosine 394 phosphorylation in kinase activity.

    PubMed Central

    D'Oro, U; Sakaguchi, K; Appella, E; Ashwell, J D

    1996-01-01

    The CD45 tyrosine phosphatase has been reported to activate the src family tyrosine kinases Lck and Fyn by dephosphorylating regulatory COOH-terminal tyrosine residues 505 and 528, respectively. However, recent studies with CD45- T-cell lines have found that despite the fact that Lck and Fyn were constitutively hyperphosphorylated, the tyrosine kinase activity of both enzymes was actually increased. In the present study, phosphoamino acid analysis revealed that the increased phosphorylation of Lck in CD45- YAC-1 T cells was restricted to tyrosine residues. To understand the relationship between tyrosine phosphorylation and Lck kinase activity, CD45- YAC-1 cells were transfected with forms of Lck in which tyrosines whose phosphorylation is thought to regulate enzyme activity (Tyr-192, Tyr-394, Tyr-505, or both Tyr-394 and Tyr-505) were replaced with phenylalanine. While the Y-to-F mutation at position 192 (192-Y-->F) had little effect, the 505-Y-->F mutation increased enzymatic activity. In contrast, the 394-Y-->F mutation decreased the kinase activity to very low levels, an effect that the double mutation, 394-Y-->F and 505Y-->F, could not reverse. Phosphopeptide analysis of tryptic digests of Lck from CD45- YAC-1 cells revealed that it is hyperphosphorylated on two tyrosine residues, Tyr-505 and, to a lesser extent, Tyr-394. The purified and enzymatically active intracellular portion of CD45 dephosphorylated Lck Tyr-394 in vitro. These results demonstrate that in addition to Tyr-505, CD45 can dephosphorylate Tyr-394, and that in the absence of CD45 the hyperphosphorylation of Tyr-394 can cause an increase in the kinase activity of Lck despite the inhibitory hyperphosphorylation of Tyr-505. Therefore, Lck kinase activity is determined by the balance of activating and inhibitory tyrosine phosphorylations that are, in turn, regulated by CD45. PMID:8756658

  8. Haloperoxidases: Enzymatic Synthesis of ?,?-Halohydrins from Gaseous Alkenes

    PubMed Central

    Geigert, John; Neidleman, Saul L.; Dalietos, Demetrios J.; DeWitt, Susanne K.

    1983-01-01

    The enzymatic synthesis of ?,?-halohydrins from gaseous alkenes is described. The enzymatic reaction required an alkene, a halide ion, dilute hydrogen peroxide, and a haloperoxidase enzyme. A wide range of gaseous alkenes were suitable for this reaction, including those containing isolated, conjugated, and cumulative carbon-carbon double bonds. Chlorohydrins, bromohydrins, and iodohydrins could be formed. The combining of this enzymatic synthesis with a previously described enzymatic synthesis of epoxides from ?,?-halohydrins provides an alternate pathway, other than the well-known enzymatic direct epoxidation pathway, from alkene to an epoxide. PMID:16346188

  9. KIDs rule: regulatory phosphorylation of RTKs.

    PubMed

    Locascio, Lauren E; Donoghue, Daniel J

    2013-02-01

    Receptor tyrosine kinases (RTKs) are mediators of multiple cell signaling networks linked to cell growth and differentiation. In general, they exhibit similar overall structure with a ligand-binding extracellular domain and a conserved intracellular tyrosine kinase domain. In many RTKs, the kinase domain is interrupted by a sequence known as the kinase insert domain (KID). In addition to phosphorylation sites within the kinase domain, regulatory phosphorylation also occurs within the KID of several RTKs important in human health and disease. Phosphorylation of specific Tyr or Ser residues within the KID of some RTKs triggers distinct cellular signaling outcomes. Here, we review the functionality of KIDs throughout all RTK families, and provide justification for further study of this often-overlooked domain. PMID:23312584

  10. Src kinase regulation by phosphorylation and dephosphorylation

    SciTech Connect

    Roskoski, Robert [Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1100 Florida Avenue, New Orleans, LA 70119 (United States)]. E-mail: biocrr@lsuhsc.edu

    2005-05-27

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTP{alpha}, PTP{epsilon}, and PTP{lambda}. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.

  11. Focus Issue: Systems Analysis of Protein Phosphorylation

    NSDL National Science Digital Library

    Nancy R. Gough (American Association for the Advancement of Science; Science Signaling REV)

    2010-08-31

    Kinases and phosphatases are key regulatory molecules that participate in most cell signaling pathways. Systems-level analyses are providing new insights into phosphorylation sites and kinase specificity, and phosphoproteomic analyses are creating not just a wealth of data, but are also revealing rich revelations about cellular behavior, cellular responses to changing environmental conditions, and mechanisms of disease. Phosphatases are not merely signal terminators, but play active roles in signal transduction and are especially important in redox signaling. In this Focus Issue, Science Signaling highlights protein phosphorylation as the center of the signaling universe.

  12. SPONTANEOUS AND CATALYZED HYDROGEN SHIFTS IN RADICAL CATIONS HAVING A PHOSPHORYL OR CARBONYL GROUP

    Microsoft Academic Search

    Lisa Natasha Heydorn

    2003-01-01

    Intermolecular and intramolecular hydrogen shifts represent a key component of a vast number of chemical reactions. This is particularly true for radical cations, whose high reactivity makes them prone to isomerization and dissociation reactions. In the context of the experimental work in this thesis, hydrogen transfers involved in both the intra-and inter-molecular isomerization of radical cations containing a phosphoryl (P=O)

  13. Biofunctional Properties of Enzymatic Squid Meat Hydrolysate

    PubMed Central

    Choi, Joon Hyuk; Kim, Kyung-Tae; Kim, Sang Moo

    2015-01-01

    Squid is one of the most important commercial fishes in the world and is mainly utilized or consumed as sliced raw fish or as processed products. The biofunctional activities of enzymatic squid meat hydrolysate were determined to develop value-added products. Enzymatic squid hydrolysate manufactured by Alcalase effectively quenched 1,1-diphenyl-2-picrylhydrazyl radical, hydroxyl radical, and hydrogen peroxide radical with IC50 values of 311, 3,410, and 111.5 ?g/mL, respectively. Angiotensin I-converting enzyme inhibitory activity of squid hydrolysate was strong with an IC50 value of 145.1 ?g/mL, while tyrosinase inhibitory activity with an IC50 value of 4.72 mg/mL was moderately low. Overall, squid meat hydrolysate can be used in food or cosmetic industries as a bioactive ingredient and possibly be used in the manufacture of seasoning, bread, noodle, or cosmetics. PMID:25866752

  14. Enzymatic de Novo Pyrimidine Nucleotide Synthesis

    PubMed Central

    Schultheisz, Heather L.; Szymczyna, Blair R.; Scott, Lincoln G.

    2011-01-01

    The use of stable isotope labeling has revolutionized NMR studies of nucleic acids, and there is a need for methods of incorporation of specific isotope labels to facilitate specific NMR experiments and applications. Enzymatic synthesis offers an efficient and flexible means to synthesize nucleoside triphosphates from a variety of commercially available specifically labeled precursors, permitting isotope labeling of RNAs prepared by in vitro transcription. Here, we recapitulate de novo pyrimidine biosynthesis in vitro, using recombinantly expressed enzymes to perform efficient single-pot syntheses of UTP and CTP that bear a variety of stable isotope labeling patterns. Filtered NMR experiments on 13C, 15N, 2H-labeled HIV-2 TAR RNA demonstrate the utility and value of this approach. This flexible enzymatic synthesis will make implementing detailed and informative RNA stable isotope labeling schemes substantially more cost effective and efficient, providing advanced tools for the study of structure and dynamics of RNA molecules. PMID:21166398

  15. Enzymatic intracrine regulation of white adipose tissue

    PubMed Central

    DiSilvestro, David; Petrosino, Jennifer; Aldoori, Ayat; Melgar-Bermudez, Emiliano; Wells, Alexandra; Ziouzenkova, Ouliana

    2015-01-01

    Abdominal fat formation has become a permanent risk factor for metabolic syndrome and various cancers in one-third of the world's population of obese and even lean patients. Formation of abdominal fat involves additional mechanisms beyond an imbalance in energy intake and expenditure, which explains systemic obesity. In this review, we briefly summarized autonomous regulatory circuits that locally produce hormones from inactive precursors or nutrients for intra-/auto-/paracrine signaling in white adipose depots. Enzymatic pathways activating steroid and thyroid hormones in adipose depots were compared with enzymatic production of retinoic acid from vitamin A. We discussed the role of intracrine circuits in fat-depot functions and strategies to reduce abdominal adiposity through thermogenic adipocytes with interrupted generation of retinoic acid. PMID:25390015

  16. Enzymatic degradation of polycaprolactone–gelatin blend

    NASA Astrophysics Data System (ADS)

    Banerjee, Aditi; Chatterjee, Kaushik; Madras, Giridhar

    2015-04-01

    Blends of polycaprolactone (PCL), a synthetic polymer and gelatin, natural polymer offer a optimal combination of strength, water wettability and cytocompatibility for use as a resorbable biomaterial. The enzymatic degradation of PCL, gelatin and PCL–gelatin blended films was studied in the presence of lipase (Novozym 435, immobilized) and lysozyme. Novozym 435 degraded the PCL films whereas lysozyme degraded the gelatin. Though Novozym 435 and lysozyme individually could degrade PCL–gelatin blended films, the combination of these enzymes showed the highest degradation of these blended films. Moreover, the enzymatic degradation was much faster when fresh enzymes were added at regular intervals. The changes in physico-chemical properties of polymer films due to degradation were studied by scanning electron microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. These results have important implications for designing resorbable biomedical implants.

  17. Enzymatic transformation of nonfood biomass to starch

    PubMed Central

    You, Chun; Chen, Hongge; Myung, Suwan; Sathitsuksanoh, Noppadon; Ma, Hui; Zhang, Xiao-Zhou; Li, Jianyong; Zhang, Y.-H. Percival

    2013-01-01

    The global demand for food could double in another 40 y owing to growth in the population and food consumption per capita. To meet the world’s future food and sustainability needs for biofuels and renewable materials, the production of starch-rich cereals and cellulose-rich bioenergy plants must grow substantially while minimizing agriculture’s environmental footprint and conserving biodiversity. Here we demonstrate one-pot enzymatic conversion of pretreated biomass to starch through a nonnatural synthetic enzymatic pathway composed of endoglucanase, cellobiohydrolyase, cellobiose phosphorylase, and alpha-glucan phosphorylase originating from bacterial, fungal, and plant sources. A special polypeptide cap in potato alpha-glucan phosphorylase was essential to push a partially hydrolyzed intermediate of cellulose forward to the synthesis of amylose. Up to 30% of the anhydroglucose units in cellulose were converted to starch; the remaining cellulose was hydrolyzed to glucose suitable for ethanol production by yeast in the same bioreactor. Next-generation biorefineries based on simultaneous enzymatic biotransformation and microbial fermentation could address the food, biofuels, and environment trilemma. PMID:23589840

  18. Enzymatic glycoprotein synthesis: Preparation of ribonuclease glycoforms via enzymatic glycopeptide condensation and glycosylation

    SciTech Connect

    Witte, K.; Sears, P.; Martin, R.; Wong, C.H. [Scripps Research Inst., La Jolla, CA (United States)] [Scripps Research Inst., La Jolla, CA (United States)

    1997-03-05

    In order to study the effects carbohydrates have on glycoprotein structure and funciton, it is imperative to be able to synthesize the appropriate natural and non-natural glycoprotein variants in a single form. Because the available in vivo techniques provide only heterogeneous mixtures of different glycoforms, enzymatic in vitro methodologies have been pursued. Using the N-glycoprotein RNase B as a model system, the oligosaccharide was removed leaving only the N-acetylglucosamine as a `tag` to the site of glycosylation. Glycosyltransferases were then used to build a unique carbohydrate moiety. A new RNase glycoform containing the branched oligosccharide, sialyl Lewis X or the Hg derivative, was synthesized enzymatically to demonstrate the feasibility of the method. In addition, the monoglycosylated protein was digested into several smaller pieces by subtilisin BPN`. These fragments were religated by subtilisin 8397 to the full length RNase by addition glycerol; this method points to a new chemical-enzymatic process for the synthesis of glycoproteins using synthetic peptides and glycopeptides as substrates for enzymatic ligation followed by further enzymatic glycosylations. 29 refs., 6 figs.

  19. Interactive effect of oxytetracycline and lead on soil enzymatic activity and microbial biomass.

    PubMed

    Gao, Minling; Song, Wenhua; Zhou, Qian; Ma, Xiaojun; Chen, Xiaoying

    2013-09-01

    Interactive effect of oxytetracyline (OTC) and lead on soil enzymatic activity and population of microbes was studied in the paper. The results showed effect of pollutants on bacteria, actinomycetes and enzymatic activity increased in the order: (OTC+Pb)>Pb>OTC, (OTC+Pb)>Pb>OTC and (OTC+Pb)>OTC>Pb, respectively. However, impact of pollutants on fungi decreased in the order: (OTC+Pb)transfer, resulting from oxygen and carbon atoms coordinated with metal ions, other chemicals or partial decomposition. Thus, the findings of this study can provide a better insight into the interaction of both pollutants and their impacts on soil quality. PMID:23892283

  20. Ion channels, phosphorylation and mammalian sperm capacitation

    PubMed Central

    Visconti, Pablo E; Krapf, Dario; de la Vega-Beltrán, José Luis; Acevedo, Juan José; Darszon, Alberto

    2011-01-01

    Sexually reproducing animals require an orchestrated communication between spermatozoa and the egg to generate a new individual. Capacitation, a maturational complex phenomenon that occurs in the female reproductive tract, renders spermatozoa capable of binding and fusing with the oocyte, and it is a requirement for mammalian fertilization. Capacitation encompasses plasma membrane reorganization, ion permeability regulation, cholesterol loss and changes in the phosphorylation state of many proteins. Novel tools to study sperm ion channels, image intracellular ionic changes and proteins with better spatial and temporal resolution, are unraveling how modifications in sperm ion transport and phosphorylation states lead to capacitation. Recent evidence indicates that two parallel pathways regulate phosphorylation events leading to capacitation, one of them requiring activation of protein kinase A and the second one involving inactivation of ser/thr phosphatases. This review examines the involvement of ion transporters and phosphorylation signaling processes needed for spermatozoa to achieve capacitation. Understanding the molecular mechanisms leading to fertilization is central for societies to deal with rising male infertility rates, to develop safe male gamete-based contraceptives and to preserve biodiversity through better assisted fertilization strategies. PMID:21540868

  1. Cold enzymatic bleaching of fluid whey.

    PubMed

    Campbell, R E; Drake, M A

    2013-12-01

    Chemical bleaching of fluid whey and retentate with hydrogen peroxide (HP) alone requires high concentrations (100-500 mg of HP/kg) and recent studies have demonstrated that off-flavors are generated during chemical bleaching that carry through to spray-dried whey proteins. Bleaching of fluid whey and retentate with enzymes such as naturally present lactoperoxidase or an exogenous commercial peroxidase (EP) at cold temperatures (4°C) may be a viable alternative to traditional chemical bleaching of whey. The objective of this study was to determine the optimum level of HP for enzymatic bleaching (both lactoperoxidase and EP) at 4°C and to compare bleaching efficacy and sensory characteristics to HP chemical bleaching at 4°C. Selected treatments were subsequently applied for whey protein concentrate with 80% protein (WPC80) manufacture. Fluid Cheddar whey and retentate (80% protein) were manufactured in triplicate from pasteurized whole milk. The optimum concentration of HP (0 to 250 mg/kg) to activate enzymatic bleaching at 4°C was determined by quantifying the loss of norbixin. In subsequent experiments, bleaching efficacy, descriptive sensory analysis, and volatile compounds were monitored at selected time points. A control with no bleaching was also evaluated. Enzymatic bleaching of fluid whey and retentate at 4°C resulted in faster bleaching and higher bleaching efficacy (color loss) than bleaching with HP alone at 250 mg/kg. Due to concentrated levels of naturally present lactoperoxidase, retentate bleached to completion (>80% norbixin destruction in 30 min) faster than fluid whey at 4°C (>80% norbixin destruction in 12h). In fluid whey, the addition of EP decreased bleaching time. Spray-dried WPC80 from bleached wheys, regardless of bleaching treatment, were characterized by a lack of sweet aromatic and buttery flavors, and the presence of cardboard flavor concurrent with higher relative abundance of 1-octen-3-ol and 1-octen-3-one. Among enzymatically bleached WPC80, lactoperoxidase-bleached WPC80 contained higher relative abundance of 2,3-octadienone, 2-pentyl furan, and hexanal than those bleached with added EP. Bleach times, bleaching efficacy, and flavor results suggest that enzymatic bleaching may be a viable and desirable alternative to HP bleaching of fluid whey or retentate. PMID:24140314

  2. PKA regulates calcineurin function through the phosphorylation of RCAN1: identification of a novel phosphorylation site.

    PubMed

    Kim, Seon Sook; Lee, Eun Hye; Lee, Kooyeon; Jo, Su-Hyun; Seo, Su Ryeon

    2015-04-17

    Calcineurin is a calcium/calmodulin-dependent phosphatase that has been implicated in T cell activation through the induction of nuclear factors of activated T cells (NFAT). We have previously suggested that endogenous regulator of calcineurin (RCAN1, also known as DSCR1) is targeted by protein kinase A (PKA) for the control of calcineurin activity. In the present study, we characterized the PKA-mediated phosphorylation site in RCAN1 by mass spectrometric analysis and revealed that PKA directly phosphorylated RCAN1 at the Ser 93. PKA-induced phosphorylation and the increase in the half-life of the RCAN1 protein were prevented by the substitution of Ser 93 with Ala (S93A). Furthermore, the PKA-mediated phosphorylation of RCAN1 at Ser 93 potentiated the inhibition of calcineurin-dependent pro-inflammatory cytokine gene expression by RCAN1. Our results suggest the presence of a novel phosphorylation site in RCAN1 and that its phosphorylation influences calcineurin-dependent inflammatory target gene expression. PMID:25753203

  3. Protein phosphorylation in isolated human adipocytes - Adrenergic control of the phosphorylation of hormone-sensitive lipase

    SciTech Connect

    Smiley, R.M. (Rockefeller Univ., New York, NY (USA) Columbia Univ College of Physicians and Surgeons, New York, NY (USA)); Paul, S.; Browning, M.D.; Leibel, R.L.; Hirsch, J. (Rockefeller Univ., New York, NY (USA))

    1990-01-01

    The effect of adrenergic agents on protein phosphorylation in human adipocytes was examined. Freshly isolated human fat cells were incubated with {sup 32}PO{sub 4} in order to label intracellular ATP, then treated with a variety of adrenergic and other pharmacologic agents. Treatment with the {beta}-adrenergic agonist isoproterenol led to a significant increase in phosphate content of at least five protein bands (M{sub r} 52, 53, 63, 67, 84 kDa). The increase in phosphorylation was partially inhibited by the {alpha}-2 agonist clonidine. Epinephrine, a combined {alpha} and {beta} agonist, was less effective at increasing phosphate content of the proteins than was isoproterenol. Neither insulin nor the {alpha}-1 agonist phenylephrine had any discernible effect on the pattern of protein phosphorylation. The 84 kDa phosphorylated peptide band appears to contain hormone-sensitive lipase, a key enzyme in the lipolytic pathway which is activated by phosphorylation. These results are somewhat different than previously reported results for rat adipocytes, and represent the first report of overall pattern and adrenergic modulation of protein phosphorylation in human adipocytes.

  4. Nuclear tyrosine phosphorylation: the beginning of a map

    Microsoft Academic Search

    Christophe Cans; Raffaella Mangano; Daniela Barilá; Gitte Neubauer; Giulio Superti-Furga

    2000-01-01

    Tyrosine phosphorylation is usually associated with cytoplasmic events. Yet, over the years, many reports have accumulated on tyrosine phosphorylation of individual molecules in the nucleus, and several tyrosine kinases and phosphatases have been found to be at least partially nuclear. The question arises as to whether nuclear tyrosine phosphorylation represents a collection of loose ends of events originating in the

  5. The significance of regulatory light chain phosphorylation in cardiac physiology

    Microsoft Academic Search

    Sarah B. Scruggs; R. John Solaro

    2011-01-01

    It has been over 35years since the first identification of phosphorylation of myosin light chains in skeletal and cardiac muscle. Yet only in the past few years has the role of these phosphorylations in cardiac dynamics been more fully understood. Advances in this understanding have come about with further evidence on the control mechanisms regulating the level of phosphorylation by

  6. A systematic approach to the analysis of protein phosphorylation

    Microsoft Academic Search

    Huilin Zhou; Julian D. Watts; Ruedi Aebersold

    2001-01-01

    Reversible protein phosphorylation has been known for some time to control a wide range of biological functions and activities. Thus determination of the site(s) of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. However, direct determination of individual phosphorylation sites occurring on phosphoproteins in vivo has been difficult to date, typically

  7. Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane

    PubMed Central

    Takagishi, Teruhisa; Oda, Masataka; Kabura, Michiko; Kurosawa, Mie; Tominaga, Kaori; Urano, Shiori; Ueda, Yoshibumi; Kobayashi, Keiko; Kobayashi, Toshihide; Sakurai, Jun; Terao, Yutaka; Nagahama, Masahiro

    2015-01-01

    Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC) hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG) with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-? (EYFP-C1AB). DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase C?-1 (PLC?-1), a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLC?-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step) and activation of endogenous PLC?-1 (second step) leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA. PMID:25910247

  8. Dysfunctional conformational dynamics of protein kinase A induced by a lethal mutant of phospholamban hinder phosphorylation.

    PubMed

    Kim, Jonggul; Masterson, Larry R; Cembran, Alessandro; Verardi, Raffaello; Shi, Lei; Gao, Jiali; Taylor, Susan S; Veglia, Gianluigi

    2015-03-24

    The dynamic interplay between kinases and substrates is crucial for the formation of catalytically committed complexes that enable phosphoryl transfer. However, a clear understanding on how substrates modulate kinase structural dynamics to control catalytic efficiency is still missing. Here, we used solution NMR spectroscopy to study the conformational dynamics of two complexes of the catalytic subunit of the cAMP-dependent protein kinase A with WT and R14 deletion phospholamban, a lethal human mutant linked to familial dilated cardiomyopathy. Phospholamban is a central regulator of heart muscle contractility, and its phosphorylation by protein kinase A constitutes a primary response to ?-adrenergic stimulation. We found that the single deletion of arginine in phospholamban's recognition sequence for the kinase reduces its binding affinity and dramatically reduces phosphorylation kinetics. Structurally, the mutant prevents the enzyme from adopting conformations and motions committed for catalysis, with concomitant reduction in catalytic efficiency. Overall, these results underscore the importance of a well-tuned structural and dynamic interplay between the kinase and its substrates to achieve physiological phosphorylation levels for proper Ca(2+) signaling and normal cardiac function. PMID:25775607

  9. Global Quantitative SILAC Phosphoproteomics Reveals Differential Phosphorylation Is Widespread between the Procyclic and

    E-print Network

    Schnaufer, Achim

    Global Quantitative SILAC Phosphoproteomics Reveals Differential Phosphorylation Is Widespread in this organism. KEYWORDS: phosphorylation, SILAC, Trypanosoma brucei, quantitative proteomics, phosphoproteomics

  10. Nature-inspired enzymatic cascades to build valuable compounds.

    PubMed

    Sigrist, Renata; Costa, Bruna Zucoloto da; Marsaioli, Anita Jocelyne; de Oliveira, Luciana Gonzaga

    2015-01-01

    Biocatalysis currently is focusing on enzymatic and multi-enzymatic cascade processes instead of single steps imbedded into chemical pathways. Alongside this scientific revolution, this review provides an overview on multi-enzymatic cascades that are responsible for the biosynthesis of some terpenes, alkaloids and polyethers, which are important classes of natural products. Herein, we illustrate the development of studies inspired by multi- and chemo-enzymatic approaches to build the core moieties of polyethers, polypeptide alkaloids, piperidines and pyrrolidines promoted by the joint action of oxidoreductases, hydrolases, cyclases, transaminases and imine reductases. PMID:25795056

  11. Monoclonal Antibodies Distinguish Several Differentially Phosphorylated States of the Two Largest Rat Neurofilament Subunits (NF-H and NFM) and Demonstrate Their Existence in the Normal Nervous System of Adult Rats

    Microsoft Academic Search

    Virginia M.-Y. Lee; Martin J. Carden; William W. Schlaepfer; John Q. Trojanowski

    A new panel of >300 monoclonal antibodies (mAbs) was prepared to the high, middle, and low M. rat neurofilament (NF) subunits (NF-H, NF-M and NF-L, respectively). NF pro- teins were purified both from native, i.e., phosphorylated rat NFs and from enzymatically dephosphorylated rat NFs. The resulting mAbs were used to biochemically and immuno- chemically distinguish and characterize distinct and differ-

  12. Effect of Ser-129 Phosphorylation on Interaction of ?-Synuclein with Synaptic and Cellular Membranes*

    PubMed Central

    Visanji, Naomi P.; Wislet-Gendebien, Sabine; Oschipok, Loren W.; Zhang, Gang; Aubert, Isabelle; Fraser, Paul E.; Tandon, Anurag

    2011-01-01

    In the healthy brain, less than 5% of ?-synuclein (?-syn) is phosphorylated at serine 129 (Ser(P)-129). However, within Parkinson disease (PD) Lewy bodies, 89% of ?-syn is Ser(P)-129. The effects of Ser(P)-129 modification on ?-syn distribution and solubility are poorly understood. As ?-syn normally exists in both membrane-bound and cytosolic compartments, we examined the binding and dissociation of Ser(P)-129 ?-syn and analyzed the effects of manipulating Ser(P)-129 levels on ?-syn membrane interactions using synaptosomal membranes and neural precursor cells from ?-syn-deficient mice or transgenic mice expressing human ?-syn. We first evaluated the recovery of the Ser(P)-129 epitope following either ?-syn membrane binding or dissociation. We demonstrate a rapid turnover of Ser(P)-129 during both binding to and dissociation from synaptic membranes. Although the membrane binding of WT ?-syn was insensitive to modulation of Ser(P)-129 levels by multiple strategies (the use of phosphomimic S129D and nonphosphorylated S129A ?-syn mutants; by enzymatic dephosphorylation of Ser(P)-129 or proteasome inhibitor-induced elevation in Ser(P)-129; or by inhibition or stable overexpression of PLK2), PD mutant Ser(P)-129 ?-syn showed a preferential membrane association compared with WT Ser(P)-129 ?-syn. Collectively, these data suggest that phosphorylation at Ser-129 is dynamic and that the subcellular distribution of ?-syn bearing PD-linked mutations, A30P or A53T, is influenced by the phosphorylation state of Ser-129. PMID:21849493

  13. Short-term TNF? shedding is independent of cytoplasmic phosphorylation or furin cleavage of ADAM17.

    PubMed

    Schwarz, Jeanette; Broder, Claudia; Helmstetter, Ansgard; Schmidt, Stefanie; Yan, Isabell; Müller, Miryam; Schmidt-Arras, Dirk; Becker-Pauly, Christoph; Koch-Nolte, Friedrich; Mittrücker, Hans-Willi; Rabe, Björn; Rose-John, Stefan; Chalaris, Athena

    2013-12-01

    Proteolysis of transmembrane molecules is an irreversible post-translational modification enabling autocrine, paracrine and endocrine signaling of many cytokines. The pro-inflammatory activities of membrane bound TNF? (pro-TNF?) strongly depend on ectodomain shedding mediated by the A Disintegrin And Metalloprotease family member ADAM17. Despite the well-documented role of ADAM17 in pro-TNF? cleavage during inflammation, little is known about its regulation. Mitogen-activated protein kinase-induced phosphorylation of the ADAM17 cytoplasmic tail has been described to be required for proper activation. To address, if pro-TNF? shedding depends on cytosolic phosphorylation we analyzed ADAM17 mutants lacking the cytoplasmic domain. ADAM17 mediated shedding of pro-TNF? was induced by PMA, Anisomycin and the phosphatase inhibitors Cantharidin and Calyculin A. Deletion of the entire cytoplasmic portion of ADAM17 abolished furin-dependent proteolytic maturation and pro-TNF? cleavage. Interestingly, we could exclude that resistance to proconvertase processing is the reason for the enzymatic inactivity of ADAM17 lacking the cytoplasmic portion as furin-resistant ADAM17 mutants rescued genetic ADAM17 deficiency after mitogen-activated protein kinase activation. Adding only 6 cytoplasmic amino acids completely restored ADAM17 maturation and shedding of pro-TNF? as well as of both TNF-receptors Finally, we showed that a pro-TNF? mutant lacking the cytoplasmic portion was also shed from the cell surface. We conclude that pro-TNF? cleavage by its major sheddase ADAM17 does not depend on cytosolic phosphorylation and/or interaction. These results have general implications on understanding the activation mechanism controlling the activity of ADAM17. PMID:24135057

  14. Enzymatic surface modification of acrylonitrile fibers

    NASA Astrophysics Data System (ADS)

    Battistel, Ezio; Morra, Marco; Marinetti, Massimo

    2001-06-01

    The surface of polyacrylonitrile polymer (containing 10% acetate groups) as fibers and finely ground powder have been modified by enzymatic treatment. The enzyme used was a nitrile hydratase, member of the class of nitrile converting enzymes, present in the microorganisms Brevibacterium imperiale and Corynebacterium nitrilophilus. The pendant nitrile groups were selectively converted into the corresponding amides as assessed by XPS analysis. As indicated by the increase of the O/C atomic ratio, the fiber surface showed a significant increase in hydrophilicity. The newly formed amide groups were then able to react with the acid dyes typically used to stain natural fibers, conferring the coloring properties to the otherwise inert polymer surface.

  15. Phosphorylation of Kraft fibers with phosphate esters.

    PubMed

    Shi, Ying; Belosinschi, Dan; Brouillette, François; Belfkira, Ahmed; Chabot, Bruno

    2014-06-15

    Phosphate esters, derived from two different long-chain aliphatic alcohols, were used as phosphorylating reagents for Kraft pulp fibers. High phosphorus contents and almost non-degraded fibers were obtained by following this pathway. The phosphorylation efficiency was influenced by the alkyl chain length of PEs since the phosphorus content in modified fibers was higher for the shorter chain reagent. Due to the heterogeneous reaction environment, the amount of grafted phosphorus was found to be almost three times higher at the surface than in the bulk of the fibers. Analyses also indicated that the phosphorus was bonded to fibers as a phosphate-like structure. Furthermore, the situation seemed to be different for the fiber surface where significant amounts of phosphorus were present in more complex structures like pyrophosphate or even oligo-phosphate. PMID:24721058

  16. Exocyst function regulated by effector phosphorylation

    Microsoft Academic Search

    Xiao-Wei Chen; Dara Leto; Junyu Xiao; John Goss; Qian Wang; Jordan A. Shavit; Tingting Xiong; Genggeng Yu; David Ginsburg; Derek Toomre; Zhaohui Xu; Alan R. Saltiel

    2011-01-01

    The exocyst complex tethers vesicles at sites of fusion through interactions with small GTPases. The G protein RalA resides on Glut4 vesicles, and binds to the exocyst after activation by insulin, but must then disengage to ensure continuous exocytosis. Here we report that, after recognition of the exocyst by activated RalA, disengagement occurs through phosphorylation of its effector Sec5, rather

  17. MAP kinase phosphorylation of plant profilin

    Microsoft Academic Search

    Apinun Limmongkon; Concetta Giuliani; Rudolf Valenta; Irene Mittermann; Erwin Heberle-Bors; Cathal Wilson

    2004-01-01

    Profilin is a small actin-binding protein and is expressed at high levels in mature pollen where it is thought to regulate actin filament dynamics upon pollen germination and tube growth. The majority of identified plant profilins contain a MAP kinase phosphorylation motif, P–X–T–P, and a MAP kinase interaction motif (KIM). In in vitro kinase assays, the tobacco MAP kinases p45Ntf4

  18. Hormonal regulation of mitochondrial oxidative phosphorylation

    E-print Network

    Djavadi, Gholam Reza

    1969-01-01

    techniques, Sarkissian and NcDaniel (1966) observed enhanced phosphorylative activity in corn mitochondria utilizing n ? ketoglutarate (n-KGA) as substrate when the Warburg vessel contained catalytic amount of IAA. The present study is a continuation... have extensively been used: manometric and polarographic techniques. In spite of the fact that in previous reports from this laboratory (HcDaniel and Sarkissian 1966; Sarkissian and NcDaniel 1966) manometric techniques had been used, author chose...

  19. Multistep phosphorylation systems: tunable components of biological signaling circuits.

    PubMed

    Valk, Evin; Venta, Rainis; Ord, Mihkel; Faustova, Ilona; Kõivomägi, Mardo; Loog, Mart

    2014-11-01

    Multisite phosphorylation of proteins is a powerful signal processing mechanism that plays crucial roles in cell division and differentiation as well as in disease. We recently demonstrated a novel phenomenon in cell cycle regulation by showing that cyclin-dependent kinase-dependent multisite phosphorylation of a crucial substrate is performed sequentially in the N-to-C terminal direction along the disordered protein. The process is controlled by key parameters, including the distance between phosphorylation sites, the distribution of serines and threonines in sites, and the position of docking motifs. According to our model, linear patterns of phosphorylation along disordered protein segments determine the signal-response function of a multisite phosphorylation switch. Here we discuss the general advantages and engineering principles of multisite phosphorylation networks as processors of kinase signals. We also address the idea of using the mechanistic logic of linear multisite phosphorylation networks to design circuits for synthetic biology applications. PMID:25368420

  20. Imaging phosphorylated peptide distribution in human lens by MALDI MS.

    PubMed

    Jiao, Jing; Miao, Aizhu; Zhang, Ying; Fan, Qi; Lu, Yi; Lu, Haojie

    2015-06-21

    Phosphorylation plays vital roles in complex biological processes such as cellular growth, division and signaling transduction. However, due to the low ionization efficiency of phosphorylated peptides, it is still a huge challenge to obtain region-specific phosphorylated peptide distribution by imaging mass spectrometry. To achieve the on-tissue analysis of phosphorylated peptides, we took advantage of a graphene oxide-immobilized enzyme reactor to conduct the in situ digestion, followed by dephosphorylation treatment that removed the phosphate groups and thereby helped to improve the signal intensity of phosphorylated peptides. A visual representation of the phosphoproteome of a human lens was successfully mapped. Results showed that phosphorylated peptides localized mainly in the nucleus region of a healthy lens while the outer cortex is the dominant region for phosphorylated peptides of a cataractous lens. PMID:25943257

  1. Mixed mechanisms of multi-site phosphorylation.

    PubMed

    Suwanmajo, Thapanar; Krishnan, J

    2015-06-01

    Multi-site phosphorylation is ubiquitous in cell biology and has been widely studied experimentally and theoretically. The underlying chemical modification mechanisms are typically assumed to be distributive or processive. In this paper, we study the behaviour of mixed mechanisms that can arise either because phosphorylation and dephosphorylation involve different mechanisms or because phosphorylation and/or dephosphorylation can occur through a combination of mechanisms. We examine a hierarchy of models to assess chemical information processing through different mixed mechanisms, using simulations, bifurcation analysis and analytical work. We demonstrate how mixed mechanisms can show important and unintuitive differences from pure distributive and processive mechanisms, in some cases resulting in monostable behaviour with simple dose-response behaviour, while in other cases generating new behaviour-like oscillations. Our results also suggest patterns of information processing that are relevant as the number of modification sites increases. Overall, our work creates a framework to examine information processing arising from complexities of multi-site modification mechanisms and their impact on signal transduction. PMID:25972433

  2. Phosphorylation Stoichiometries of Human Eukaryotic Initiation Factors

    PubMed Central

    Andaya, Armann; Villa, Nancy; Jia, Weitao; Fraser, Christopher S.; Leary, Julie A.

    2014-01-01

    Eukaryotic translation initiation factors are the principal molecular effectors regulating the process converting nucleic acid to functional protein. Commonly referred to as eIFs (eukaryotic initiation factors), this suite of proteins is comprised of at least 25 individual subunits that function in a coordinated, regulated, manner during mRNA translation. Multiple facets of eIF regulation have yet to be elucidated; however, many of the necessary protein factors are phosphorylated. Herein, we have isolated, identified and quantified phosphosites from eIF2, eIF3, and eIF4G generated from log phase grown HeLa cell lysates. Our investigation is the first study to globally quantify eIF phosphosites and illustrates differences in abundance of phosphorylation between the residues of each factor. Thus, identification of those phosphosites that exhibit either high or low levels of phosphorylation under log phase growing conditions may aid researchers to concentrate their investigative efforts to specific phosphosites that potentially harbor important regulatory mechanisms germane to mRNA translation. PMID:24979134

  3. Phosphorylation of erythrocyte membrane liberates calcium

    SciTech Connect

    Chauhan, V.P.S.; Brockerhoff, H.

    1986-05-01

    Phosphorylation of permeabilized erythrocyte ghost membranes with ATP results in an increase free calcium level as measured with the help of Ca/sup 2 +/ electrode and /sup 45/Ca. This effect could not be observed in the presence of p/sup -/ chloromercuric benzoate, an inhibitor of kinases. The rise in the free calcium due to phosphorylation of the membrane was accompanied by a decrease in the level of phosphatidylinositol (PI) and an increase in phosphatidylinositolmonophosphate (PIP) and phosphatidylinositolbisphosphate (PIP/sub 2/). These results support the proposal that an inositol shuttle, PI in equilibrium PIP in equilibrium PIP/sub 2/, operates to maintain the intracellular calcium concentration. The cation is believed to be sequestered in a cage formed by the head groups of two acidic phospholipid molecules, e.g., phosphatidylserine and phosphatidylinositol, with the participation of both PO and fatty acid ester CO groups. When the inositol group of such a cage is phosphorylated, inter-headgroup hydrogen bonding between the lipids is broken. As a result the cage opens and calcium is released.

  4. Phosphorylation network rewiring by gene duplication

    PubMed Central

    Freschi, Luca; Courcelles, Mathieu; Thibault, Pierre; Michnick, Stephen W; Landry, Christian R

    2011-01-01

    Elucidating how complex regulatory networks have assembled during evolution requires a detailed understanding of the evolutionary dynamics that follow gene duplication events, including changes in post-translational modifications. We compared the phosphorylation profiles of paralogous proteins in the budding yeast Saccharomyces cerevisiae to that of a species that diverged from the budding yeast before the duplication of those genes. We found that 100 million years of post-duplication divergence are sufficient for the majority of phosphorylation sites to be lost or gained in one paralog or the other, with a strong bias toward losses. However, some losses may be partly compensated for by the evolution of other phosphosites, as paralogous proteins tend to preserve similar numbers of phosphosites over time. We also found that up to 50% of kinase–substrate relationships may have been rewired during this period. Our results suggest that after gene duplication, proteins tend to subfunctionalize at the level of post-translational regulation and that even when phosphosites are preserved, there is a turnover of the kinases that phosphorylate them. PMID:21734643

  5. The Oniom Method and its Applications to Enzymatic Reactions

    NASA Astrophysics Data System (ADS)

    Lundberg, Marcus; Morokuma; Keiji

    ONIOM is a flexible hybrid scheme that can combine the most suitable computational methods for a given system without previous parameterization. The reason for its flexibility is that all calculations are performed on complete molecular systems, and the total energy is obtained from an extrapolation scheme. Most commonly used is the combination of a quantum mechanics and a molecular mechanics method (ONIOM QM:MM), and we describe applications of this method to several enzymatic systems, e.g., glutathione peroxidase and methylmalonyl-CoA mutase. The role of the protein is highlighted by comparing models with and without explicit inclusion of the protein matrix. We also outline future directions for the application of ONIOM to enzymes. One of the major deficiencies of QM/MM models in general, including ONIOM QM:MM, is the poor description of electrostatic interactions between the QM and the MM region. An attractive alternative to QM:MM is to take advantage of the multi-layer capability of ONIOM and design three-layer QM:QM’:MM models. In this scheme QM’ is a relatively fast molecular orbital method that can describe charge transfer and mutual polarization between the reactive region and the protein surroundings

  6. Enzymatic labeling of proteins: techniques and approaches.

    PubMed

    Rashidian, Mohammad; Dozier, Jonathan K; Distefano, Mark D

    2013-08-21

    Site-specific modification of proteins is a major challenge in modern chemical biology due to the large number of reactive functional groups typically present in polypeptides. Because of its importance in biology and medicine, the development of methods for site-specific modification of proteins is an area of intense research. Selective protein modification procedures have been useful for oriented protein immobilization, for studies of naturally occurring post-translational modifications, for creating antibody–drug conjugates, for the introduction of fluorophores and other small molecules on to proteins, for examining protein structure, folding, dynamics, and protein–protein interactions, and for the preparation of protein–polymer conjugates. One of the most important approaches for protein labeling is to incorporate bioorthogonal functionalities into proteins at specific sites via enzymatic reactions. The incorporated tags then enable reactions that are chemoselective, whose functional groups not only are inert in biological media, but also do not occur natively in proteins or other macromolecules. This review article summarizes the enzymatic strategies, which enable site-specific functionalization of proteins with a variety of different functional groups. The enzymes covered in this review include formylglycine generating enzyme, sialyltransferases, phosphopantetheinyltransferases, O-GlcNAc post-translational modification, sortagging, transglutaminase, farnesyltransferase, biotin ligase, lipoic acid ligase, and N-myristoyltransferase. PMID:23837885

  7. Mapping the Reaction Coordinates of Enzymatic Defluorination

    PubMed Central

    Chan, Peter W. Y.; Yakunin, Alexander F.; Edwards, Elizabeth A.; Pai, Emil F.

    2011-01-01

    The carbon-fluorine bond is the strongest covalent bond in organic chemistry, yet fluoroacetate dehalogenases can readily hydrolyze this bond under mild physiological conditions. Elucidating the molecular basis of this rare biocatalytic activity will provide the fundamental chemical insights of how this formidable feat is achieved. Here, we present a series of high-resolution (1.15–1.80 Å) crystal structures of a fluoroacetate dehalogenase, capturing snapshots along the defluorination reaction: the free enzyme, enzyme-fluoroacetate Michaelis complex, glycolyl-enzyme covalent intermediate and enzyme-product complex. We demonstrate that enzymatic defluorination requires a halide pocket that not only supplies three hydrogen bonds to stabilize the fluoride ion, but is also finely tailored for the smaller fluorine halogen atom to establish selectivity towards fluorinated substrates. We have further uncovered dynamics near the active site which may play pivotal roles in enzymatic defluorination. These findings may ultimately lead to the development of novel defluorinases that will enable the biotransformation of more complex fluorinated organic compounds, which in turn will assist the synthesis, detoxification, biodegradation, disposal, recycling and regulatory strategies for the growing markets of organofluorines across major industrial sectors. PMID:21510690

  8. Palm date fibers: analysis and enzymatic hydrolysis.

    PubMed

    Shafiei, Marzieh; Karimi, Keikhosro; Taherzadeh, Mohammad J

    2010-01-01

    Waste palm dates were subjected to analysis for composition and enzymatic hydrolysis of their flesh fibers. The fruit contained 32% glucose and 30% fructose, while the water-insoluble fibers of its flesh consisted of 49.9% lignin and 20.9% polysaccharides. Water-insoluble fibers were settled to 55% of its initial volume in 12 h. The presence of skin and flesh colloidal fibers results in high viscosity and clogging problems during industrial processes. The settling velocity of the fibers was improved by enzymatic hydrolysis. Hydrolysis resulted in 84.3% conversion of the cellulosic part of the fibers as well as reducing the settling time to 10 minutes and the final settled volume to 4% of the initial volume. It implies easier separation of the fibers and facilitates fermentation processes in the corresponding industries. Two kinds of high- and low-lignin fibers were identified from the water-insoluble fibers. The high-lignin fibers (75% lignin) settled easily, while the low-lignin fibers (41.4% lignin) formed a slurry suspension which settled very slowly. The hydrophilicity of these low-lignin fibers is the major challenge of the industrial processes. PMID:21151438

  9. A sensitive and robust method for automated on-line monitoring of enzymatic activities in water and water resources.

    PubMed

    Ryzinska-Paier, G; Lendenfeld, T; Correa, K; Stadler, P; Blaschke, A P; Mach, R L; Stadler, H; Kirschner, A K T; Farnleitner, A H

    2014-01-01

    The realisation of a novel concept for automated on-line monitoring of enzymatic activities in water was successfully demonstrated by long-term field testing at two remote Austrian ground water resources. The ?-D-glucuronidase (GLUC) activity was selected as a representative enzymatic model parameter for the on-line determination. But the device can be adapted for any enzymatic reaction with diagnostic relevance for microbial water quality monitoring, as demonstrated for the ?-D-galactosidase activity. Automated filtration of volumes up to 5 litres supports sensitive quantification of enzymatic activities. Internet-based data transfer, using internal control parameters for verification and a dynamic determination of the limit of quantification, enabled robust enzymatic on-line monitoring during a 2-year period. A proportion of 5,313 out of 5,506 GLUC activity measurements (96.5%) could be positively verified. Hydrological (discharge, gauge, turbidity, temperature, pH, electric conductivity, spectral absorbance coefficient at 254 nm) as well as microbiological parameters (Escherichia coli, coliforms) were concurrently determined to characterise the investigated ground water resources. The enzymatic on-line measurements closely reflected the different hydrological conditions and contamination patterns of the test sites. Contrary to expectations, GLUC did not qualify as a proxy-parameter for the occurrence of cultivation-based E. coli contamination and warrants further detailed investigations on its indication capacity as a rapid means for microbial faecal pollution detection in such aquatic habitats. Microbial on-line monitoring is likely to become more important in the future, complementing existing surveillance strategies for water safety management. Further perspectives on the application of such analytical on-line technologies, such as their connection with event-triggered sampling and standardised diagnostics, are discussed. PMID:24647204

  10. Enzymatic Products from Modified Soybean Oil Containing Hydrazinoester

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We use soybean oil to produce new, non-petroleum based products. The starting material is the ene reaction product of soybean oil and diethyl azodicarboxylate (DEAD), which can then be hydrolyzed chemically and enzymatically. Chemical hydrolysis gives hydrazino-fatty acids, whereas enzymatic hydro...

  11. Scleral Reinforcement Through Host Tissue Integration with Biomimetic Enzymatically Degradable

    E-print Network

    Healy, Kevin Edward

    , including chemical- and irradiation- induced scleral collagen crosslinking,17 systemic delivery ofScleral Reinforcement Through Host Tissue Integration with Biomimetic Enzymatically Degradable Semi. Wildsoet, O.D., Ph.D.1 Enzymatically degradable semi-interpenetrating polymer networks (edsIPNs) were

  12. Enzymatic hydrolysis of rawhide using papain and neutrase

    Microsoft Academic Search

    Siriporn Damrongsakkul; Kongpob Ratanathammapan; Kittinan Komolpis; Wiwut Tanthapanichakoon

    2008-01-01

    Rawhide split was hydrolysed separately by two proteolytic enzymes, papain and neutrase. The effects of enzymatic conditions of the hydrolysis reaction were investigated. During the first 10min of the enzymatic hydrolysis, the yield of the hydrolysed protein increased sharply, then it slowly increased or became essentially constant due to the limited availability of the substrate. The optimum hydrolysis conditions of

  13. Fractionation and Molecular Characteristics of Cellulose During Enzymatic Hydrolysis

    Microsoft Academic Search

    T. Eremeeva; T. Bikova; M. Eisimonte; U. Viesturs; A. Treimanis

    2001-01-01

    The effect of enzymatic hydrolysis (EH) on the molecular characteristics as well as fractional composition of cellulose was studied using the direct size exclusion chromatography (SEC) analysis in sodium hydroxide. Bleached hardwood pulp was subjected to the action of the cellulase complex Celluclast™ supplemented with Novozyme 188™. The residues after the enzymatic treatment were fractionated by dissolution in 10% NaOH

  14. Effect of Src Kinase Phosphorylation on Disordered C-terminal Domain of N-Methyl-d-aspartic Acid (NMDA) Receptor Subunit GluN2B Protein*

    PubMed Central

    Choi, Ucheor B.; Xiao, Shifeng; Wollmuth, Lonnie P.; Bowen, Mark E.

    2011-01-01

    NMDA receptors are ligand-gated ion channels with a regulatory intracellular C-terminal domain (CTD). In GluN2B, the CTD is the largest domain in the protein but is intrinsically disordered. The GluN2B subunit is the major tyrosine-phosphorylated protein in synapses. Src kinase phosphorylates the GluN2B CTD, but it is unknown how this affects channel activity. In disordered proteins, phosphorylation can tip the balance between order and disorder. Transitions can occur in both directions, so it is not currently possible to predict the effects of phosphorylation. We used single molecule fluorescence to characterize the effects of Src phosphorylation on GluN2B. Scanning fluorescent labeling sites throughout the domain showed no positional dependence of the energy transfer. Instead, efficiency only scaled with the separation between labeling sites suggestive of a relatively featureless conformational energy landscape. Src phosphorylation led to a general expansion of the polypeptide, which would result in greater exposure of known protein-binding sites and increase the physical separation between contiguous sites. Phosphorylation makes the CTD more like a random coil leaving open the question of how Src exerts its effects on the NMDA receptor. PMID:21712388

  15. Mitotic phosphorylation of histone H3 threonine 80.

    PubMed

    Hammond, Sharra L; Byrum, Stephanie D; Namjoshi, Sarita; Graves, Hillary K; Dennehey, Briana K; Tackett, Alan J; Tyler, Jessica K

    2014-01-01

    The onset and regulation of mitosis is dependent on phosphorylation of a wide array of proteins. Among the proteins that are phosphorylated during mitosis is histone H3, which is heavily phosphorylated on its N-terminal tail. In addition, large-scale mass spectrometry screens have revealed that histone H3 phosphorylation can occur at multiple sites within its globular domain, yet detailed analyses of the functions of these phosphorylations are lacking. Here, we explore one such histone H3 phosphorylation site, threonine 80 (H3T80), which is located on the nucleosome surface. Phosphorylated H3T80 (H3T80ph) is enriched in metazoan cells undergoing mitosis. Unlike H3S10 and H3S28, H3T80 is not phosphorylated by the Aurora B kinase. Further, mutations of T80 to either glutamic acid, a phosphomimetic, or to alanine, an unmodifiable residue, result in an increase in cells in prophase and an increase in anaphase/telophase bridges, respectively. SILAC-coupled mass spectrometry shows that phosphorylated H3T80 (H3T80ph) preferentially interacts with histones H2A and H4 relative to non-phosphorylated H3T80, and this result is supported by increased binding of H3T80ph to histone octamers in vitro. These findings support a model where H3T80ph, protruding from the nucleosome surface, promotes interactions between adjacent nucleosomes to promote chromatin compaction during mitosis in metazoan cells. PMID:24275038

  16. Derepression of RNA polymerase III transcription by phosphorylation and nuclear export of its negative regulator, Maf1.

    PubMed

    Towpik, Joanna; Graczyk, Damian; Gajda, Anna; Lefebvre, Olivier; Boguta, Magdalena

    2008-06-20

    Maf1 is the global repressor of RNA polymerase III (Pol III) in yeast Saccharomyces cerevisiae. Transcription regulation by Maf1 is important under stress conditions and during the switch between fermentation and respiration. Under repressive conditions on nonfermentable carbon sources, Maf1 is dephosphorylated and located predominantly in the nucleus. When cells were shifted to glucose medium, Maf1 became phosphorylated and concomitantly relocated to the cytoplasm. This relocation was dependent on Msn5, a carrier responsible for export of several other phosphoproteins out of the nucleus. Using coimmunoprecipitation, Maf1 was found to interact with Msn5. When msn5-Delta cells were transferred to glucose, Maf1 remained in the nucleus. Remarkably, despite constitutive presence in the nucleus, Maf1 was dephosphorylated and phosphorylated normally in the msn5-Delta mutant, and Pol III was under proper regulation. That phosphorylation of Maf1 and Pol III derepression are tightly linked was shown by studying tRNA transcription in Maf1 mutants with an altered pattern of phosphorylation. In summary, we conclude that phosphorylation of Maf1 inside the nucleus acts both directly by decreasing of Maf1-mediated repression of Pol III and indirectly by stimulation of Msn5 binding and export of nuclear Maf1 to the cytoplasm. PMID:18445601

  17. A Novel In Vitro Assay to Assess Phosphorylation of 3?-[18F]fluoro-3?-Deoxythymidine

    PubMed Central

    Guo, Ning; Xie, Jingping; Manning, H. Charles; Deane, Natasha G.; Ansari, M. Sib; Coffey, Robert J.; Gore, John; Price, Ronald R.; Baldwin, Ronald M.; McIntyre, J. Oliver

    2014-01-01

    Purpose 3?-[18F]fluoro-3’-deoxythymidine ([18F]FLT) is phosphorylated by thymidine kinase 1 (TK-1), a cell cycle regulated enzyme. Appropriate use of [18F]FLT tracer requires validation of the TK-1 activity. Here, we report development of a novel phosphoryl-transfer assay to assess phosphorylation of [18F]FLT both in tumor cell lysates and tumor cells. Procedures The intrinsic F-18 radioactivity was used to quantify both substrate and phosphorylated products using a rapid thin layer chromatography method. Phosphorylation kinetics of [18F]FLT in SW480 and DiFi tumor cell lysates and cellular uptake were measured. Results The apparent Michaelis–Menten kinetic parameters for [18F]FLT are Km = 4.8 ± 0.3 ?M and Vmax=7.4 pmol min?1per 1×106 cells with ~2-fold higher TK-1 activity in DiFi versus SW480 lysates. Conclusions The apparent Km of [18F]FLT was comparable to the value reported with purified recombinant TK-1. The uptake of [18F]FLT by SW480 cells is inhibited by nitrobenzylthioinosine or dipyridamole indicating that uptake is mediated predominantly by the equilibrative nucleoside transporters in these tumor cells. PMID:20532643

  18. The UL12 Protein of Herpes Simplex Virus 1 Is Regulated by Tyrosine Phosphorylation

    PubMed Central

    Fujii, Hikaru; Kato, Akihisa; Mugitani, Michio; Kashima, Yukie; Oyama, Masaaki; Kozuka-Hata, Hiroko; Arii, Jun

    2014-01-01

    ABSTRACT The herpes simplex virus 1 (HSV-1) UL12 protein (pUL12) is a nuclease that is critical for viral replication in vitro and neurovirulence in vivo. In this study, mass spectrometric analysis of pUL12 and phosphate-affinity SDS-polyacrylamide gel electrophoresis analysis identified tyrosine at pUL12 residue 371 (Tyr-371) as a pUL12 phosphorylation site: Tyr-371 is conserved in pUL12 homologs in herpesviruses in all Herpesviridae subfamilies. Replacement of Tyr-371 with phenylalanine (Y371F) in pUL12 (i) abolished its exonuclease activity in HSV-1-infected Vero, HEL, and A549 cells, (ii) reduced viral replication, cell-cell spread, and pUL12 expression in infected cells in a cell type-dependent manner, (iii) led to aberrant subcellular localization of pUL12 in infected cells in a cell type-dependent manner, and (iv) reduced HSV-1 neurovirulence in mice. The effects of the pUL12 Y371F mutation in cell cultures and mice were similar to those of a nuclease-dead double mutation in pUL12, although the Y371F mutation reduced viral replication severalfold more than the nuclease-dead double mutation in a cell type- and multiplicity-of-infection-dependent manner. Replacement of Tyr-371 with glutamic acid, which mimics constitutive phosphorylation, restored the wild-type phenotype in cell cultures and mice. These results suggested that phosphorylation of pUL12 Tyr-371 was essential for pUL12 to express its nuclease activity in HSV-1-infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice mainly by upregulating pUL12 nuclease activity and, in part, by regulating the subcellular localization and expression of pUL12 in HSV-1-infected cells. IMPORTANCE Herpesviruses encode a considerable number of enzymes for their replication. Like cellular enzymes, the viral enzymes need to be properly regulated in infected cells. Although the functional aspects of herpesvirus enzymes have gradually been clarified, information on how most of these enzymes are regulated in infected cells is lacking. In the present study, we report that the enzymatic activity of the herpes simplex virus 1 alkaline nuclease pUL12 was regulated by phosphorylation of pUL12 Tyr-371 in infected cells and that this phosphorylation promoted viral replication and cell-cell spread in cell cultures and neurovirulence in mice, mainly by upregulating pUL12 nuclease activity. Interestingly, pUL12 and tyrosine at pUL12 residue 371 appeared to be conserved in all herpesviruses in the family Herpesviridae, raising the possibility that the herpesvirus pUL12 homologs may also be regulated by phosphorylation of the conserved tyrosine residue. PMID:24991005

  19. Fungal biodegradation and enzymatic modification of lignin

    PubMed Central

    Dashtban, Mehdi; Schraft, Heidi; Syed, Tarannum A.; Qin, Wensheng

    2010-01-01

    Lignin, the most abundant aromatic biopolymer on Earth, is extremely recalcitrant to degradation. By linking to both hemicellulose and cellulose, it creates a barrier to any solutions or enzymes and prevents the penetration of lignocellulolytic enzymes into the interior lignocellulosic structure. Some basidiomycetes white-rot fungi are able to degrade lignin efficiently using a combination of extracellular ligninolytic enzymes, organic acids, mediators and accessory enzymes. This review describes ligninolytic enzyme families produced by these fungi that are involved in wood decay processes, their molecular structures, biochemical properties and the mechanisms of action which render them attractive candidates in biotechnological applications. These enzymes include phenol oxidase (laccase) and heme peroxidases [lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP)]. Accessory enzymes such as H2O2-generating oxidases and degradation mechanisms of plant cell-wall components in a non-enzymatic manner by production of free hydroxyl radicals (·OH) are also discussed. PMID:21968746

  20. Cascade enzymatic reactions for efficient carbon sequestration.

    PubMed

    Xia, Shunxiang; Zhao, Xueyan; Frigo-Vaz, Benjamin; Zheng, Wenyun; Kim, Jungbae; Wang, Ping

    2015-04-01

    Thermochemical processes developed for carbon capture and storage (CCS) offer high carbon capture capacities, but are generally hampered by low energy efficiency. Reversible cascade enzyme reactions are examined in this work for energy-efficient carbon sequestration. By integrating the reactions of two key enzymes of RTCA cycle, isocitrate dehydrogenase and aconitase, we demonstrate that intensified carbon capture can be realized through such cascade enzymatic reactions. Experiments show that enhanced thermodynamic driving force for carbon conversion can be attained via pH control under ambient conditions, and that the cascade reactions have the potential to capture 0.5 mol carbon at pH 6 for each mole of substrate applied. Overall it manifests that the carbon capture capacity of biocatalytic reactions, in addition to be energy efficient, can also be ultimately intensified to approach those realized with chemical absorbents such as MEA. PMID:25708541

  1. Zinc oxide inverse opal enzymatic biosensor

    NASA Astrophysics Data System (ADS)

    You, Xueqiu; Pikul, James H.; King, William P.; Pak, James J.

    2013-06-01

    We report ZnO inverse opal- and nanowire (NW)-based enzymatic glucose biosensors with extended linear detection ranges. The ZnO inverse opal sensors have 0.01-18 mM linear detection range, which is 2.5 times greater than that of ZnO NW sensors and 1.5 times greater than that of other reported ZnO sensors. This larger range is because of reduced glucose diffusivity through the inverse opal geometry. The ZnO inverse opal sensors have an average sensitivity of 22.5 ?A/(mM cm2), which diminished by 10% after 35 days, are more stable than ZnO NW sensors whose sensitivity decreased by 10% after 7 days.

  2. Heavy atom isotope effects on enzymatic reactions

    NASA Astrophysics Data System (ADS)

    Paneth, Piotr

    1994-05-01

    The theory of isotope effects, which has proved to be extremely useful in providing geometrical details of transition states in a variety of chemical reactions, has recently found an application in studies of enzyme-catalyzed reactions. These reactions are multistep in nature with few steps being partially rate-limiting, thus interpretation of these isotope effects is more complex. The theoretical framework of heavy-atom isotope effects on enzymatic reactions is critically analyzed on the basis of recent results of: carbon kinetic isotope effects on carbonic anhydrase and catalytic antibodies; multiple carbon, deuterium isotope effects on reactions catalyzed by formate decarboxylase; oxygen isotope effects on binding processes in reactions catalyzed by pyruvate kinase; and equilibrium oxygen isotope effect on binding an inhibitor to lactate dehydrogenase. The advantages and disadvantages of reaction complexity in learning details of formal and molecular mechanisms are discussed in the examples of reactions catalyzed by phosphoenolpyruvate carboxylase, orotidine decarboxylase and glutamine synthetase.

  3. ?-cyclodextrin assistant flavonoid glycosides enzymatic hydrolysis

    PubMed Central

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

    2013-01-01

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

  4. Can You Hear Me Now? Regulating Transcriptional Activators by Phosphorylation

    NSDL National Science Digital Library

    Kevin H. Gardner (University of Texas Southwestern Medical Center; Departments of Biochemistry and Pharmacology REV)

    2005-09-13

    Extracellular signals often modulate the expression of specific genetic programs by triggering the phosphorylation of relevant transcription factors (TFs). Phosphorylation in turn regulates such TFs by altering their cellular localization, DNA binding affinity, or transcriptional activity. Structural approaches have revealed how phosphorylation turns some TFs on or off; but less is known about how phosphorylation regulates other transcription factors in a graded manner that depends on signal intensity. A recent paper by Graves and colleagues reveals how a group of phosphorylation sites in Ets-1 regulates its DNA binding activity. Their studies provide new insight into the importance of multisite phosphorylation for the graded regulation of transcription and highlight the involvement of allosteric mechanisms in this process.

  5. Regulation of elongation factor-2 by multisite phosphorylation.

    PubMed

    Redpath, N T; Price, N T; Severinov, K V; Proud, C G

    1993-04-15

    We have studied the phosphorylation of protein synthesis elongation factor eEF-2, the effects of phosphorylation on its activity and the dephosphorylation of phosphorylated eEF-2 by protein phosphatases-2A and -2C. Extensive analysis of phosphopeptides generated from eEF-2 phosphorylated in vitro by subsequent digestion with CNBr and trypsin indicated that Thr56 and Thr58 are the only residues significantly phosphorylated, consistent with our earlier report. They are also the only two residues to be significantly phosphorylated in reticulocyte lysates: in this system monophosphorylated eEF-2 corresponded only to phosphorylation of Thr56, no factor phosphorylated at only Thr58 being detected. Phosphorylation of Thr56 and Thr58 was found to be an ordered process, modification of Thr56 preceding, and apparently being required for, phosphorylation of Thr58. This presumably explains why the only species of mono-phosphorylated eEF-2 detected are phosphorylated at Thr56. The eEF-2 kinase could phosphorylate a synthetic peptide based on residues 49-60 of eEF-2 (RAGETRFTDTRK), albeit only at a very low rate, and with a very high Km, compared to eEF-2 itself. The kinase phosphorylated the residues corresponding to Thr56 and Thr58, apparently in a random manner, but not Thr53. In the light of the existence of two phosphorylation sites in eEF-2, the relationship between phosphorylation and activity was investigated. Activity was measured in the poly(U)-directed synthesis of polyphenylalanine, where both the bis- and mono-phosphorylated (mono at Thr56) forms of the factor were found to be completely inactive. Indeed, the phosphorylated species appeared to be able to impair the activity of non-phosphorylated eEF-2 in this system. Experiments using reticulocyte lysates also indicated that both phosphorylated forms of eEF-2 were inactive in the translation of physiological templates, but no evidence for dominant inhibition by these species was obtained. Protein phosphatases-2A and -2C (PP-2A and PP-2C) can each efficiently dephosphorylate phosphorylated eEF-2. While bis-phosphorylated eEF-2 was a better substrate for PP-2A than monophosphorylated factor (phosphorylated at Thr56), the converse was true for PP-2C. This seemed to be due, at least in part, to the inhibition of dephosphorylation of Thr56 by PP-2C by the presence of phosphate on Thr58. Nevertheless, PP-2C exhibited a preference for dephosphorylation of Thr56 in bis-phosphorylated eEF-2, while PP-2A showed no such preference. These findings are discussed in terms of current knowledge of the specificity of these two protein phosphatases. PMID:8386634

  6. High-sensitivity determination of tyrosine-phosphorylated peptides by on-line enzyme reactor and electrospray ionization mass spectrometry.

    PubMed Central

    Amankwa, L. N.; Harder, K.; Jirik, F.; Aebersold, R.

    1995-01-01

    We describe a simple, fast, sensitive, and nonisotopic bioanalytical technique for the detection of tyrosine-phosphorylated peptides and the determination of sites of protein tyrosine phosphorylation. The technique employs a protein tyrosine phosphatase micro enzyme reactor coupled on-line to either capillary electrophoresis or liquid chromatography and electrospray ionization mass spectrometry instruments. The micro enzyme reactor was constructed by immobilizing genetically engineered, metabolically biotinylated human protein tyrosine phosphatase beta onto the inner surface of a small piece of a 50-microns inner diameter, 360-microns outer diameter fused silica capillary or by immobilization of the phosphatase onto 40-90-microns avidin-activated resins. By coupling these reactors directly to either a capillary electrophoresis column or a liquid chromatography column, we were able to rapidly perform enzymatic dephosphorylation and separation of the reaction products. Detection and identification of the components of the reaction mixture exiting these reactors were done by mass analysis with an on-line electrospray ionization mass spectrometer. Tyrosine-phosphorylated peptides, even if present in a complex peptide mixture, were identified by subtractive analysis of peptide patterns generated with or without phosphatase treatment. Two criteria, namely a phosphatase-induced change in hydropathy and charge, respectively, and a change in molecular mass by 80 Da, were used jointly to identify phosphopeptides. We demonstrate that, with this technique, low picomole amounts of a tyrosine-phosphorylated peptide can be detected in a complex peptide mixture generated by proteolysis of a protein and that even higher sensitivities can be realized if more sensitive detection systems are applied. PMID:7539661

  7. Nuclear receptors: integration of multiple signalling pathways through phosphorylation

    Microsoft Academic Search

    Cécile Rochette-Egly

    2003-01-01

    Nuclear receptors (NRs) orchestrate the transcription of specific gene networks in response to binding of their cognate ligand. They also act as mediators in a variety of signalling pathways through integrating diverse phosphorylation events. NR phosphorylation concerns all three major domains, the N-terminal activation function (AF-1), the ligand-binding and the DNA binding domains. Often, phosphorylation of NRs by kinases that

  8. Constitutive phosphorylation of cardiac Myosin regulatory light chain in vivo.

    PubMed

    Chang, Audrey N; Battiprolu, Pavan K; Cowley, Patrick M; Chen, Guohua; Gerard, Robert D; Pinto, Jose R; Hill, Joseph A; Baker, Anthony J; Kamm, Kristine E; Stull, James T

    2015-04-24

    In beating hearts, phosphorylation of myosin regulatory light chain (RLC) at a single site to 0.45 mol of phosphate/mol by cardiac myosin light chain kinase (cMLCK) increases Ca(2+) sensitivity of myofilament contraction necessary for normal cardiac performance. Reduction of RLC phosphorylation in conditional cMLCK knock-out mice caused cardiac dilation and loss of cardiac performance by 1 week, as shown by increased left ventricular internal diameter at end-diastole and decreased fractional shortening. Decreased RLC phosphorylation by conventional or conditional cMLCK gene ablation did not affect troponin-I or myosin-binding protein-C phosphorylation in vivo. The extent of RLC phosphorylation was not changed by prolonged infusion of dobutamine or treatment with a ?-adrenergic antagonist, suggesting that RLC is constitutively phosphorylated to maintain cardiac performance. Biochemical studies with myofilaments showed that RLC phosphorylation up to 90% was a random process. RLC is slowly dephosphorylated in both noncontracting hearts and isolated cardiac myocytes from adult mice. Electrically paced ventricular trabeculae restored RLC phosphorylation, which was increased to 0.91 mol of phosphate/mol of RLC with inhibition of myosin light chain phosphatase (MLCP). The two RLCs in each myosin appear to be readily available for phosphorylation by a soluble cMLCK, but MLCP activity limits the amount of constitutive RLC phosphorylation. MLCP with its regulatory subunit MYPT2 bound tightly to myofilaments was constitutively phosphorylated in beating hearts at a site that inhibits MLCP activity. Thus, the constitutive RLC phosphorylation is limited physiologically by low cMLCK activity in balance with low MLCP activity. PMID:25733667

  9. Characterization of kinases involved in the phosphorylation of aggregated ?-synuclein

    PubMed Central

    Waxman, Elisa A.; Giasson, Benoit I.

    2015-01-01

    ?-Synuclein (?-syn) is the major component of pathological inclusions characteristic of several neurodegenerative disorders, such as Parkinson disease. The major post-translational modification of ?-syn is phosphorylation at S129, and previous studies estimate that approximately 90% of ?-syn in proteinaceous, pathological inclusions is phosphorylated at this site. ?-Syn can be phosphorylated by polo-like kinases (PLKs) 1-3 and casein kinases (CK) 1 and 2; however the kinases associated with the hyperphosphorylation of aggregated ?-syn is still under debate. Using a high-efficiency cellular model of ?-syn aggregate formation, we found that selective inhibitors for CK2 and PLKs each partially inhibited S129 phosphorylation of soluble (non-aggregated) ?-syn, but only PLK inhibitors modestly attenuated the phosphorylation of aggregated ?-syn. In addition, none of kinase inhibitors used had a substantial effect on the propensity of ?-syn to aggregate. Overexpression of PLKs each promoted robust phosphorylation of soluble ?-syn, but none altered the propensity of ?-syn to aggregate. Overexpression of only PLK2 increased phosphorylation of aggregated ?-syn at S129, which is likely due to increased phosphorylation of soluble ?-syn, which then incorporated into aggregates. Overexpression of PLK1 and treatment with BI2536 resulted in a significant reduction of phosphorylated, aggregated ?-syn protein, beyond that of BI2536 treatment alone. These studies suggest that phosphorylation of ?-syn is independent of ?-syn aggregate formation, that PLK1 is involved in the phosphorylation of aggregated ?-syn at S129 in this system, and that mechanisms resulting in hyperphosphorylation of aggregated ?-syn appear independent from those responsible for the phosphorylation of soluble ?-syn. PMID:21162130

  10. TNF?- and IKK?-mediated TANK/I-TRAF phosphorylation: implications for interaction with NEMO/IKK? and NF-?B activation

    PubMed Central

    Bonif, Marianne; Meuwis, Marie-Alice; Close, Pierre; Benoit, Valérie; Heyninck, Karen; Chapelle, Jean-Paul; Bours, Vincent; Merville, Marie-Paule; Piette, Jacques; Beyaert, Rudi; Chariot, Alain

    2005-01-01

    Pro-inflammatory cytokines trigger signalling cascades leading to NF-?B (nuclear factor-?B)-dependent gene expression through IKK [I?B (inhibitory ?B) kinase]-dependent phosphorylation and subsequent degradation of the I?B proteins and via induced phosphorylation of p65. These signalling pathways rely on sequentially activated kinases which are assembled by essential and non-enzymatic scaffold proteins into functional complexes. Here, we show that the pro-inflammatory cytokine TNF? (tumour necrosis factor ?) promotes TANK [TRAF (TNF receptor-associated factor) family member associated NF-?B activator] recruitment to the IKK complex via a newly characterized C-terminal zinc finger. Moreover, we show that TANK is phosphorylated by IKK? upon TNF? stimulation and that this modification negatively regulates TANK binding to NEMO (NF-?B essential modulator). Interestingly, reduced TANK expression by RNA interference attenuates TNF?-mediated induction of a subset of NF-?B target genes through decreased p65 transactivation potential. Therefore the scaffold protein TANK is required for the cellular response to TNF? by connecting upstream signalling molecules to the IKKs and p65, and its subsequent IKK?-mediated phosphorylation may be a mechanism to terminate the TANK-dependent wave of NF-?B activation. PMID:16336209

  11. Rho-associated kinase phosphorylates MARCKS in human neuronal cells.

    PubMed

    Nagumo, H; Ikenoya, M; Sakurada, K; Furuya, K; Ikuhara, T; Hiraoka, H; Sasaki, Y

    2001-01-26

    Myristoylated alanine-rich C kinase substrate (MARCKS) is a filamentous actin bundling protein and has multiple sites for phosphorylation, by which the biochemical function is negatively regulated. However, the role of such phosphorylation in physiological functions, particularly in neuronal functions, is not well understood. Using a phosphorylation-site specific antibody, we detected the phosphorylation of MARCKS at Ser159 by various protein kinases. Rho-kinase, protein kinase A, and protein kinase C, could introduce (32)P into human recombinant MARCKS in vitro and the phosphorylation site was confirmed to be the Ser159 residue. In human neuronal teratoma (NT-2) cells, lysophosphatidic acid (LPA) induced MARCKS phosphorylation dose- and time-dependently. This phosphorylation was sensitive to Rho-kinase inhibitor HA1077. However, the phosphorylation induced by PDBu was lesser sensitive. In a skinned NTera-2 cell system, Ca(2+)-independent and GTP gamma S/ATP-stimulated phosphorylation at Ser159 was also sensitive to pre-treatment C3 toxin and HA1077. These findings suggest that the Ser159 residue of MARCKS is a target of LPA-stimulated Rho-kinase in neuronal cells. PMID:11162562

  12. On the enzymatic response to injury and its mediators.

    PubMed

    Raekallio, J; Nieminen, L

    1979-08-01

    The enzymatic response to injury appears as an increase in enzymatic activity in the periphery of burns and other injuries. The following processes constitute the enzymatic response: 1) release, 2) activation and 3) synthesis of enzymes. Processes 2) and 3) are dependent upon the fibroblast, which is an activated fibrocyte. Among the fibrocyte activators, and thus among the mediators of the enzymatic response, are histamine, serotonin, kinins, prostaglandins etc. The effects of non-steroidal anti-inflammatory drugs on the enzymatic response to burn injury were studied. Indomethacin, mefenamic acid or aspirin, suspended in carboxymethylcellulose, were given to rats by stomach tube. Controls received carboxymethylcellulose only. Circular burns were inflicted on anaesthetized animals which were killed 30 min, 2 h or 4 h after burning. The burns were studied histologically and enzyme histochemically by using the methods for prostaglandin synthetase, esterases, and adenosine triphosphatase. Aspirin had no effect on the enzymatic response. Mefenamic acid and indomethacin caused a less severe enzymatic response in the 4-h groups as compared to control rats. PMID:159985

  13. Phosphorylated silica nanotubes: preparation and characterization

    NASA Astrophysics Data System (ADS)

    Zhang, Yuqing; Xu, Yan; Lu, Yiren; Zhao, Lili; Song, Lixin

    2013-08-01

    Recently, the strategy of doping inorganic particles into polymer membranes to modify them has been studied intensively. However, these inorganic particles have a disadvantage without being in good compatibility with the polymers. To enhance the compatibility between inorganic particles and polymers, phosphorylated silica nanotubes (PSNTs) with specific high ratios of length to diameter are prepared. Silica nanotubes (SNTs) are prepared through the hydrolysis of tetraethyl orthosilicate in a mixture of aqueous ammonia and dl-tartaric acid, then PSNTs are obtained by silylation and phosphorylation modifications. The optimum synthesis conditions of PSNTs are explored; in addition, the as-prepared PSNTs are characterized by Fourier transform infrared, transmission electron microscope, BET, x-ray photoelectron spectroscopy analysis and thermogravimetric analysis. The results indicate that the ratio of length to diameter of the PSNTs is approximately 20, the thickness of the tube wall is 20 nm, the specific surface area of the PSNTs is 460.2 m2 g-1, the inner diameter of the PSNTs is 76 nm, many mesopores are distributed in the tube walls of the PSNTs, and the PSNTs have numerous hydroxyl active sites along their length direction. Therefore, PSNTs are desirable as suitable fillers of polymer membranes.

  14. Phosphorylation regulates coilin activity and RNA association

    PubMed Central

    Broome, Hanna J.; Carrero, Zunamys I.; Douglas, Heather E.; Hebert, Michael D.

    2013-01-01

    Summary The Cajal body (CB) is a domain of concentrated components found within the nucleus of cells in an array of species that is functionally important for the biogenesis of telomerase and small nuclear ribonucleoproteins. The CB is a dynamic structure whose number and size change during the cell cycle and is associated with other nuclear structures and gene loci. Coilin, also known as the marker protein for the CB, is a phosphoprotein widely accepted for its role in maintaining CB integrity. Recent studies have been done to further elucidate functional activities of coilin apart from its structural role in the CB in an attempt to explore the rationale for coilin expression in cells that have few CBs or lack them altogether. Here we show that the RNA association profile of coilin changes in mitosis with respect to that during interphase. We provide evidence of transcriptional and/or processing dysregulation of several CB-related RNA transcripts as a result of ectopic expression of both wild-type and phosphomutant coilin proteins. We also show apparent changes in transcription and/or processing of these transcripts upon coilin knockdown in both transformed and primary cell lines. Additionally, we provide evidence of specific coilin RNase activity regulation, on both U2 and hTR transcripts, by phosphorylation of a single residue, serine 489. Collectively, these results point to additional functions for coilin that are regulated by phosphorylation. PMID:23616925

  15. Conformational rearrangements upon Syk auto-phosphorylation

    PubMed Central

    Arias-Palomo, Ernesto; Recuero-Checa, María A.; Bustelo, Xosé R.; Llorca, Oscar

    2009-01-01

    Syk is a cytoplasmic tyrosine kinase that is activated after recruitment to immune receptors, triggering the phopshorylation of downstream targets. The kinase activity of Syk is controlled by an auto-inhibited conformation consisting of a regulatory region that contains two N-terminal Src homology 2 (SH2) domains inhibiting the catalytic activity of the kinase domain located at the C-terminus. The atomic structure of the related Zap-70 kinase and an electron microscopy (EM) model of Syk have revealed the structural mechanism of this auto-inhibition based on the formation of a compact conformation sustained by interactions between the regulatory and catalytic domains. On the other hand, the structural basis of Syk activation is not fully understood due to the lack of a 3D structure of full length Syk in an active conformation. Here, we have used single particle electron microscopy to analyse the conformational changes taken place in an activated form of Syk induced by auto-phosphorylation. The conformation of phosphorylated Syk is reminiscent of the compact structure of the inhibited protein but significant conformational changes are observed in the regulatory region. These rearrangements could be sufficient to disrupt the inhibitory interactions, contributing to Syk activation. These results suggest that the regulation of the activation of Syk might be modulated by subtle changes in the positioning of the regulatory domains rather than a full opening mechanism as proposed for the Src kinases. PMID:19409513

  16. The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

    PubMed Central

    Aké, Francine Moussan Désirée; Derkaoui, Meriem; Zébré, Arthur Constant; Cao, Thanh Nguyen; Bouraoui, Houda; Kentache, Takfarinas; Mokhtari, Abdelhamid; Milohanic, Eliane; Joyet, Philippe

    2014-01-01

    SUMMARY The bacterial phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) carries out both catalytic and regulatory functions. It catalyzes the transport and phosphorylation of a variety of sugars and sugar derivatives but also carries out numerous regulatory functions related to carbon, nitrogen, and phosphate metabolism, to chemotaxis, to potassium transport, and to the virulence of certain pathogens. For these different regulatory processes, the signal is provided by the phosphorylation state of the PTS components, which varies according to the availability of PTS substrates and the metabolic state of the cell. PEP acts as phosphoryl donor for enzyme I (EI), which, together with HPr and one of several EIIA and EIIB pairs, forms a phosphorylation cascade which allows phosphorylation of the cognate carbohydrate bound to the membrane-spanning EIIC. HPr of firmicutes and numerous proteobacteria is also phosphorylated in an ATP-dependent reaction catalyzed by the bifunctional HPr kinase/phosphorylase. PTS-mediated regulatory mechanisms are based either on direct phosphorylation of the target protein or on phosphorylation-dependent interactions. For regulation by PTS-mediated phosphorylation, the target proteins either acquired a PTS domain by fusing it to their N or C termini or integrated a specific, conserved PTS regulation domain (PRD) or, alternatively, developed their own specific sites for PTS-mediated phosphorylation. Protein-protein interactions can occur with either phosphorylated or unphosphorylated PTS components and can either stimulate or inhibit the function of the target proteins. This large variety of signal transduction mechanisms allows the PTS to regulate numerous proteins and to form a vast regulatory network responding to the phosphorylation state of various PTS components. PMID:24847021

  17. Enzymatic treatment of duck hepatitis B virus: Topology of the surface proteins for virions and noninfectious subviral particles

    SciTech Connect

    Franke, Claudia [Heinrich-Pette-Institut fuer Experimentelle Virologie und Immunologie an der Universitaet Hamburg, Martinistrasse 52, D-20251 Hamburg (Germany); Matschl, Urte [Heinrich-Pette-Institut fuer Experimentelle Virologie und Immunologie an der Universitaet Hamburg, Martinistrasse 52, D-20251 Hamburg (Germany); Bruns, Michael [Heinrich-Pette-Institut fuer Experimentelle Virologie und Immunologie an der Universitaet Hamburg, Martinistrasse 52, D-20251 Hamburg (Germany)]. E-mail: mbruns@hpi.uni-hamburg.de

    2007-03-01

    The large surface antigen L of duck hepatitis B virus exhibits a mixed topology with the preS domains of the protein alternatively exposed to the particles' interior or exterior. After separating virions from subviral particles (SVPs), we compared their L topologies and showed that both particle types exhibit the same amount of L with the following differences: 1-preS of intact virions was enzymatically digested with chymotrypsin, whereas in SVPs only half of preS was accessible, 2-phosphorylation of L at S118 was completely removed by phosphatase treatment only in virions, 3-iodine-125 labeling disclosed a higher ratio of exposed preS to S domains in virions compared to SVPs. These data point towards different surface architectures of virions and SVPs. Because the preS domain acts in binding to a cellular receptor of hepatocytes, our findings implicate the exclusion of SVPs as competitors for the receptor binding and entry of virions.

  18. Enzymatic synthesis of N- and O-linked glycopeptides

    SciTech Connect

    Wong, C.H.; Schuster, M.; Wang, P.; Sears, P. (Scripps Research Inst., La Jolla, CA (United States))

    1993-07-14

    This paper describes the study of kinetically controlled enzymatic coupling of N- and O-glycopeptide fragments using subtilisin BPN' and two of its variants developed for use in high concentrations of dimethylformamide and in aqueous solution, respectively. Glycosyl amino acids were exploited as the P[sub 1], P[sub 2], P[sub 3], P'[sub 1], P'[sub 2], or P'[sub 3] residue in the enzymatic coupling. Glycosyltransferase-mediated glycosylation of the glycopeptide fragments obtained prior to or after enzymatic peptide bond formation is demonstrated. 19 refs., 5 figs., 1 tab.

  19. Anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue in lipopolysaccharide-stimulated RAW 264.7 macrophages and in vivo zebrafish model

    PubMed Central

    Ko, Seok-Chun

    2015-01-01

    BACKGROUND/OBJECTIVES In this study, potential anti-inflammatory effect of enzymatic hydrolysates from Styela clava flesh tissue was assessed via nitric oxide (NO) production in lipopolysaccahride (LPS) induced RAW 264.7 macrophages and in vivo zebrafish model. MATERIALS/METHODS We investigated the ability of enzymatic hydrolysates from Styela clava flesh tissue to inhibit LPS-induced expression of pro-inflammatory mediators in RAW 264.7 macrophages, and the molecular mechanism through which this inhibition occurred. In addition, we evaluated anti-inflammatory effect of enzymatic hydrolysates against a LPS-exposed in in vivo zebrafish model. RESULTS Among the enzymatic hydrolysates, Protamex-proteolytic hydrolysate exhibited the highest NO inhibitory effect and was fractionated into three ranges of molecular weight by using ultrafiltration (UF) membranes (MWCO 5 kDa and 10 kDa). The above 10 kDa fraction down-regulated LPS-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), thereby reducing production of NO and prostaglandin E2 (PGE2) in LPS-activated RAW 264.7 macrophages. The above 10 kDa fraction suppressed LPS-induced production of pro-inflammatory cytokines, including interleukin (IL)-1?, IL-6, and tumor necrosis factor (TNF)-?. In addition, the above 10 kDa fraction inhibited LPS-induced phosphorylation of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinase (JNK), and p38. Furthermore, NO production in live zebrafish induced by LPS was reduced by addition of the above 10 kDa fraction from S. clava enzymatic hydrolysate. CONCLUSION The results of this study suggested that hydrolysates derived from S. clava flesh tissue would be new anti-inflammation materials in functional resources.

  20. A Grammar Inference Approach for Predicting Kinase Specific Phosphorylation Sites

    PubMed Central

    Datta, Sutapa; Mukhopadhyay, Subhasis

    2015-01-01

    Kinase mediated phosphorylation site detection is the key mechanism of post translational mechanism that plays an important role in regulating various cellular processes and phenotypes. Many diseases, like cancer are related with the signaling defects which are associated with protein phosphorylation. Characterizing the protein kinases and their substrates enhances our ability to understand the mechanism of protein phosphorylation and extends our knowledge of signaling network; thereby helping us to treat such diseases. Experimental methods for predicting phosphorylation sites are labour intensive and expensive. Also, manifold increase of protein sequences in the databanks over the years necessitates the improvement of high speed and accurate computational methods for predicting phosphorylation sites in protein sequences. Till date, a number of computational methods have been proposed by various researchers in predicting phosphorylation sites, but there remains much scope of improvement. In this communication, we present a simple and novel method based on Grammatical Inference (GI) approach to automate the prediction of kinase specific phosphorylation sites. In this regard, we have used a popular GI algorithm Alergia to infer Deterministic Stochastic Finite State Automata (DSFA) which equally represents the regular grammar corresponding to the phosphorylation sites. Extensive experiments on several datasets generated by us reveal that, our inferred grammar successfully predicts phosphorylation sites in a kinase specific manner. It performs significantly better when compared with the other existing phosphorylation site prediction methods. We have also compared our inferred DSFA with two other GI inference algorithms. The DSFA generated by our method performs superior which indicates that our method is robust and has a potential for predicting the phosphorylation sites in a kinase specific manner. PMID:25886273

  1. Hits, Fhits and Nits: beyond enzymatic function.

    PubMed

    Huebner, Kay; Saldivar, Joshua C; Sun, Jin; Shibata, Hidetaka; Druck, Teresa

    2011-01-01

    We have briefly summarized what is known about these proteins, but in closing wish to feature the outstanding questions. Hint1 was discovered mistakenly as an inhibitor of Protein Kinase C and designated Pkci, a designation that still confuses the literature. The other Hint family members were discovered by homology to Hint1. Aprataxin was discovered as a result of the hunt for a gene responsible for AOA1. Fhit was discovered through cloning of a familial chromosome translocation breakpoint on chromosome 3 that interrupts the large FHIT gene within an intron, in the FRA3B chromosome region (Ohta et al., 1996), now known to be the region of the human genome most susceptible to DNA damage due to replication stress (Durkin et al., 2008). The NitFhit fusion genewas discovered during searches for Fhit homologs in flies and worms because the fly/worm Nit polypeptide is fused to the 5'-end of the Fhit gene; the mammalian Nit gene family was discovered because of the NitFhit fusion gene, in searches for homologs to the Nit polypeptide of the NitFhit gene. Each of the Hit family member proteins is reported to have enzymatic activities toward putative substrates involving nucleosides or dinucleosides. Most surprisingly, each of the Hit family proteins discussed has been implicated in important DNA damage response pathways and/or tumor suppression pathways. And for each of them it has been difficult to assign definite substrates, to know if the substrates and catalytic products have biological functions, to know if that function is related to the DNA damage response and suppressor functions, and to precisely define the pathways through which tumor suppression occurs. When the fly Nit sequence was found at the 5'-end of the fly Fhit gene, this gene was hailed as a Rosetta stone gene/protein that would help in discovery of the function of Fhit, because the Nit protein should be in the same signal pathway (Pace et al., 2000). However, the mammalian Nit family proteins have turned out to be at least as mysterious as the Fhit proteins, with the Nit1 substrate still unknown and the surprising finding that Nit proteins also appear to behave as tumor suppressor proteins. Whether the predicted enzymatic functions of these proteins are relevant to the observed biological functions, remain among the outstanding unanswered puzzles and raise the question: have these mammalian proteins evolved beyond the putative original enzymatic purpose, such that the catalytic function is now vestigial and subservient to signal pathways that use the protein-substrate complexes in pathways that signal apoptosis or DNA damage response? Or can these proteins be fulfilling catalytic functions independently but in parallel with signal pathway functions, as perhaps observed for Aprataxin? Or is the catalytic function indeed part of the observed biological functions, such as apoptosis and tumor suppression? Perhaps the recent, post-genomic focus on metabolomics and genome-wide investigations of signal pathway networks will lead to answers to some of these outstanding questions. PMID:21035495

  2. Engineering Kinases to Phosphorylate Nucleoside Analogs for Antiviral and Cancer Therapy.

    PubMed

    Lutz, Stefan; Liu, Lingfeng; Liu, Yichen

    2009-11-01

    Enzyme engineering by directed evolution presents a powerful strategy for tailoring the function and physicochemical properties of biocatalysts to therapeutic and industrial applications. Our laboratory's research focuses on developing novel molecular tools for protein engineering, as well as on utilizing these methods to customize enzymes and to study fundamental aspects of their structure and function. Specifically, we are interested in nucleoside and nucleotide kinases which are responsible for the intracellular phosphorylation of nucleoside analog (NA) prodrugs to their biologically active triphosphates. The high substrate specificity of the cellular kinases often interferes with prodrug activation and consequently lowers the potency of NAs as antiviral and cancer therapeutics. A working solution to the problem is the co-adminstration of a promiscuous kinase from viruses, bacteria, and other mammals. However, further therapeutic enhancements of NAs depend on the selective and efficient prodrug phosphorylation. In the absence of true NA kinases in nature, we are pursuing laboratory evolution strategies to generate efficient phosphoryl-transfer catalysts. This review summarizes some of our recent work in the field and outlines future challenges. PMID:20305804

  3. Enzymatic detection of troponin C and melittin bee

    NASA Astrophysics Data System (ADS)

    Jeetender, Amritsar; Stiharu, Ion; Packirisamy, Muthukumaran

    2005-04-01

    One of the major goals of biosensor technology is to detect and quantify in detail analytes with very high accuracy. To achieve this, much of the emphasis in sensor fabrication has been laid on antibody-antigen interaction. The consequence of this focus of enzyme biosensor studies is the development of critical techniques which can be extended in the detection of Acute Myocardial Infarction (AMI). Biosensors for AMI have attracted considerable interest in the last few years since the monitoring of a specific substance is central in enzymatic reactions. This interest has led to the investigation of biochemical markers of myocardial injury. These biomarkers facilitate the diagnosis and treatment of patients with AMI. Serial measurements of biochemical markers are now universally accepted as an important determinant in AMI diagnosis. Due to their high sensitivity and specificity over other biomarkers, the troponins are the markers of choice for the diagnosis or exclusion of AMI. The present techniques used in the identification of the troponins are lengthy and require large amount of specimen solution. The present research is directed towards the identification of optical detection procedures that are compatible to the miniaturization. In the present study an effort has been made to study the antigen-antibody reaction of rabbit skeletal muscle troponin C (TnC) and bee venom melittin (ME). Fluorescence energy transfer experiments were done to investigate the Ca 2+ -dependant interaction of TnC-ME in a 1:1 complex. Experiments were also conducted on TnC-ME binding at different ratios. These results validate the biosensor technology and illustrate how a biosensor can be developed based on the study of interaction between monoclonal antibody and antigen reaction in real time. The reported experimental results provide valuable information that will be useful in the development of a biosensor for the detection of AMI.

  4. The importance of intrinsic disorder for protein phosphorylation

    E-print Network

    Radivojac, Predrag

    The importance of intrinsic disorder for protein phosphorylation Lilia M. Iakoucheva, Predrag for Information Science and Technology, Temple University, Philadelphia, PA 19122, USA Received November 12, 2003-based tool for the prediction of protein phos- phorylation sites, DISPHOS (DISorder-enhanced PHOSphorylation

  5. Molecular Cell TFIIH Phosphorylation of the Pol II CTD

    E-print Network

    Molecular Cell Article TFIIH Phosphorylation of the Pol II CTD Stimulates Mediator Dissociation by RNA polymerase (Pol) II is associated with phosphorylation of its C-terminal tail (CTD). Depletion of Kin28, the TFIIH subunit that phosphor- ylates the CTD, does not affect elongation but causes Pol II

  6. Phosphorylation and functions of the RNA polymerase II CTD

    Microsoft Academic Search

    Hemali P. Phatnani; Arno L. Greenleaf

    2008-01-01

    The C-terminal repeat domain (CTD), an unusual exten- sion appended to the C terminus of the largest subunit of RNA polymerase II, serves as a flexible binding scaffold for numerous nuclear factors; which factors bind is de- termined by the phosphorylation patterns on the CTD repeats. Changes in phosphorylation patterns, as poly- merase transcribes a gene, are thought to orchestrate

  7. A simple enzymatic quantitative analysis of triglycerides in tissues.

    PubMed

    Danno, H; Jincho, Y; Budiyanto, S; Furukawa, Y; Kimura, S

    1992-10-01

    We determined a method to measure the triglyceride levels in tissues by using a modified enzymatic kit. This enzymatic kit was originally designed to be used to measure the triglyceride levels in plasma. Our method of triglyceride level determination includes dissolving the tissue lipid extracts in an alcohol. Before using the enzymatic kit directly, the lipids were dissolved in tert-butyl alcohol, then a Triton X-100/methyl alcohol mixture was added (1/1 by volume). The presence of organic surfactants such as tert-butyl alcohol and methyl alcohol, and of a surfactant such as Triton X-100, did not interfere with the enzymatic activity. This method enabled us to determine triglyceride levels between 10 and 90 nmol, by using a spectrophotometer to measure the absorbances. PMID:1294711

  8. Bioinspired Associative Memory System Based on Enzymatic Cascades

    E-print Network

    MacVittie, Kevin; Privman, Vladimir; Katz, Evgeny

    2013-01-01

    A biomolecular system representing the first realization of associative memory based on enzymatic reactions in vitro has been designed. The system demonstrated "training" and "forgetting" features characteristic of memory in biological systems, but presently realized in simple biocatalytic cascades.

  9. The Participation of Calponin in the Cross Talk between 20-Hydroxyecdysone and Juvenile Hormone Signaling Pathways by Phosphorylation Variation

    PubMed Central

    Liu, Peng-Cheng; Wang, Jin-Xing; Song, Qi-Sheng; Zhao, Xiao-Fan

    2011-01-01

    20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways interact to mediate insect development, but the mechanism of this interaction is poorly understood. Here, a calponin homologue domain (Chd) containing protein (HaCal) is reported to play a key role in the cross talk between 20E and JH signaling by varying its phosphorylation. Chd is known as an actin binding domain present in many proteins including some signaling proteins. Using an epidermal cell line (HaEpi), HaCal was found to be up-regulated by either 20E or the JH analog methoprene (JHA). 20E induced rapid phosphorylation of HaCal whereas no phosphorylation occurred with JHA. HaCal could be quickly translocated into the nuclei through 20E or JH signaling but interacted with USP1 only under the mediation of JHA. Knockdown of HaCal by RNAi blocked the 20E inducibility of USP1, PKC and HR3, and also blocked the JHA inducibility of USP1, PKC and JHi. After gene silencing of HaCal by ingestion of dsHaCal expressed by Escherichia coli, the larval development was arrested and the gene expression of USP1, PKC, HR3 and JHi were blocked. These composite data suggest that HaCal plays roles in hormonal signaling by quickly transferring into nucleus to function as a phosphorylated form in the 20E pathway and as a non-phosphorylated form interacting with USP1 in the JH pathway to facilitate 20E or JH signaling cascade, in short, by switching its phosphorylation status to regulate insect development. PMID:21625546

  10. The enzymatic synthesis of rubber polymer

    SciTech Connect

    Venkatachalam, K.V.; Wooten, L.; Benedict, C.R. (Texas A M Univ., College Station (USA))

    1990-05-01

    Washed rubber particles (WRP) isolated from stem homogenates of Parthenium argentatum by ultracentrifugation and gel filtration on columns of LKB Ultrogel AcA34 contain a tightly bound rubber transferase which catalyzes the polymerization of IPP into rubber polymer. The polymerization reaction requires Mg{sup 2+}, IPP and an allylic-PP. The Km values for Mg{sup 2+}, IPP and DMAPP are 5.2{times}10{sup {minus}4}M, 8.3{times}10{sup {minus}5} M and 9.6{times}10{sup {minus}5}M respectively. Gel permeation chromatography of the enzymatic polymer product on 3 linear columns of 1{times}10{sup 6} to 500 {angstrom} Ultrastyragel shows that the in vitro formed polymer has a similar mol wt to natural rubber. Over 90% of the in vitro formation of the rubber polymer was a de novo polymerization reaction from DMAPP initiator and IPP monomers. The bound rubber polymerase substantially differs from cytosolic rubber transferase which catalyzes only chain lengthening reactions. Treatment of the WRP with Chaps solubilized the bound rubber transferase which was further purified by DEAE-cellulose chromatography. The purified preparation primarily consists of a 52 kD polypeptide which binds to a photolabile substrate analog. The soluble rubber transferase catalyzes the synthesis of a 1{times}10{sup 5} mol wt rubber polymer from Mg{sup 2+}, DMAPP, IPP and detergent.

  11. Non-eluting Enzymatic Antibiofilm Coatings

    PubMed Central

    Pavlukhina, Svetlana V.; Kaplan, Jeffrey B.; Xu, Li; Chang, Wei; Yu, Xiaojun; Madhyastha, Srinivasa; Yakandawala, Nandadeva; Mentbayeva, Almagul; Khan, Babar; Sukhishvili, Svetlana A.

    2012-01-01

    We developed a highly efficient, biocompatible surface coating that disperses bacterial biofilms through enzymatic cleavage of the extracellular biofilm matrix. The coating was fabricated by binding the naturally existing enzyme dispersin B (DspB) to surface-attached polymer matrices constructed via a layer-by-layer (LbL) deposition technique. LbL matrices were assembled through electrostatic interactions of poly(allylamine hydrochloride) (PAH) and poly(methacrylic acid) (PMAA), followed by chemical crosslinking with glutaraldehyde and pH triggered removal of PMAA, producing a stable PAH hydrogel matrix used for DspB loading. The amount of DspB loaded increased linearly with the number of PAH layers in surface hydrogels. DspB was retained within these coatings in the pH range from 4 to 7.5. DspB-loaded coatings inhibited biofilm formation by two clinical strains of Staphylococcus epidermidis. Biofilm inhibition was ? 98% compared to mock-loaded coatings as determined by CFU enumeration. In addition, DspB-loaded coatings did not inhibit attachment or growth of cultured human osteoblast cells. We suggest that the use of DspB-loaded multilayer coatings presents a promising method for creating biocompatible surfaces with high antibiofilm efficiency, especially when combined with conventional antimicrobial treatment of dispersed bacteria. PMID:22909396

  12. Enzymatic production of hydrogen from glucose

    NASA Astrophysics Data System (ADS)

    Woodward, J.; Mattingly, S. M.

    The objective of this research is to optimize conditions for the enzymatic production of hydrogen gas from biomass-derived glucose. This new project is funded at 0.5 PY level of effort for FY 1995. The rationale for the work is that cellulose is, potentially, a vast source of hydrogen and that enzymes offer a specific and efficient method for its extraction with minimal environmental impact. This work is related to the overall hydrogen program goal of technology development and validation. The approach is based on knowledge that glucose is oxidized by the NADP(sup +) requiring enzyme glucose dehydrogenase (GDH) and that the resulting NADPH can donate its electrons to hydrogenase (H2ase) which catalyzes the evolution of H2. Thus hydrogen production from glucose was achieved using calf liver GDH and Pyrococcus furiosus H2ase yielding 17% of theoretical maximum expected. The cofactor NADP(sup +) for this reaction was regenerated and recycled. Current and future work includes understanding the rate limiting steps of this process and the stabilization/immobilization of the enzymes for long term hydrogen production. Cooperative interactions with the Universities of Georgia and Bath for obtaining thermally sta

  13. Comparison of alternative MS/MS and bioinformatics approaches for confident phosphorylation site localization.

    PubMed

    Wiese, Heike; Kuhlmann, Katja; Wiese, Sebastian; Stoepel, Nadine S; Pawlas, Magdalena; Meyer, Helmut E; Stephan, Christian; Eisenacher, Martin; Drepper, Friedel; Warscheid, Bettina

    2014-02-01

    Over the past years, phosphoproteomics has advanced to a prime tool in signaling research. Since then, an enormous amount of information about in vivo protein phosphorylation events has been collected providing a treasure trove for gaining a better understanding of the molecular processes involved in cell signaling. Yet, we still face the problem of how to achieve correct modification site localization. Here we use alternative fragmentation and different bioinformatics approaches for the identification and confident localization of phosphorylation sites. Phosphopeptide-enriched fractions were analyzed by multistage activation, collision-induced dissociation and electron transfer dissociation (ETD), yielding complementary phosphopeptide identifications. We further found that MASCOT, OMSSA and Andromeda each identified a distinct set of phosphopeptides allowing the number of site assignments to be increased. The postsearch engine SLoMo provided confident phosphorylation site localization, whereas different versions of PTM-Score integrated in MaxQuant differed in performance. Based on high-resolution ETD and higher collisional dissociation (HCD) data sets from a large synthetic peptide and phosphopeptide reference library reported by Marx et al. [Nat. Biotechnol. 2013, 31 (6), 557-564], we show that an Andromeda/PTM-Score probability of 1 is required to provide an false localization rate (FLR) of 1% for HCD data, while 0.55 is sufficient for high-resolution ETD spectra. Additional analyses of HCD data demonstrated that for phosphotyrosine peptides and phosphopeptides containing two potential phosphorylation sites, PTM-Score probability cutoff values of <1 can be applied to ensure an FLR of 1%. Proper adjustment of localization probability cutoffs allowed us to significantly increase the number of confident sites with an FLR of <1%.Our findings underscore the need for the systematic assessment of FLRs for different score values to report confident modification site localization. PMID:24364495

  14. Enzymatic profile of clinical isolates of Acinetobacter calcoaceticus

    Microsoft Academic Search

    C. L. Poh; G. K. Loh

    1985-01-01

    The enzymatic profiles of 109 clinical isolates ofAcinetobacter calcoaceticus subsp.anitratus andlwoffi were determined with conventional plate tests and the rapid API ZYM system (Analytab Products, Plainview, N.Y.). The majority of strains tested lacked DNase, hemolysin, protease, elastase and gelatinase. Strong enzymatic activities of butyrate esterase (C4), caprylate esterase (C8) and leucine arylamidase were detected in all isolates. No trypsin, chymotrypsin,

  15. Supercritical CO 2 pretreatment of lignocellulose enhances enzymatic cellulose hydrolysis

    Microsoft Academic Search

    Kyoung Heon Kim; Juan Hong

    2001-01-01

    The supercritical carbon dioxide (SC–CO2) pretreatment of lignocellulose for enzymatic hydrolysis of cellulose was investigated. Aspen (hardwood) and southern yellow pine (softwood) with moisture contents in the range of 0–73% (w\\/w) were pretreated with SC–CO2 at 3100 and 4000 psi and at 112–165°C for 10–60 min. Each pretreated lignocellulose was hydrolyzed with commercial cellulase to assess its enzymatic digestibility. Untreated

  16. An enzymatic membrane reactor for extracorporeal blood oxygenation

    Microsoft Academic Search

    Federico Cioci; Roberto Lavecchia; Pierfrancesco Mazzocchi

    1999-01-01

    The potential use of the enzyme catalase to oxygenate blood was investigated. A microcalorimetric technique was used to study the kinetics of oxygen generation in human blood serum at 25 and 37°C. Experiments carried out in serum and in phosphate buffer provided the apparent rate constants for the enzymatic and non-enzymatic oxygen-generation reactions. A simplified mathematical model was then developed

  17. Modulation of enzymatic activity of Src-family kinases in bovine T cells transformed by Theileria parva.

    PubMed

    Fich, C; Klauenberg, U; Fleischer, B; Bröker, B M

    1998-08-01

    After infection with sporozoites of the protozoon Theileria parva (Tp) bovine T cells are readily transformed to permanent growth in vivo and in vitro. Their transformed state depends on the constant presence of the parasite but membrane signals remain important. Non-receptor tyrosine kinases play a critical role in the transduction of membrane signals in haematopoietic cells. We have investigated Src-family kinases in bovine T cells transformed by Tp. The T cell receptor-associated tyrosine kinase p60fyn had high activity in all cell lines tested. In addition, weak phosphorylation of 2 novel bands was observed associated with Fyn. In contrast to Fyn, enzymatic activity of p56lck, which in T cells has an essential role in signalling, was low. Furthermore, 1 of 3 Tp transformed cell lines was completely devoid of p56lck indicating that the enzyme is not necessary for the Tp dependent growth of the T cells. In addition to p60fyn and p56lck weak enzymatic activity of 1 splice variant of p53/56lyn was observed after infection of T cells with Tp. These data show that growth transformation by Tp influences kinase activity in bovine T cells. However, they also prove that p56lck does not play an essential role in the transformation mechanism. PMID:9778632

  18. Proinflammatory actions of visfatin/nicotinamide phosphoribosyltransferase (Nampt) involve regulation of insulin signaling pathway and Nampt enzymatic activity.

    PubMed

    Jacques, Claire; Holzenberger, Martin; Mladenovic, Zvezdana; Salvat, Colette; Pecchi, Emilie; Berenbaum, Francis; Gosset, Marjolaine

    2012-04-27

    Visfatin (also termed pre-B-cell colony-enhancing factor (PBEF) or nicotinamide phosphoribosyltransferase (Nampt)) is a pleiotropic mediator acting on many inflammatory processes including osteoarthritis. Visfatin exhibits both an intracellular enzymatic activity (nicotinamide phosphoribosyltransferase, Nampt) leading to NAD synthesis and a cytokine function via the binding to its hypothetical receptor. We recently reported the role of visfatin in prostaglandin E(2) (PGE(2)) synthesis in chondrocytes. Here, our aim was to characterize the signaling pathways involved in this response in exploring both the insulin receptor (IR) signaling pathway and Nampt activity. IR was expressed in human and murine chondrocytes, and visfatin triggered Akt phosphorylation in murine chondrocytes. Blocking IR expression with siRNA or activity using the hydroxy-2-naphthalenyl methyl phosphonic acid tris acetoxymethyl ester (HNMPA-(AM)(3)) inhibitor diminished visfatin-induced PGE(2) release in chondrocytes. Moreover, visfatin-induced IGF-1R(-/-) chondrocytes released higher concentration of PGE(2) than IGF-1R(+/+) cells, a finding confirmed with an antibody that blocked IGF-1R. Using RT-PCR, we found that visfatin did not regulate IR expression and that an increased insulin release was also unlikely to be involved because insulin was unable to increase PGE(2) release. Inhibition of Nampt activity using the APO866 inhibitor gradually decreased PGE(2) release, whereas the addition of exogenous nicotinamide increased it. We conclude that the proinflammatory actions of visfatin in chondrocytes involve regulation of IR signaling pathways, possibly through the control of Nampt enzymatic activity. PMID:22399297

  19. Impact of Phosphorylation on Structure and Thermodynamics of the Interaction between the N-terminal Domain of Enzyme I and the Histidine Phosphocarrier Protein of the Bacterial Phosphotransferase System*

    PubMed Central

    Suh, Jeong-Yong; Cai, Mengli; Clore, G. Marius

    2008-01-01

    The structural and thermodynamic impact of phosphorylation on the interaction of the N-terminal domain of enzyme I (EIN) and the histidine phosphocarrier protein (HPr), the two common components of all branches of the bacterial phosphotransferase system, have been examined using NMR spectroscopy and isothermal titration calorimetry. His-189 is located at the interface of the ? and ?? domains of EIN, resulting in rather widespread chemical shift perturbation upon phosphorylation, in contrast to the highly localized perturbations seen for HPr, where His-15 is fully exposed to solvent. Residual dipolar coupling measurements, however, demonstrate unambiguously that no significant changes in backbone conformation of either protein occur upon phosphorylation: for EIN, the relative orientation of the ? and ?? domains remains unchanged; for HPr, the backbone ?/? torsion angles of the active site residues are unperturbed within experimental error. His ? Glu/Asp mutations of the active site histidines designed to mimic the phosphorylated states reveal binding equilibria that favor phosphoryl transfer from EIN to HPr. Although binding of phospho-EIN to phospho-HPr is reduced by a factor of ?21 relative to the unphosphorylated complex, residual dipolar coupling measurements reveal that the structures of the unphosphorylated and biphosphorylated complexes are the same. Hence, the phosphorylation states of EIN and HPr shift the binding equilibria predominantly by modulating intermolecular electrostatic interactions without altering either the backbone scaffold or binding interface. This facilitates highly efficient phosphoryl transfer between EIN and HPr, which is estimated to occur at a rate of ?850 s-1 from exchange spectroscopy. PMID:18445588

  20. In situ modification of a semiconductor surface by an enzymatic process: a general strategy for photoelectrochemical bioanalysis.

    PubMed

    Zhao, Wei-Wei; Ma, Zheng-Yuan; Xu, Jing-Juan; Chen, Hong-Yuan

    2013-09-17

    Usually, the photoelectrochemical (PEC) bioanalysis necessitates ready photoactive materials as signal sources to convert the specific biological events into electrical signals. Herein, the first PEC bioanalysis without the necessity of ready visible-light-active species was demonstrated. We use an enzyme catalytic process to couple with the unique surface chemistry of semiconductive nanocrystalline, whereby its electronic properties could be modified spontaneously during the enzymatic reaction. Specifically, the enzymatic hydrolysis of ascorbic acid 2-phosphate by alkaline phosphatase is allowed to interact on the TiO2 nanoparticles (NPs) matrix. PEC tests reveal that the self-coordination of the biocatalyzed enediol-ligands onto the undercoordinated surface defect sites would in situ form a ligand-to-metal charge transfer (CT) complex, endowing the inert semiconductor with strong absorption bands in the visible region, and hence underlying a novel and general PEC bioanalysis strategy. PMID:23984980

  1. Phosphate ions and glutaminyl cyclases catalyze the cyclization of glutaminyl residues by facilitating synchronized proton transfers.

    PubMed

    Seifert, Franziska; Demuth, Hans-Ulrich; Weichler, Teresa; Ludwig, Hans-Henning; Tittmann, Kai; Schilling, Stephan

    2015-06-01

    Phosphate ions and glutaminyl cyclase (QC) both catalyze the formation of pyroglutamate (pE, pGlu) from N-terminal glutamine residues of peptides and proteins. Here, we studied the mechanism of glutamine cyclization using kinetic secondary deuterium and solvent isotope effects. The data suggest that proton transfer(s) are rate determining for the spontaneous reaction, and that phosphate and QC are accelerating the reaction by promoting synchronized proton transfers in a concerted mechanism. Thus, non-enzymatic and enzymatic catalysis of pyroglutamate formation exploit a similar mode of transition-state stabilization. PMID:25981125

  2. Effect of non-enzymatic proteins on enzymatic hydrolysis and simultaneous saccharification and fermentation of different lignocellulosic materials.

    PubMed

    Wang, Hui; Kobayashi, Shinichi; Mochidzuki, Kazuhiro

    2015-08-01

    Non-enzymatic proteins were added during hydrolysis of cellulose and simultaneous saccharification and fermentation (SSF) of different biomass materials. Bovine serum albumin (BSA), a model non-enzymatic protein, increased cellulose and xylose conversion efficiency and also enhanced the ethanol yield during SSF of rice straw subjected to varied pretreatments. Corn steep liquor, yeast extract, and peptone also exerted a similar effect as BSA and enhanced the enzymatic hydrolysis of rice straw. Compared to the glucose yields obtained after enzymatic hydrolysis of rice straw in the absence of additives, the glucose yields after 72h of hydrolysis increased by 12.7%, 13.5%, and 13.7% after addition of the corn steep liquor, yeast extract, and peptone, respectively. This study indicated the use of BSA as an alternative to intensive pretreatment of lignocellulosic materials for enhancing enzymatic digestibility. The utilization of non-enzymatic protein additives is promising for application in glucose and ethanol production from lignocellulosic materials. PMID:25974351

  3. Enzymatic production of ceramide from sphingomyelin.

    PubMed

    Zhang, Long; Hellgren, Lars I; Xu, Xuebing

    2006-05-01

    Due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potentials in cosmetic and pharmaceutical industries such as in hair and skin care products. Chemical synthesis of ceramide is a costly process, and developments of alternative cost-efficient production methods are of great interest. Present study was the first attempt to perform a systematic study on the production of ceramide through enzymatic hydrolysis of sphingomyelin. Sphingomyelin hydrolysis proved to be more efficient in two-phase (water:organic solvent) system than in one-phase (water-saturated organic solvent) system. Among the screened phospholipase C, the Clostridium perfringens enzyme had the highest sphingomyelin conversion rate, with very small temperature dependence. Addition of ethanol to the system markedly enhanced the rate of ceramide formation, and a mixture of ethylacetate:hexane (50:50) was the best organic solvent tested. Other factors such as (NH(4))(2)SO(4), NaCl and CaCl(2) were also tested but excluded for further consideration. On the basis of the initial experiments, the reaction system was optimized using response surface methodology including five factors (enzyme amount, water amount, ethanol amount, reaction time and the hexane ratio of organic solvent). Water content and enzyme amount was shown to have the most significant influence on the hydrolysis reaction in the fitted quadratic model. The efficiency of sphingomyelin hydrolysis was dramatically improved through system evaluation and optimization, with the optimal conditions at 75 min reaction time, 3 Uml(-1) enzyme amount, 6% water amount, 1.8% ethanol amount and 46% hexane in ethylacetate. PMID:16337303

  4. Mouse Class III myosins: Kinase activity and phosphorylation sites

    PubMed Central

    Dalal, J.S.; Stevens, S. M.; Alvarez, S.; Munoz, N.; Kempler, K.E.; Dose, A.C.; Burnside, B.; Battelle, B-A.

    2011-01-01

    Since class III unconventional myosins are motor proteins with an N-terminal kinase domain, it seems likely they play a role in both signaling and actin based transport. A growing body of evidence indicates that the motor functions of human class IIIA myosin, which has been implicated in progressive hearing loss, are modulated by intermolecular autophosphorylation. However, the phosphorylation sites have not been identified. We studied the kinase activity and phosphorylation sites of mouse class III myosins, mMyo3A and 3B, which are highly similar to their human orthologs. We demonstrate that the kinase domains of mMyo3A and 3B are active kinases, and that they have similar, if not identical, substrate specificities. We show that the kinase domains of these proteins autophosphorylate, and that they can phosphorylate sites within their myosin and tail domains. Using liquid chromatography-mass spectrometry, we identified phosphorylated sites in the kinase, myosin motor and tail domains of both mMyo3A and 3B. Most of the phosphorylated sites we identified and their consensus phosphorylation motifs are highly conserved among vertebrate class III myosins, including human class III myosins. Our findings are a major step toward understanding how the functions of class III myosins are regulated by phosphorylation. PMID:21895655

  5. A Systems Model of Phosphorylation for Inflammatory Signaling Events

    PubMed Central

    Sadreev, Ildar I.; Chen, Michael Z. Q.; Welsh, Gavin I.; Umezawa, Yoshinori; Kotov, Nikolay V.; Valeyev, Najl V.

    2014-01-01

    Phosphorylation is a fundamental biochemical reaction that modulates protein activity in cells. While a single phosphorylation event is relatively easy to understand, multisite phosphorylation requires systems approaches for deeper elucidation of the underlying molecular mechanisms. In this paper we develop a mechanistic model for single- and multi-site phosphorylation. The proposed model is compared with previously reported studies. We compare the predictions of our model with experiments published in the literature in the context of inflammatory signaling events in order to provide a mechanistic description of the multisite phosphorylation-mediated regulation of Signal Transducer and Activator of Transcription 3 (STAT3) and Interferon Regulatory Factor 5 (IRF-5) proteins. The presented model makes crucial predictions for transcription factor phosphorylation events in the immune system. The model proposes potential mechanisms for T cell phenotype switching and production of cytokines. This study also provides a generic framework for the better understanding of a large number of multisite phosphorylation-regulated biochemical circuits. PMID:25333362

  6. Systematic Phosphorylation Analysis of Human Mitotic Protein Complexes

    PubMed Central

    Hegemann, Björn; Hutchins, James R.A.; Hudecz, Otto; Novatchkova, Maria; Rameseder, Jonathan; Sykora, Martina M.; Liu, Sihan; Mazanek, Michael; Lénárt, Péter; Hériché, Jean-Karim; Poser, Ina; Kraut, Norbert; Hyman, Anthony A.; Yaffe, Michael B.; Mechtler, Karl; Peters, Jan-Michael

    2014-01-01

    Progression through mitosis depends on a large number of protein complexes that regulate the major structural and physiological changes necessary for faithful chromosome segregation. Most, if not all, of the mitotic processes are regulated by a set of mitotic protein kinases that control protein activity by phosphorylation. Although many mitotic phosphorylation events have been identified in proteome-scale mass spectrometry studies, information on how these phosphorylation sites are distributed within mitotic protein complexes and which kinases generate these phosphorylation sites is largely lacking. We used systematic protein-affinity purification combined with mass spectrometry to identify 1818 phosphorylation sites in more than 100 mitotic protein complexes. In many complexes the phosphorylation sites were concentrated on a few subunits, suggesting that these subunits serve as “switchboards” to relay the kinase-regulatory signals within the complexes. Consequent bioinformatic analyses identified potential kinase – substrate relationships for most of these sites. In a subsequent in-depth analysis of key mitotic regulatory complexes using the Aurora kinase B (AURKB) inhibitor Hesperadin and a new Pololike kinase (PLK1) inhibitor, BI 4834, we determined the kinase-dependency for 172 phosphorylation sites on 41 proteins. Combination of the results of the cellular studies with Scansite motif prediction enabled us to identify 14 sites on 6 proteins as direct candidate substrates of AURKB or PLK1. PMID:22067460

  7. Comprehensive Analysis of Phosphorylated Proteins of E. coli Ribosomes

    PubMed Central

    Soung, George Y.; Miller, Jennifer L.; Koc, Hasan; Koc, Emine C.

    2009-01-01

    Phosphorylation of bacterial ribosomal proteins has been known for decades; however, there is still very limited information available on specific locations of the phosphorylation sites in ribosomal proteins and the role they might play in protein synthesis. In this study, we have mapped the specific phosphorylation sites in twenty-four E. coli ribosomal proteins by tandem mass spectrometry. Specific detection of phosphorylation was achieved by either phosphorylation specific visualization techniques, ProQ staining and antibodies for phospho-Ser, Thr, and Tyr, or by mass spectrometry equipped with a capability to detect addition and the loss of the phosphate moiety. Enrichment by immobilized metal affinity and/or strong cation exchange chromatography was used to improve the success of detection of the low abundance phosphopeptides. We found the small subunit (30S) proteins S3, S4, S5, S7, S11, S12, S13, S18, and S21 and the large subunit (50S) proteins L1, L2, L3, L5, L6, L7/L12, L13, L14, L16, L18, L19, L21, L22, L28, L31 to be phosphorylated at one or more residues. Potential roles for each specific site in ribosome function were deduced through careful evaluation of the given site of the phosphorylation in 3D-crystal structure models of ribosomes and the previous mutational studies of E. coli ribosomal proteins. PMID:19469554

  8. Pramipexole reduces phosphorylation of ?-synuclein at serine-129.

    PubMed

    Chau, Kai-Yin; Cooper, J Mark; Schapira, Anthony Henry V

    2013-10-01

    ?-Synuclein is a central component of the pathogenesis of Parkinson's disease (PD). Phosphorylation at serine-129 represents an important post-translational modification and constitutes the major form of the protein in Lewy bodies. Several kinases have been implicated in the phosphorylation of ?-synuclein. The targeting of kinase pathways as a potential to influence the pathogenesis of PD is an important focus of attention, given that mutations of specific kinases (LRRK2 and PINK1) are causes of familial PD. Pramipexole (PPX) is a dopamine agonist developed for the symptomatic relief of PD. Several in vitro and in vivo laboratory studies have demonstrated that PPX exerts neuroprotective properties in model systems of relevance to PD. The present study demonstrates that PPX inhibits the phosphorylation of ?-synuclein and that this is independent of dopamine receptor activation. PPX blocks the increase in phosphorylated ?-synuclein induced by inhibition of the ubiquitin proteasomal system. The phosphorylation of ?-synuclein occurs in part at least through casein kinase 2, and PPX in turn reduces the phosphorylation of this enzyme, thereby inhibiting its activity. Thus, PPX decreases the phosphorylation of ?-synuclein, and this mechanism may contribute to its protective properties in PD models. PMID:23681749

  9. Phosphorylation regulates FOXC2-mediated transcription in lymphatic endothelial cells.

    PubMed

    Ivanov, Konstantin I; Agalarov, Yan; Valmu, Leena; Samuilova, Olga; Liebl, Johanna; Houhou, Nawal; Maby-El Hajjami, Hélène; Norrmén, Camilla; Jaquet, Muriel; Miura, Naoyuki; Zangger, Nadine; Ylä-Herttuala, Seppo; Delorenzi, Mauro; Petrova, Tatiana V

    2013-10-01

    One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in the human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. We report here that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild-type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development. PMID:23878394

  10. Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

    PubMed Central

    Ivanov, Konstantin I.; Agalarov, Yan; Valmu, Leena; Samuilova, Olga; Liebl, Johanna; Houhou, Nawal; Maby-El Hajjami, Hélène; Norrmén, Camilla; Jaquet, Muriel; Miura, Naoyuki; Zangger, Nadine; Ylä-Herttuala, Seppo; Delorenzi, Mauro

    2013-01-01

    One of the key mechanisms linking cell signaling and control of gene expression is reversible phosphorylation of transcription factors. FOXC2 is a forkhead transcription factor that is mutated in the human vascular disease lymphedema-distichiasis and plays an essential role in lymphatic vascular development. However, the mechanisms regulating FOXC2 transcriptional activity are not well understood. We report here that FOXC2 is phosphorylated on eight evolutionarily conserved proline-directed serine/threonine residues. Loss of phosphorylation at these sites triggers substantial changes in the FOXC2 transcriptional program. Through genome-wide location analysis in lymphatic endothelial cells, we demonstrate that the changes are due to selective inhibition of FOXC2 recruitment to chromatin. The extent of the inhibition varied between individual binding sites, suggesting a novel rheostat-like mechanism by which expression of specific genes can be differentially regulated by FOXC2 phosphorylation. Furthermore, unlike the wild-type protein, the phosphorylation-deficient mutant of FOXC2 failed to induce vascular remodeling in vivo. Collectively, our results point to the pivotal role of phosphorylation in the regulation of FOXC2-mediated transcription in lymphatic endothelial cells and underscore the importance of FOXC2 phosphorylation in vascular development. PMID:23878394

  11. The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A

    PubMed Central

    Romero-Brey, Inés; Radujkovic, Danijela; Klein, Rahel; Ruggieri, Alessia; Rebhan, Ilka; Bartenschlager, Ralf; Lohmann, Volker

    2013-01-01

    The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIII?) is an essential host factor of hepatitis C virus (HCV) replication. PI4KIII? catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P) accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A). This study describes the interaction between PI4KIII? and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIII? interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIII? functional interaction site (PFIS) encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIII? binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIII?. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58), indicating that PI4KIII? affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIII? or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIII? increased relative abundance of basally phosphorylated NS5A (p56). PI4KIII? therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIII? in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIII? activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIII? in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis. PMID:23675303

  12. Global proteomic profiling of phosphopeptides using electron transfer dissociation tandem mass spectrometry

    PubMed Central

    Molina, Henrik; Horn, David M.; Tang, Ning; Mathivanan, Suresh; Pandey, Akhilesh

    2007-01-01

    Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique that provides a more comprehensive coverage of peptide sequences and posttranslational modifications. Here, we evaluated the use of ETD for a global phosphoproteome analysis. In all, we identified a total of 1,435 phosphorylation sites from human embryonic kidney 293T cells, of which 1,141 (?80%) were not previously described. A detailed comparison of ETD and collision-induced dissociation (CID) modes showed that ETD identified 60% more phosphopeptides than CID, with an average of 40% more fragment ions that facilitated localization of phosphorylation sites. Although our data indicate that ETD is superior to CID for phosphorylation analysis, the two methods can be effectively combined in alternating ETD and CID modes for a more comprehensive analysis. Combining ETD and CID, from this single study, we were able to identify 80% of the known phosphorylation sites in >1,000 phosphorylated peptides analyzed. A hierarchical clustering of the identified phosphorylation sites allowed us to discover 15 phosphorylation motifs that have not been reported previously. Overall, ETD is an excellent method for localization of phosphorylation sites and should be an integral component of any strategy for comprehensive phosphorylation analysis. PMID:17287340

  13. Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

    NASA Astrophysics Data System (ADS)

    Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao

    2014-07-01

    Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin ?v?3, but not in those by integrin ?5?1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

  14. Highly ordered Ni-Ti-O nanotubes for non-enzymatic glucose detection.

    PubMed

    Hang, Ruiqiang; Liu, Yanlian; Gao, Ang; Bai, Long; Huang, Xiaobo; Zhang, Xiangyu; Lin, Naiming; Tang, Bin; Chu, Paul K

    2015-06-01

    Anodization is used to fabricate Ni-Ti-O nanotube (NT) electrodes for non-enzymatic glucose detection. The morphology, microstructure and composition of the materials are characterized by field emission scanning electron microscopy (FE-SEM), high resolution transmission electron microscopy (HR-TEM) and X-ray photoelectron spectroscopy (XPS). Our results show amorphous and highly ordered NTs with diameter of 50nm, length of 800nm, and Ni/Ti ratio (at %) of 0.35 can be fabricated in ethylene glycol electrolyte supplemented with 0.2 wt.% NH4F and 0.5 vol.% H2O at 30°C and 25V for 1h. Electrochemical experiments indicate that at an applied potential of 0.60V vs. Ag/AgCl, the electrode exhibits a linear response window for glucose concentrations from 0.002mM to 0.2mM with a response time of 10s, detection limit of 0.13?M (S/N=3), and sensitivity of 83?AmM(-1)cm(-2). The excellent performance of the electrode is attributed to its large specific area and fast electron transfer between the NT walls. The good electrochemical performance of the Ni-Ti-O NTs as well as their simple and low-cost preparation method make the strategy promising in non-enzymatic glucose detection. PMID:25842105

  15. Impaired oxidative phosphorylation in overtrained rat myocardium

    PubMed Central

    Kadaja, Lumme; Eimre, Margus; Paju, Kalju; Roosimaa, Mart; Põdramägi, Taavi; Kaasik, Priit; Pehme, Ando; Orlova, Ehte; Mudist, Margareeta; Peet, Nadezhda; Piirsoo, Andres; Seene, Teet; Gellerich, Frank N; Seppet, Enn K

    2010-01-01

    The present study was undertaken to characterize and review the changes in energy metabolism in rat myocardium in response to chronic exhaustive exercise. It was shown that a treadmill exercise program applied for six weeks led the rats into a state characterized by decreased performance, loss of body weight and enhanced muscle catabolism, indicating development of overtraining syndrome. Electron microscopy revealed disintegration of the cardiomyocyte structure, cellular swelling and appearance of peroxisomes. Respirometric assessment of mitochondria in saponin-permeabilized cells in situ revealed a decreased rate of oxidative phosphorylation (OXPHOS) due to diminished control over it by ADP and impaired functional coupling of adenylate kinase to OXPHOS. In parallel, reduced tissue content of cytochrome c was observed, which could limit the maximal rate of OXPHOS. The results are discussed with respect to relationships between the volume of work and corresponding energy metabolism. It is concluded that overtraining syndrome is not restricted to skeletal muscle but can affect cardiac muscle as well. PMID:21264069

  16. Subcellular localization and phosphorylation of antizyme 2.

    PubMed

    Murai, Noriyuki; Shimizu, Akihiro; Murakami, Yasuko; Matsufuji, Senya

    2009-11-01

    Antizymes (AZs) are polyamine-induced proteins that negatively regulate cellular polyamine synthesis and uptake. Three antizyme isoforms are conserved among mammals. AZ1 and AZ2 have a broad tissue distribution, while AZ3 is testis specific. Both AZ1 and AZ2 inhibit ornithine decarboxylase (ODC) activity by binding to ODC monomer and target it to the 26S proteasome at least in vivo. Both also inhibit extra-cellular polyamine uptake. Despite their being indistinguishable by these criteria, we show here using enhanced green fluorescent protein (EGFP)-AZ2 fusion protein that in mammalian cells, the subcellular location of AZ2 is mainly in the nucleus, and is different from that of AZ1. The C-terminal part of AZ2 is necessary for the nuclear distribution. Within a few hours, a shift in the distribution of EGFP-AZ2 fusion protein from cytoplasm to the nucleus or from nucleus to cytoplasm is observable in NIH3T3 cells. In addition, we found that in cells a majority of AZ2, but not AZ1, is phosphorylated at Ser-186, likely by protein kinase CK2. There may be a specific function of AZ2 in the nucleus. PMID:19725046

  17. Redox polymer mediation for enzymatic biofuel cells

    NASA Astrophysics Data System (ADS)

    Gallaway, Joshua

    Mediated biocatalytic cathodes prepared from the oxygen-reducing enzyme laccase and redox-conducting osmium hydrogels were characterized for use as cathodes in enzymatic biofuel cells. A series of osmium-based redox polymers was synthesized with redox potentials spanning the range from 0.11 V to 0.85 V (SHE), and the resulting biocatalytic electrodes were modeled to determine reaction kinetic constants using the current response, measured osmium concentration, and measured apparent electron diffusion. As in solution-phase systems, the bimolecular rate constant for mediation was found to vary greatly with mediator potential---from 250 s-1M-1 when mediator and enzyme were close in potential to 9.4 x 10 4 s-1M-1 when this overpotential was large. Optimum mediator potential for a cell operating with a non-limiting platinum anode and having no mass transport limitation from bulk solution was found to be 0.66 V (SHE). Redox polymers were synthesized under different concentrations, producing osmium variation. An increase from 6.6% to 7.2% osmium increased current response from 1.2 to 2.1 mA/cm2 for a planar film in 40°C oxygen-saturated pH 4 buffer, rotating at 900 rpm. These results translated to high surface area electrodes, nearly doubling current density to 13 mA/cm2, the highest to date for such an electrode. The typical fungal laccase from Trametes versicolor was replaced by a bacterially-expressed small laccase from Streptomyces coelicolor, resulting in biocatalytic films that reduced oxygen at increased pH, with full functionality at pH 7, producing 1.5 mA/cm 2 in planar configuration. Current response was biphasic with pH, matching the activity profile of the free enzyme in solution. The mediated enzyme electrode system was modeled with respect to apparent electron diffusion, mediator concentration, and transport of oxygen from bulk solution, all of which are to some extent controlled by design. Each factor was found to limit performance in certain circumstances. In systems relying on stagnant solution, oxygen transport was found to dominate. However, if mass transport was efficient, differences in mediator design greatly affected performance.

  18. Enzymatic accessibility of fiber hemp is enhanced by enzymatic or chemical removal of pectin.

    PubMed

    Pakarinen, A; Zhang, J; Brock, T; Maijala, P; Viikari, L

    2012-03-01

    Pectinolytic enzymes, steam explosion and alkaline treatment were used to assess the role of pectin for the accessibility of hydrolytic enzymes in the enzymatic hydrolysis of biomass. Hemp (Cannabis sativa L.), a potential energy crop especially in boreal climate with a low need of fertilizers, was used in the study either as untreated or anaerobically preserved raw material. Addition of pectinases increased the hydrolysis yield by 26%, 54%, and 64% from the theoretical carbohydrates of untreated, acid, and alkali-preserved materials, respectively. Steam explosion and hot alkali treatment increased the conversion of the total carbohydrates by 78% and 60%, respectively, compared to the untreated hemp. Elevated separation of cells within the hemp stalk tissues and an increased surface area was revealed after hot alkali or pectinase treatments, contributing to the increased conversion to sugars by commercial enzymes. PMID:22237172

  19. Computational enzymatic catalysis--clarifying enzymatic mechanisms with the help of computers.

    PubMed

    Sousa, Sérgio Filipe; Fernandes, Pedro Alexandrino; Ramos, Maria João

    2012-09-28

    Enzymes play a biologically essential role in performing and controlling an important share of the chemical processes occurring in life. However, despite their critical role in nature, attaining a clear understanding of the way an enzyme acts, i.e. its catalytic mechanism, is a cumbersome task that requires the cooperative efforts of a large number of different scientific techniques. Computational methods offer a particularly insightful way to study such mechanisms, always beautifully complementing the information arising from experimental techniques and working as an excellent alternative for assessing the viability of different mechanistic proposals. This review highlights two important computational strategies to study enzymatic catalysis - the cluster modeling approach and the hybrid quantum mechanical/molecular mechanical (QM/MM) method - complemented with a selection of hand-picked examples of our own work. PMID:22870506

  20. Stereochemistry and Mechanism of Enzymatic and Non-Enzymatic Hydrolysis of Benzylic sec-Sulfate Esters

    PubMed Central

    Toesch, Michael; Schober, Markus; Breinbauer, Rolf; Faber, Kurt

    2014-01-01

    The substrate scope of inverting alkylsulfatase Pisa1 was extended towards benzylic sec-sulfate esters by suppression of competing non-enzymatic autohydrolysis by addition of dimethyl sulfoxide as co-solvent. Detailed investigation of the mechanism of autohydrolysis in 18O-labeled buffer by using an enantiopure sec-benzylic sulfate ester as substrate revealed that from the three possible pathways (i) inverting SN2-type nucleophilic attack of [OH–] at the benzylic carbon represents the major pathway, whereas (ii) SN1-type formation of a planar benzylic carbenium ion leading to racemization was a minor event, and (iii) Retaining SN2-type nucleophilic attack at sulfur took place at the limits of detection. The data obtained are interpreted by analysis of Hammett constants of meta substituents. PMID:25232289

  1. Binding of phosphorylated histone H1 to DNA.

    PubMed Central

    Knippers, R; Otto, B; Böhme, R

    1978-01-01

    A chromatin associated protein kinase was used to add 3 moles of phosphate to seryl side chains of 1 mole of histone H1. The DNA binding properties of this in vitro phosphorylated H1 were compared with those of unmodified H1. Considerably more radioactive superhelical DNA was retained on nitrocellulose filters at 20mM-40mM NaCl by phosphorylated H1 than by unmodified H1. However, zone velocity sedimentation analysis of histone-DNA complexes indicated that similar amounts of phosphorylated and unmodified H1 are bound to DNA. It is therefore concluded that phosphorylated H1 binds distributively to many or all DNA molecules available (depending on the histone/DNA ratio) while unmodified H1 binds cooperatively to a fraction of the DNA molecules in the reaction mixture. Images PMID:209406

  2. HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO

    EPA Science Inventory

    HALOACETIC ACIDS PERTURB PROTEIN PHOSPHORYLATION IN MOUSE EMBRYOS IN VITRO. MR Blanton and ES Hunter. Reproductive Toxicology Division, NHEERL, ORD, US EPA, RTP, NC, USA. Sponsor: JM Rogers. Haloacetic Acids (HAAs) formed during the disinfection process are present in drin...

  3. Fission Yeast Rad52 Phosphorylation Restrains Error Prone Recombination Pathways

    PubMed Central

    Bellini, Angela; Girard, Pierre-Marie; Tessier, Ludovic; Sage, Evelyne; Francesconi, Stefania

    2014-01-01

    Rad52 is a key protein in homologous recombination (HR), a DNA repair pathway dedicated to double strand breaks and recovery of blocked or collapsed replication forks. Rad52 allows Rad51 loading on single strand DNA, an event required for strand invasion and D-loop formation. In addition, Rad52 functions also in Rad51 independent pathways because of its ability to promote single strand annealing (SSA) that leads to loss of genetic material and to promote D-loops formation that are cleaved by Mus81 endonuclease. We have previously reported that fission yeast Rad52 is phosphorylated in a Sty1 dependent manner upon oxidative stress and in cells where the early step of HR is impaired because of lack of Rad51. Here we show that Rad52 is also constitutively phosphorylated in mus81 null cells and that Sty1 partially impinges on such phosphorylation. As upon oxidative stress, the Rad52 phosphorylation in rad51 and mus81 null cells appears to be independent of Tel1, Rad3 and Cdc2. Most importantly, we show that mutating serine 365 to glycine (S365G) in Rad52 leads to loss of the constitutive Rad52 phosphorylation observed in cells lacking Rad51 and to partial loss of Rad52 phosphorylation in cells lacking Mus81. Contrariwise, phosphorylation of Rad52-S365G protein is not affected upon oxidative stress. These results indicate that different Rad52 residues are phosphorylated in a Sty1 dependent manner in response to these distinct situations. Analysis of spontaneous HR at direct repeats shows that mutating serine 365 leads to an increase in spontaneous deletion-type recombinants issued from mitotic recombination that are Mus81 dependent. In addition, the recombination rate in the rad52-S365G mutant is further increased by hydroxyurea, a drug to which mutant cells are sensitive. PMID:24748152

  4. Rapid and Reproducible Deactivation of Rhodopsin Requires Multiple Phosphorylation Sites

    Microsoft Academic Search

    Ana Mendez; Marie E. Burns; Angela Roca; Janis Lem; Lan-Wing Wu; Melvin I. Simon; Denis A. Baylor; Jeannie Chen

    2000-01-01

    Efficient single-photon detection by retinal rod photoreceptors requires timely and reproducible deactivation of rhodopsin. Like other G protein–coupled receptors, rhodopsin contains multiple sites for phosphorylation at its COOH-terminal domain. Transgenic and electrophysiological methods were used to functionally dissect the role of the multiple phosphorylation sites during deactivation of rhodopsin in intact mouse rods. Mutant rhodopsins bearing zero, one (S338), or

  5. Smad3 phosphorylation by cyclin-dependent kinases

    Microsoft Academic Search

    Fang Liu

    2006-01-01

    Smad3, a key mediator for TGF-? antiproliferative responses, is phosphorylated by both CDK4 and CDK2 in vivo and in vitro. Except for the Rb family members, Smad3 is the only CDK4 substrate demonstrated so far. CDK phosphorylation of Smad3 inhibits its transcriptional activity and antiproliferative function. Because cancer cells often contain high levels of CDK activity, inhibition of Smad activity

  6. Tyrosine phosphorylation of Grb14 by Tie2

    Microsoft Academic Search

    Celina Sturk; Daniel J Dumont

    2010-01-01

    BACKGROUND: Growth factor receptor bound (Grb) proteins 7, 10 and 14 are a family of structurally related multi-domain adaptor proteins involved in a variety of biological processes. Grb7, 10 and 14 are known to become serine and\\/or threonine phosphorylated in response to growth factor (GF) stimulation. Grb7 and 10 have also been shown to become tyrosine phosphorylated under certain conditions.

  7. ACTH, cyclic nucleotides, and brain protein phosphorylation in vitro

    Microsoft Academic Search

    H. Zwiers; H. D. Veldhuis; P. Schotman; W. H. Gispen

    1976-01-01

    Endogenous phosphorylation of proteins from rat brain synaptosomal plasma membranes was studied in vitro. Cyclic AMP (cAMP) markedly stimulated32P incorporation in three protein bands with molecular weights of 75,000, 57,000, and 54,000, respectively. The effect of the behaviorally active peptide ACTH1–24 on this endogenous phosphorylation in vitro was studied using peptide concentrations from 10-10 to 10-4 M. In a number

  8. AMP-activated protein kinase phosphorylation of endothelial NO synthase

    Microsoft Academic Search

    Zhi-Ping Chen; Ken I Mitchelhill; Belinda J Michell; David Stapleton; Ignacio Rodriguez-Crespo; Lee A Witters; David A Power; Paul R Ortiz de Montellano; Bruce E Kemp

    1999-01-01

    The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK co-immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca2+-calmodulin (CaM) to activate eNOS both

  9. Optimal control strategy for fed-batch enzymatic hydrolysis of lignocellulosic biomass based on epidemic modeling.

    PubMed

    Tai, Chao; Keshwani, Deepak R; Voltan, Diego S; Kuhar, Pankaj S; Engel, Aaron J

    2015-07-01

    A mathematical optimal control strategy for feeding operation was developed for fed-batch enzymatic hydrolysis of dilute acid pretreated lignocellulosic biomass based on a modified epidemic model. Cellulose conversion was maximized and glucose concentration achieved highest possible value over a fixed hydrolysis time. Boundaries of feeding rate and lignin content were set for feasible controls. Using the optimal control feeding strategy, glucose concentration and accumulated cellulose conversion reached up to 77.31?g/L and 72.08% in 100?h, which are 108.76% and 37.50% higher than in batch hydrolysis with same amount of enzyme consumption. Solids content in feeding source has a significant interference on system mass transfer. Optimal control is a useful tool for guiding operations in fed-batch and continuous processes as it enables process optimization through clear objective functions and feasible controls. Biotechnol. Bioeng. 2015;112: 1376-1382. © 2015 Wiley Periodicals, Inc. PMID:25656971

  10. Recent progress and continuing challenges in bio-fuel cells. Part I: enzymatic cells.

    PubMed

    Osman, M H; Shah, A A; Walsh, F C

    2011-03-15

    Recent developments in bio-fuel cell technology are reviewed. A general introduction to bio-fuel cells, including their operating principles and applications, is provided. New materials and methods for the immobilisation of enzymes and mediators on electrodes, including the use of nanostructured electrodes are considered. Fuel, mediator and enzyme materials (anode and cathode), as well as cell configurations are discussed. A detailed summary of recently developed enzymatic fuel cell systems, including performance measurements, is conveniently provided in tabular form. The current scientific and engineering challenges involved in developing practical bio-fuel cell systems are described, with particular emphasis on a fundamental understanding of the reaction environment, the performance and stability requirements, modularity and scalability. In a companion review (Part II), new developments in microbial fuel cell technologies are reviewed in the context of fuel sources, electron transfer mechanisms, anode materials and enhanced O(2) reduction. PMID:21295964

  11. Micro-electro-mechanical systems (MEMS) for enzymatic detection

    NASA Astrophysics Data System (ADS)

    Jeetender, Amritsar; Packirisamy, Muthukumaran; Stiharu, Ion G.; Balagopal, Ganesharam

    2004-08-01

    Early enzymatic identification and confirmation is essential for diagnosis and prevention as in the case of Acute Myocardial Infarction (AMI). Biochemical markers continue to be an important clinical tool for the enzymatic detection. The advent of MEMS devices can enable the use of various microstructures for the detection of enzymes. In this study, the concept of MEMS is applied for the detection of enzyme reaction, in which microcantilevers undergo changes in mechanical behavior that can be optically detected when enzyme molecules adsorb on their surface. This paper presents the static behavior of microcantilevers under Horse Radish Peroxide (HRP) enzyme reaction. The reported experimental results provide valuable information that will be useful in the development of MEMS sensors for enzymatic detection. The surface stress produced due to enzyme reactions results in the bending of cantilevers as similar to the influencing of thermal stress in the cantilevers. This paper also reports the influence of thermal gradient on the microcantilevers.

  12. Enzymatic hydrolysis of Russian-VX by organophosphorus hydrolase.

    PubMed

    Rastogi, V K; DeFrank, J J; Cheng, T C; Wild, J R

    1997-12-18

    The Russian-VX (R-VX) is the principle V-type nerve agent in the chemical warfare (CW) arsenal of the Former Soviet Union. We here report the enzymatic hydrolysis of the P-S bond of Russian-VX by organophosphorus hydrolase (OPH) from Pseudomonas diminuta. While the Michaelis constant, K(m) for R-VX (474 microM), was similar to that for VX (434 microM), the Vmax for R-VX (2.1 mumoles/mg/min) was about four-fold higher compared to that for VX (0.56 mumoles/mg/min). A 50% inhibition in the rate of the enzymatic hydrolysis of R-VX was observed in the presence of 0.5% ethanol, isoamyl-alcohol, or isopropanol. The presence of acetonitrile, diethylene glycol, or methanol had marginal effects. These results comprise the first demonstration of enzymatic detoxification of R-VX. PMID:9425265

  13. In vivo regulation of Yorkie phosphorylation and localization.

    PubMed

    Oh, Hyangyee; Irvine, Kenneth D

    2008-03-01

    Yorkie (Yki), a transcription factor of the Fat and Hippo signaling pathways, is negatively regulated by the Warts kinase. Here, we use Phos-tag gels to characterize Warts-dependent phosphorylation of Yki in vivo, and show that Warts promotes phosphorylation of Yki at multiple sites. We also show that Warts inhibits Yki nuclear localization in vivo, and can promote binding of Yki to 14-3-3 proteins in cultured cells. In vivo assessment of the influence of individual upstream regulators of Warts reveals that some mutants (e.g. fat) have only partial effects on Yki phosphorylation, and weak effects on Yki localization, whereas other genotypes (e.g. ex fat double mutants) have stronger effects on both Yki phosphorylation and localization. We also identify serine 168 as a critical site through which negative regulation of Yki by Warts-mediated phosphorylation occurs, but find that this site is not sufficient to explain effects of Hippo signaling on Yki in vivo. These results identify modulation of subcellular localization as a mechanism of Yki regulation, and establish that this regulation occurs in vivo through multiple sites of Warts-dependent phosphorylation on Yki. PMID:18256197

  14. Phosphorylation of Mutant Huntingtin at Serine 116 Modulates Neuronal Toxicity

    PubMed Central

    Waldron-Roby, Elaine; O'Meally, Robert; Ratovitski, Tamara; Cole, Robert N.; Ross, Christopher A.

    2014-01-01

    Phosphorylation has been shown to have a significant impact on expanded huntingtin-mediated cellular toxicity. Several phosphorylation sites have been identified on the huntingtin (Htt) protein. To find new potential therapeutic targets for Huntington's Disease (HD), we used mass spectrometry to identify novel phosphorylation sites on N-terminal Htt, expressed in HEK293 cells. Using site-directed mutagenesis we introduced alterations of phosphorylation sites in a N586 Htt construct containing 82 polyglutamine repeats. The effects of these alterations on expanded Htt toxicity were evaluated in primary neurons using a nuclear condensation assay and a direct time-lapse imaging of neuronal death. As a result of these studies, we identified several novel phosphorylation sites, validated several known sites, and discovered one phospho-null alteration, S116A, that had a protective effect against expanded polyglutamine-mediated cellular toxicity. The results suggest that S116 is a potential therapeutic target, and indicate that our screening method is useful for identifying candidate phosphorylation sites. PMID:24505464

  15. Whose Entropy: A Maximal Entropy Analysis of Phosphorylation Signaling

    NASA Astrophysics Data System (ADS)

    Remacle, F.; Graeber, T. G.; Levine, R. D.

    2011-07-01

    High throughput experiments, characteristic of studies in systems biology, produce large output data sets often at different time points or under a variety of related conditions or for different patients. In several recent papers the data is modeled by using a distribution of maximal information-theoretic entropy. We pose the question: `whose entropy' meaning how do we select the variables whose distribution should be compared to that of maximal entropy. The point is that different choices can lead to different answers. Due to the technological advances that allow for the system-wide measurement of hundreds to thousands of events from biological samples, addressing this question is now part of the analysis of systems biology datasets. The analysis of the extent of phosphorylation in reference to the transformation potency of Bcr-Abl fusion oncogene mutants is used as a biological example. The approach taken seeks to use entropy not simply as a statistical measure of dispersion but as a physical, thermodynamic, state function. This highlights the dilemma of what are the variables that describe the state of the signaling network. Is what matters Boolean, spin-like, variables that specify whether a particular phosphorylation site is or is not actually phosphorylated. Or does the actual extent of phosphorylation matter. Last but not least is the possibility that in a signaling network some few specific phosphorylation sites are the key to the signal transduction even though these sites are not at any time abundantly phosphorylated in an absolute sense.

  16. Protein phosphorylation differs significantly among ontogenetic phases in Malus seedlings

    PubMed Central

    2014-01-01

    Background Although protein phosphorylation is an important post-translational modification affecting protein function and metabolism, dynamic changes in this process during ontogenesis remain unexplored in woody angiosperms. Methods Phosphorylated proteins from leaves of three apple seedlings at juvenile, adult vegetative and reproductive stages were extracted and subjected to alkaline phosphatase pre-treatment. After separating the proteins by two-dimensional gel electrophoresis and phosphoprotein-specific Pro-Q Diamond staining, differentially expressed phosphoproteins were identified by MALDI-TOF-TOF mass spectrometry. Results A total of 107 phosphorylated protein spots on nine gels (three ontogenetic phases?×?three seedlings) were identified by MALDI-TOF-TOF mass spectrometry. The 55 spots of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) large-chain fragments varied significantly in protein abundance and degree of phosphorylation among ontogenetic phases. Abundances of the 27 spots corresponding to Rubisco activase declined between juvenile and reproductive phases. More extensively, phosphorylated ?-tubulin chain spots with lower isoelectric points were most abundant during juvenile and adult vegetative phases. Conclusions Protein phosphorylation varied significantly during vegetative phase change and floral transition in apple seedlings. Most of the observed changes were consistent among seedlings and between hybrid populations. PMID:24904238

  17. Neocartilage integration in temporomandibular joint discs: physical and enzymatic methods.

    PubMed

    Murphy, Meghan K; Arzi, Boaz; Prouty, Shannon M; Hu, Jerry C; Athanasiou, Kyriacos A

    2015-02-01

    Integration of engineered musculoskeletal tissues with adjacent native tissues presents a significant challenge to the field. Specifically, the avascularity and low cellularity of cartilage elicit the need for additional efforts in improving integration of neocartilage within native cartilage. Self-assembled neocartilage holds significant potential in replacing degenerated cartilage, though its stabilization and integration in native cartilage require further efforts. Physical and enzymatic stabilization methods were investigated in an in vitro model for temporomandibular joint (TMJ) disc degeneration. First, in phase 1, suture, glue and press-fit constructs were compared in TMJ disc intermediate zone defects. In phase 1, suturing enhanced interfacial shear stiffness and strength immediately; after four weeks, a 15-fold increase in stiffness and a ninefold increase in strength persisted over press-fit. Neither suture nor glue significantly altered neocartilage properties. In phase 2, the effects of the enzymatic stabilization regimen composed of lysyl oxidase, CuSO4 and hydroxylysine were investigated. A full factorial design was employed, carrying forward the best physical method from phase 1, suturing. Enzymatic stabilization significantly increased interfacial shear stiffness after eight weeks. Combined enzymatic stabilization and suturing led to a fourfold increase in shear stiffness and threefold increase in strength over press-fit. Histological analysis confirmed the presence of a collagen-rich interface. Enzymatic treatment additionally enhanced neocartilage mechanical properties, yielding a tensile modulus over 6 MPa and compressive instantaneous modulus over 1200 kPa at eight weeks. Suturing enhances stabilization of neocartilage, and enzymatic treatment enhances functional properties and integration of neocartilage in the TMJ disc. Methods developed here are applicable to other orthopaedic soft tissues, including knee meniscus and hyaline articular cartilage. PMID:25519993

  18. FEBS Letters 372 (1995)238-242 FEBS 16062 Histidine and tyrosine phosphorylation in pea mitochondria

    E-print Network

    Allen, John F.

    1995-01-01

    mitochondria: evidence for protein phosphorylation in respiratory redox signalling Gunilla H~kansson*, John F Abstract A 37 kDa protein in pea mitochondria was found to contain phosphorylated residues. Phosphorylation in plant mitochondria. We also describe the first example of tyrosine phosphorylation in plant organelles

  19. Excited singlet molecular O?(¹?g) is generated enzymatically from excited carbonyls in the dark.

    PubMed

    Mano, Camila M; Prado, Fernanda M; Massari, Júlio; Ronsein, Graziella E; Martinez, Glaucia R; Miyamoto, Sayuri; Cadet, Jean; Sies, Helmut; Medeiros, Marisa H G; Bechara, Etelvino J H; Di Mascio, Paolo

    2014-01-01

    In mammalian tissues, ultraweak chemiluminescence arising from biomolecule oxidation has been attributed to the radiative deactivation of singlet molecular oxygen [O2 ((1)?g)] and electronically excited triplet carbonyl products involving dioxetane intermediates. Herein, we describe evidence of the generation of O2 ((1)?g) in aqueous solution via energy transfer from excited triplet acetone. This involves thermolysis of 3,3,4,4-tetramethyl-1,2-dioxetane, a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal, as an enzymatic source. Both sources of excited carbonyls showed characteristic light emission at 1,270?nm, directly indicative of the monomolecular decay of O2 ((1)?g). Indirect analysis of O2 ((1)?g) by electron paramagnetic resonance using the chemical trap 2,2,6,6-tetramethylpiperidine showed the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl. Using [(18)O]-labeled triplet, ground state molecular oxygen [(18)O2 ((3)?g(-))], chemical trapping of (18)O2 ((1)?g) with disodium salt of anthracene-9,10-diyldiethane-2,1-diyl disulfate yielding the corresponding double-[(18)O]-labeled 9,10-endoperoxide, was detected through mass spectrometry. This corroborates formation of O2 ((1)?g). Altogether, photoemission and chemical trapping studies clearly demonstrate that chemically and enzymatically nascent excited carbonyl generates (18)O2 ((1)?g) by triplet-triplet energy transfer to ground state oxygen O2 ((3)?g(-)), and supports the long formulated hypothesis of O2 ((1)?g) involvement in physiological and pathophysiological events that might take place in tissues in the absence of light. PMID:25087485

  20. Alterations of oxidative phosphorylation complexes in astrocytomas.

    PubMed

    Feichtinger, René Günther; Weis, Serge; Mayr, Johannes Adalbert; Zimmermann, Franz; Geilberger, Reinhard; Sperl, Wolfgang; Kofler, Barbara

    2014-04-01

    The shift in cellular energy production from oxidative phosphorylation (OXPHOS) to glycolysis, even under aerobic conditions, called the Warburg effect, is a feature of most solid tumors. The activity levels of OXPHOS complexes and citrate synthase were determined in astrocytomas. A gradual decrease of citrate synthase and OXPHOS complexes was observed depending on tumor grade. In low-grade astrocytomas (WHO grade II), enzyme activities of citrate synthase, complex I, and complex V were comparable to those of normal brain tissue. A trend to reduced activities was observed for complexes II-IV. In glioblastoma (WHO grade IV), activities of citrate synthase and complexes I-IV were decreased by 56-92% as compared with normal brain. Immunohistochemical staining for porin revealed that the tumorpil of low-grade astrocytomas displays characteristics of the mitochondria-rich neuropil of normal brain tissue. In high-grade tumors (WHO grades III and IV), the tumorpil was characterized by severe morphologic alterations as well as loss of "pilem" structures. Specific alterations of OXPHOS complexes were observed in all astrocytic tumors by immunohistochemical analysis: 80% of astrocytomas exhibited severe deficiency of complex IV; complex I showed a gradual reduction in amount with increasing tumor grade, whereas complex II showed reduced levels only in high-grade (WHO grade IV) tumors (9/12); complexes III and V did not show significant alterations compared with normal brain tissue. OXPHOS defects were present not only in the cell bodies of tumor cells but also in the pilem structures, indicating that the ramifications/protuberances (tumorpil) in general originate from tumor cells. PMID:24446254

  1. Enzymatic Processing of Bioactive Glycosides from Natural Sources

    NASA Astrophysics Data System (ADS)

    Weignerová, Lenka; K?en, Vladimír

    A number of biologically active natural products are glycosides. Often, the glycosidic residue is crucial for their activity. In other cases, glycosylation only improves their pharmacokinetic parameters. Enzymatic modification of these glycosides - both extension of the glycoside moiety and its selective trimming - is advantageous due to their selectivity and mildness of the reaction conditions in the presence of reactive and sensitive complex aglycones. Enzymatic reactions enable the resulting products to be used as "natural products", e.g., in nutraceuticals. This chapter concentrates on naturally occurring glycosides used in medicine but also in the food and flavor industry (e.g., sweeteners). Both "classical" and modern methods will be discussed.

  2. Enzymatic reactors for biodiesel synthesis: Present status and future prospects.

    PubMed

    Poppe, Jakeline Kathiele; Fernandez-Lafuente, Roberto; Rodrigues, Rafael C; Ayub, Marco Antônio Záchia

    2015-01-01

    Lipases are being extensively researched for the production of biodiesel as a "silver bullet" in order to avoid the drawbacks of the traditional alkaline transesterification. In this review, we analyzed the main factors involved in the enzymatic synthesis of biodiesel, focusing in the choice of the immobilization protocol, and the parameters involved in the choice and configuration of the reactors. An extensive discussion is presented about the advantages and disadvantages of each type of reactor and their mode of operation. The current scenario of the market for enzymatic biodiesel and some future prospects and necessary developments are also briefly presented. PMID:25687275

  3. Transfer Functions

    Microsoft Academic Search

    David Stephen Pollock

    2011-01-01

    In statistical time-series analysis, signal processing and control engineering, a transfer function is a mathematical relationship between a numerical input to a dynamic system and the resulting output. The theory of transfer functions describes how the input\\/output relationship is affected by the structure of the transfer function. The theory of the transfer functions of linear time-invariant (LTI) systems has been

  4. Pragmatic Transfer.

    ERIC Educational Resources Information Center

    Kasper, Gabriele

    1992-01-01

    Attempting to clarify the concept of pragmatic transfer, this article proposes as a basic distinction Leech/Thomas' dichotomy of sociopragmatics versus pragmalinguistics, presenting evidence for transfer at both levels. Issues discussed include pragmatic universals in speech act realization, conditions for pragmatic transfer, communicative…

  5. Threonine 149 Phosphorylation Enhances ?FosB Transcriptional Activity to Control Psychomotor Responses to Cocaine

    PubMed Central

    Cates, Hannah M.; Thibault, Mackenzie; Pfau, Madeline; Heller, Elizabeth; Eagle, Andrew; Gajewski, Paula; Bagot, Rosemary; Colangelo, Christopher; Abbott, Thomas; Rudenko, Gabby; Neve, Rachael; Nestler, Eric J.

    2014-01-01

    Stable changes in neuronal gene expression have been studied as mediators of addicted states. Of particular interest is the transcription factor ?FosB, a truncated and stable FosB gene product whose expression in nucleus accumbens (NAc), a key reward region, is induced by chronic exposure to virtually all drugs of abuse and regulates their psychomotor and rewarding effects. Phosphorylation at Ser27 contributes to ?FosB's stability and accumulation following repeated exposure to drugs, and our recent work demonstrates that the protein kinase CaMKII? phosphorylates ?FosB at Ser27 and regulates its stability in vivo. Here, we identify two additional sites on ?FosB that are phosphorylated in vitro by CaMKII?, Thr149 and Thr180, and demonstrate their regulation in vivo by chronic cocaine. We show that phosphomimetic mutation of Thr149 (T149D) dramatically increases AP-1 transcriptional activity while alanine mutation does not affect transcriptional activity when compared with wild-type (WT) ?FosB. Using in vivo viral-mediated gene transfer of ?FosB-T149D or ?FosB-T149A in mouse NAc, we determined that overexpression of ?FosB-T149D in NAc leads to greater locomotor activity in response to an initial low dose of cocaine than does WT ?FosB, while overexpression of ?FosB-T149A does not produce the psychomotor sensitization to chronic low-dose cocaine seen after overexpression of WT ?FosB and abrogates the sensitization seen in control animals at higher cocaine doses. We further demonstrate that mutation of Thr149 does not affect the stability of ?FosB overexpressed in mouse NAc, suggesting that the behavioral effects of these mutations are driven by their altered transcriptional properties. PMID:25143625

  6. Phosphorylation of NLRC4 is critical for inflammasome activation.

    PubMed

    Qu, Yan; Misaghi, Shahram; Izrael-Tomasevic, Anita; Newton, Kim; Gilmour, Laurie L; Lamkanfi, Mohamed; Louie, Salina; Kayagaki, Nobuhiko; Liu, Jinfeng; Kömüves, László; Cupp, James E; Arnott, David; Monack, Denise; Dixit, Vishva M

    2012-10-25

    NLRC4 is a cytosolic member of the NOD-like receptor family that is expressed in innate immune cells. It senses indirectly bacterial flagellin and type III secretion systems, and responds by assembling an inflammasome complex that promotes caspase-1 activation and pyroptosis. Here we use knock-in mice expressing NLRC4 with a carboxy-terminal 3×Flag tag to identify phosphorylation of NLRC4 on a single, evolutionarily conserved residue, Ser?533, following infection of macrophages with Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium). Western blotting with a NLRC4 phospho-Ser?533 antibody confirmed that this post-translational modification occurs only in the presence of stimuli known to engage NLRC4 and not the related protein NLRP3 or AIM2. Nlrc4(-/-) macrophages reconstituted with NLRC4 mutant S533A, unlike those reconstituted with wild-type NLRC4, did not activate caspase-1 and pyroptosis in response to S. typhimurium, indicating that S533 phosphorylation is critical for NLRC4 inflammasome function. Conversely, phosphomimetic NLRC4 S533D caused rapid macrophage pyroptosis without infection. Biochemical purification of the NLRC4-phosphorylating activity and a screen of kinase inhibitors identified PRKCD (PKC?) as a candidate NLRC4 kinase. Recombinant PKC? phosphorylated NLRC4 S533 in vitro, immunodepletion of PKC? from macrophage lysates blocked NLRC4 S533 phosphorylation in vitro, and Prkcd(-/-) macrophages exhibited greatly attenuated caspase-1 activation and IL-1? secretion specifically in response to S. typhimurium. Phosphorylation-defective NLRC4 S533A failed to recruit procaspase-1 and did not assemble inflammasome specks during S. typhimurium infection, so phosphorylation of NLRC4 S533 probably drives conformational changes necessary for NLRC4 inflammasome activity and host innate immunity. PMID:22885697

  7. Regulation of PIKfyve phosphorylation by insulin and osmotic stress.

    PubMed

    Hill, Elaine V; Hudson, Claire A; Vertommen, Didier; Rider, Mark H; Tavaré, Jeremy M

    2010-07-01

    PIKfyve is a protein and lipid kinase that plays an important role in membrane trafficking, including TGN to endosome retrograde sorting and in insulin-stimulated translocation of the GLUT4 glucose transporter from intracellular storage vesicles to the plasma membrane. We have previously demonstrated that PIKfyve is phosphorylated in response to insulin in a PI3-kinase and protein kinase B (PKB)-dependent manner. However, it has been implied that this was not due to direct phosphorylation of PIKfyve by PKB, but as a result of an insulin-induced PIKfyve autophosphorylation event. Here we demonstrate that purified PIKfyve is phosphorylated in vitro by a recombinant active PKB on two separate serine residues, S318 and S105, which flank the N-terminal FYVE domain of the protein. Only S318, however, becomes phosphorylated in intact cells stimulated with insulin. We further demonstrate that S318 is phosphorylated in response to hyperosmotic stress in a PI3-kinase- and PKB-independent manner. Importantly, the effects of insulin and sorbitol were not prevented by the presence of an ATP-competitive PIKfyve inhibitor (YM20163) or in a mutant PIKfyve lacking both lipid and protein kinase activity. Our results confirm, therefore, that PIKfyve is directly phosphorylated by PKB on a single serine residue in response to insulin and are not due to autophosphorylation of the enzyme. We further reveal that two stimuli known to promote glucose uptake in cells, both stimulate phosphorylation of PIKfyve on S318 but via distinct signal transduction pathways. PMID:20513353

  8. Acid-Induced Gelation of Enzymatically Modified, Preheated Whey Proteins

    E-print Network

    Khan, Saad A.

    Acid-Induced Gelation of Enzymatically Modified, Preheated Whey Proteins AHMED S. EISSA AND SAAD A, and gelation (1-3). The ability of whey proteins to form gels is particularly desirable as gelation plays an essential part in dictating the texture of the final food products (4-7). Typically, gelation, which

  9. Allergenic Properties of Enzymatically Hydrolyzed Peanut Flour Extracts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peanut flour is a high protein, low oil, powdered material prepared from roasted 21 peanut seed. In addition to being a well-established food ingredient, peanut flour is also the 22 active ingredient in peanut oral immunotherapy trials. Enzymatic hydrolysis was evaluated as a 23 processing strategy ...

  10. Effect of particle size on enzymatic hydrolysis of pretreated Miscanthus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Particle size reduction is a crucial factor in transportation logistics as well as cellulosic conversion. The effect of particle size on enzymatic hydrolysis of pretreated Miscanthus x giganteus was determined. Miscanthus was ground using a hammer mill equipped with screens having 0.08, 2.0 or 6.0...

  11. Homogeneous precipitation of alumina precursors via enzymatic decomposition of urea

    Microsoft Academic Search

    Hidero Unuma; Shinichi Kato; Toshitaka Ota; Minoru Takahashi

    1998-01-01

    A modified homogeneous precipitation technique involving enzymatic decomposition of urea was proposed and was applied to the synthesis of alumina precursors. Spherical aluminum hydroxide aggregates were prepared at 298 K from aqueous solutions containing aluminum sulfate, urea and urease. In contrast to the conventional technique involving thermally induced decomposition of urea, the resultant aggregates were amorphous with no trace of

  12. Enzymatic Competition: Modeling and Verification with Timed Hybrid Petri Nets

    E-print Network

    Paris-Sud XI, Université de

    Enzymatic Competition: Modeling and Verification with Timed Hybrid Petri Nets Sylvie Troncale in amphibian metamorphosis. Key words: Metamorphosis, verification, Timed hybrid Petri nets, model-checking 1, called Timed Hybrid Petri Nets (THPN) [3], we developed a model checking procedure based

  13. Kinetic Modeling of Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A semimechanistic multi-reaction kinetic model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, creeping wild ryegrass (CWR; Leymus triticoides). This model incorporated one homogeneous reaction of cellobiose-to-glucose and two heterogeneous reactions of cellulose-to...

  14. Enzymatic reactivity of glucose oxidase confined in nanochannels.

    PubMed

    Yu, Jiachao; Zhang, Yuanjian; Liu, Songqin

    2014-05-15

    The construction of nanodevices coupled with an integrated real-time detection system for evaluation of the function of biomolecules in biological processes, and enzymatic reaction kinetics occurring at the confined space or interface is a significant challenge. In this work, a nanochannel-enzyme system in which the enzymatic reaction could be investigated with an electrochemical method was constructed. The model system was established by covalently linking glucose oxidase (GOD) onto the inner wall of the nanochannels of the porous anodic alumina (PAA) membrane. An Au disc was attached at the end of the nanochannels of the PAA membrane as the working electrode for detection of H2O2 product of enzymatic reaction. The effects of ionic strength, amount of immobilized enzyme and pore diameter of the nanochannels on the enzymatic reaction kinetics were illustrated. The GOD confined in nanochannels showed high stability and reactivity. Upon addition of glucose to the nanochannel-enzyme system, the current response had a calibration range span from 0.005 to 2 mM of glucose concentration. The apparent Michaelis-Menten constant (K(m)(app)) of GOD confined in nanochannel was 0.4 mM. The presented work provided a platform for real-time monitoring of the enzyme reaction kinetics confined in nanospaces. Such a nanochannel-enzyme system could also help design future biosensors and enzyme reactors with high sensitivity and efficiency. PMID:24412427

  15. Elimination of xanthan microgels by new enzymatic treatments

    SciTech Connect

    Kohler, N.; Milas, M.; Rinaudo, M.

    1983-02-01

    Polysaccharase and particularly combined polysaccharase/protease treatments offer new alternatives for clarifying aqueous xanthan gum dispersions. Each enzyme has its specific action-the proteases on the insoluble residual bacterial cells, and the polysaccharases primarily on the microgels. Preliminary tests of these enzymatic treatments for scheduled field applications show promising improvements in filterability, especially for solutions prepared from xanthan powders.

  16. Elimination of xanthan microgels by new enzymatic treatments

    SciTech Connect

    Kohler, N.; Milas, M.; Rinaudo, M.

    1983-02-01

    Polysaccharase and particularly combined polysaccharase/protease treatments offer new alternatives for clarifying aqueous xanthan gum dispersions. Each enzyme has its specific action--the proteases on the insoluble residual bacterial cells, and the polysaccharases primarily on the microgels. Preliminary tests of these enzymatic treatments for scheduled field applications show promising improvements in filterability, especially for solutions prepared from xanthan powders.

  17. Regioselective Enzymatic Carboxylation of Phenols and Hydroxystyrene Derivatives

    PubMed Central

    2012-01-01

    The enzymatic carboxylation of phenol and styrene derivatives using (de)carboxylases in carbonate buffer proceeded in a highly regioselective fashion: Benzoic acid (de)carboxylases selectively formed o-hydroxybenzoic acid derivatives, phenolic acid (de)carboxylases selectively acted at the ?-carbon atom of styrenes forming (E)-cinnamic acids. PMID:22471935

  18. Antagonists' impact on enzymatic response in wilt infected cotton plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A number of PR-proteins possess enzymatic activity. As such, these proteins maybe indicators of defensive response of plants. Thus, we have conducted a comparative analysis of beta-1,3-glucanase, peroxidase and xylanase activity in cotton plants to determine how these enzymes are affected by the pat...

  19. Enzymatic Assay of ?-Lactamase Using Circular Dichroism Spectropolarimetry

    Microsoft Academic Search

    David M. Long

    1997-01-01

    A method for measuring the rates of enzymatic hydrolysis of ?-lactam antibiotics based on circular dichroism spectropolarimetry is described. Unhydrolyzed ?-lactam antibiotics have high molar ellipticities, but the hydrolyzed compounds are circular dichroism (CD) inactive in the case of penams or have significantly different CD spectra in the case of cephems. By measuring CD at constant wavelength as a function

  20. ENZYMATIC DEGLYCOSYLATION KIT Contains all enzymes & reagents needed to

    E-print Network

    Lebendiker, Mario

    Enzymatic Removal of N-linked Oligosaccharides O-linked Oligosaccharides Monitoring Deglycosylation/threonine-linked (O-linked) oligosaccharides are major structural components of many eukaryotic proteins. They perform, pathogenicity and many other processes. The diversity of oligosaccharide structures often results

  1. Fluctuations in the enzymatic activity of the human endometrium

    Microsoft Academic Search

    E. A. Elias; R. A. Elias; A. M. Kooistra; A. C. Roukema; J. F. Blacquiere; H. Barrowclough; A. E. F. H. Meijer

    1983-01-01

    Cyclic fluctuations were studied in the activity of oxidoreductases playing a role in the major energy metabolic pathways, lysosomal and non-lysosomal hydrolases and some non-enzymatic cytochemical components demonstrable in different developmental physiological or pathophysiological phases of human endometrium. The total scope of the study involved 170 tissues and cytological specimens. The cytological material included microcurettings, aspirates, brush preparations and tissue

  2. Improvement of protein gel by physical and enzymatic treatment

    Microsoft Academic Search

    Naofumi Kitabatake; Etsushiro Doi

    1993-01-01

    To improve the functional properties of food protein, physical and enzymatic treatments are effective and attractive. As an example of physical treatment, heat treatment of egg protein under various conditions is shown. Egg albumin and egg white usually give turbid (white) gel on heating. However, we have learned that a transparent gel can be prepared by regulating conditions of the

  3. Quantitative analysis of enzymatic fractionation of multiple substrate mixtures.

    PubMed

    Kaki, Shiva Shanker; Adlercreutz, Patrick

    2013-01-01

    The enzymatic conversion of mixtures of multiple substrates was studied quantitatively, based on established methodology used for the enzymatic kinetic resolution of racemic mixtures, involving the use of competitive factors: ratios of specificity constants (k(cat)/K(M)) of substrate pairs. The competitive factors of the substrates were defined in relation to a reference substrate. These competitive factors were used to predict the composition of the reaction mixture as a function of the degree of conversion of the reaction. The methodology was evaluated using three different lipases to hydrolyze a model mixture of four fatty acid methyl esters and for the esterification of a mixture of the same fatty acids in free form with ethanol. In most cases, the competitive factors determined from the initial phase of the reactions predicted the product composition during the rest of the reaction very well. The slowest reacting fatty acid was erucic acid (both in free form and as methyl ester), which was thus enriched in the remaining substrate fraction, while the other fatty acids: lauric acid, palmitic acid and oleic acid were converted faster. Simulations of the compositions of reaction mixtures with different values of the competitive factors were carried out to provide an overview of what could be achieved using enzymatic enrichment. Possible applications include reactions involving homologous substrates and mixtures of multiple isomers. The analysis presented provides guidelines that can be useful in the screening and development of enzymes for enzymatic enrichment applications. PMID:22811287

  4. Protection of Mesopore-Adsorbed Organic Matter from Enzymatic

    E-print Network

    Chorover, Jon

    % on average. In the case of mesoporous alumina and silica-sorbed L-DOPA, the enzyme activity was 3-40 times 16802, and Department of Soil, Water and Environmental Science, University of Arizona, Tucson, Arizona from enzymatic degradation in soils and sediments. Dihydroxyphenylalanine (L-DOPA), a model humic

  5. THE ENZYMATIC IODINATION OF THE RED CELL MEMBRANE

    Microsoft Academic Search

    ANN L. HUBBARD; ZANVIL A. COHN

    1972-01-01

    An enzymatic iodination procedure utilizing lactoperoxidase (LPO), radioactive iodide, and hydrogen peroxide generated by a glucose oxidase-glucose system has been described and utilized for a study of the red cell membrane . 97 % of the incorporated isotope is in the erythrocyte ghost and 3 % is associated with hemoglobin . No significant labeling of the red cell membrane occurs

  6. Characterization of enzymatic saccharification for acid-pretreated lignocellulosic materials with different lignin composition

    Microsoft Academic Search

    Gia-Luen Guo; Deng-Chieh Hsu; Wen-Hua Chen; Wei-Hsi Chen; Wen-Song Hwang

    2009-01-01

    The enzymatic saccharification of three different feedstocks, rice straw, bagasse and silvergrass, which had been pretreated with different dilute acid concentrations, was studied to verify how enzymatic saccharification was affected by the lignin composition of the raw materials. There was a quantitatively inverse correlation between lignin content and enzymatic digestibility after pretreatment with 1%, 2% and 4% sulfuric acid. The

  7. Activation Loop Phosphorylation of ERK3/ERK4 by Group I p21-activated Kinases (PAKs) Defines a Novel PAK-ERK3/4-MAPK-activated Protein Kinase 5 Signaling Pathway*

    PubMed Central

    Déléris, Paul; Trost, Matthias; Topisirovic, Ivan; Tanguay, Pierre-Luc; Borden, Katherine L. B.; Thibault, Pierre; Meloche, Sylvain

    2011-01-01

    Classical mitogen-activated protein (MAP) kinases are activated by dual phosphorylation of the Thr-Xxx-Tyr motif in their activation loop, which is catalyzed by members of the MAP kinase kinase family. The atypical MAP kinases extracellular signal-regulated kinase 3 (ERK3) and ERK4 contain a single phospho-acceptor site in this segment and are not substrates of MAP kinase kinases. Previous studies have shown that ERK3 and ERK4 are phosphorylated on activation loop residue Ser-189/Ser-186, resulting in their catalytic activation. However, the identity of the protein kinase mediating this regulatory event has remained elusive. We have used an unbiased biochemical purification approach to isolate the kinase activity responsible for ERK3 Ser-189 phosphorylation. Here, we report the identification of group I p21-activated kinases (PAKs) as ERK3/ERK4 activation loop kinases. We show that group I PAKs phosphorylate ERK3 and ERK4 on Ser-189 and Ser-186, respectively, both in vitro and in vivo, and that expression of activated Rac1 augments this response. Reciprocally, silencing of PAK1/2/3 expression by RNA interference (RNAi) completely abolishes Rac1-induced Ser-189 phosphorylation of ERK3. Importantly, we demonstrate that PAK-mediated phosphorylation of ERK3/ERK4 results in their enzymatic activation and in downstream activation of MAP kinase-activated protein kinase 5 (MK5) in vivo. Our results reveal that group I PAKs act as upstream activators of ERK3 and ERK4 and unravel a novel PAK-ERK3/ERK4-MK5 signaling pathway. PMID:21177870

  8. Mechanochemical Phosphorylation and Solubilisation of ?-D-Glucan from Yeast Saccharomyces cerevisiae and Its Biological Activities

    PubMed Central

    Shi, Feng; Shi, Jikui; Li, Yongfu

    2014-01-01

    To obtain a water-soluble ?-D-glucan derivative cleanly and conveniently, a highly efficient mechanochemical method, planetary ball milling, was used to phosphorylate ?-D-glucan isolated from yeast Saccharomyces cerevisiae in solid state. Soluble ?-D-glucan phosphate (GP) with a high degree of substitution (0.77–2.09) and an apparent PEAK molecular weight of 6.6–10.0 kDa was produced when ?-D-glucan was co-milled with sodium hexametaphosphate at 139.5–186.0 rad/s for 12–20 min. The energy transferred was 3.03–11.98 KJ/g. The phosphorylation of GPs was demonstrated by Fourier transform infrared spectroscopy and 13C and 31P Nuclear magnetic resonance spectroscopy. Three GP products with different degree of substitution (DS) and degree of polymerisation (DP) were able to upregulate the functional events mediated by activated murine macrophage RAW264.7 cells, among which GP-2 with a DS of 1.24 and DP of 30.5 exerted the highest immunostimulating activity. Our results indicate that mechanochemical processing is an efficient method for preparing water-soluble and biologically active GP with high DS. PMID:25075740

  9. Enzymatic fuel cells: integrating flow-through anode and air-breathing cathode into a membrane-less biofuel cell design.

    PubMed

    Rincón, Rosalba A; Lau, Carolin; Luckarift, Heather R; Garcia, Kristen E; Adkins, Emily; Johnson, Glenn R; Atanassov, Plamen

    2011-09-15

    One of the key goals of enzymatic biofuel cells research has been the development of a fully enzymatic biofuel cell that operates under a continuous flow-through regime. Here, we present our work on achieving this task. Two NAD(+)-dependent dehydrogenase enzymes; malate dehydrogenase (MDH) and alcohol dehydrogenase (ADH) were independently coupled with poly-methylene green (poly-MG) catalyst for biofuel cell anode fabrication. A fungal laccase that catalyzes oxygen reduction via direct electron transfer (DET) was used as an air-breathing cathode. This completes a fully enzymatic biofuel cell that operates in a flow-through mode of fuel supply polarized against an air-breathing bio-cathode. The combined, enzymatic, MDH-laccase biofuel cell operated with an open circuit voltage (OCV) of 0.584 V, whereas the ADH-laccase biofuel cell sustained an OCV of 0.618 V. Maximum volumetric power densities approaching 20 ?W cm(-3) are reported, and characterization criteria that will aid in future optimization are discussed. PMID:21775124

  10. Preparation of icariside II from icariin by enzymatic hydrolysis method.

    PubMed

    Xia, Quan; Xu, Dujuan; Huang, Zhaogang; Liu, Jianjun; Wang, Xinqun; Wang, Xiu; Liu, Shangquan

    2010-07-01

    It has been reported that icariin and icariside II, two flavonoid glycosides coming from herba epimedii, which have a closely structural relationship, show some pharmacological effects such as preventing osteoporosis, cancer and depression. The content of natural icariside II is very low in herba epimedii, but it is the main component in vivo after the administration of herba epimedii. More icariside II can be obtained from icariin by enzymatic hydrolysis method than by traditional isolation method. This study focuses on finding a simple and feasible method to prepare icariside II from icariin by enzymatic hydrolysis, so as to meet the request for further pharmacologic actions study. Icariin was obtained successively with 90% ethanol extraction, isolation on macroporous resin and purification on silica gel chromatography. Enzymatic hydrolysis conditions were tested for the bioconversion of icariin into icariside II by orthogonal array design. The structures of isolated icariin and produced icariside II were identified by UV, IR, ESIMS, (1)H NMR, (13)C NMR, and DEPT spectroscope. Enzymatic hydrolysis experiment showed that icariin could be transformed into icariside II with the action of beta-glucosidase and the optimum reaction conditions were determined as follows: 50 degrees C, 0.2 M disodium hydrogen phosphate and citric acid buffer system (pH6.0), the ratio of icariin/enzyme is 1:1 and reaction time 5 h. By using this enzymatic condition, 95.5 mg icariside II (with the purity of 99.1%) was obtained eventually by transforming 200 mg icariin. PMID:20026390

  11. Macrophage migration inhibitory factor (MIF) enzymatic activity and lung cancer.

    PubMed

    Mawhinney, Leona; Armstrong, Michelle E; O' Reilly, Ciaran; Bucala, Richard; Leng, Lin; Fingerle-Rowson, Gunter; Fayne, Darren; Keane, Michael P; Tynan, Aisling; Maher, Lewena; Cooke, Gordon; Lloyd, David; Conroy, Helen; Donnelly, Seamas C

    2014-01-01

    The cytokine macrophage migration inhibitory factor (MIF) possesses unique tautomerase enzymatic activity, which contributes to the biological functional activity of MIF. In this study, we investigated the effects of blocking the hydrophobic active site of the tautomerase activity of MIF in the pathogenesis of lung cancer. To address this, we initially established a Lewis lung carcinoma (LLC) murine model in Mif-KO and wild-type (WT) mice and compared tumor growth in a knock-in mouse model expressing a mutant MIF lacking enzymatic activity (Mif (P1G)). Primary tumor growth was significantly attenuated in both Mif-KO and Mif (P1G) mice compared with WT mice. We subsequently undertook a structure-based, virtual screen to identify putative small molecular weight inhibitors specific for the tautomerase enzymatic active site of MIF. From primary and secondary screens, the inhibitor SCD-19 was identified, which significantly attenuated the tautomerase enzymatic activity of MIF in vitro and in biological functional screens. In the LLC murine model, SCD-19, given intraperitoneally at the time of tumor inoculation, was found to significantly reduce primary tumor volume by 90% (p < 0.001) compared with the control treatment. To better replicate the human disease scenario, SCD-19 was given when the tumor was palpable (at d 7 after tumor inoculation) and, again, treatment was found to significantly reduce tumor volume by 81% (p < 0.001) compared with the control treatment. In this report, we identify a novel inhibitor that blocks the hydrophobic pocket of MIF, which houses its specific tautomerase enzymatic activity, and demonstrate that targeting this unique active site significantly attenuates lung cancer growth in in vitro and in vivo systems. PMID:25826675

  12. Calmodulin can modulate protein phosphorylation in rat liver cells nuclei.

    PubMed

    Bosser, R; Aligué, R; Guerini, D; Agell, N; Carafoli, E; Bachs, O

    1993-07-25

    This report describes the immunological identification of a 60-kDa calmodulin-binding protein, previously detected in the nuclei of rat liver cells (Bachs, O., Lanini, L., Serratosa, J., Coll, M.J., Bastos, R., Aligué, R., Rius, E., and Carafoli, E. (1990) J. Biol. Chem. 265, 18595-18600), as the calmodulin-dependent protein phosphatase calcineurin. Calcineurin could be extracted from the nuclei by incubation with DNase and RNase, indicating that it is associated with nuclear structures sensitive to the action of nucleases (chromatin or/and ribonucleoproteins). The presence of calcineurin in the nuclei of rat liver cells indicates that calmodulin may modulate the phosphorylation level of nuclear proteins by promoting their dephosphorylation. This report also shows that calmodulin inhibits the activity of casein kinase-2 in the nuclear fractions obtained by nuclease extraction. Phosphorylation experiments indicate that casein kinase-2 phosphorylates three major substrates of 100, 42-44, and 37 kDa as well as other minor proteins in the nuclease extracts. Calmodulin reduces the phosphorylation level of the two latter major proteins and of a minor band of 50 kDa. Thus, nuclear calmodulin in rat liver cells could regulate phosphorylation of nuclear proteins by at least two mechanisms: 1) activation of calcineurin and 2) inhibition of casein kinase-2. PMID:8393444

  13. Rock-dependent calponin 3 phosphorylation regulates myoblast fusion.

    PubMed

    Shibukawa, Yukinao; Yamazaki, Natsuko; Daimon, Etsuko; Wada, Yoshinao

    2013-03-10

    Myogenesis occurs during embryonic development as well as regeneration following postnatal muscle fiber damage. Herein, we show that acidic calponin or calponin 3 (CNN3) regulates both myoblast cell fusion and muscle-specific gene expressions. Overexpression of CNN3 impaired C2C12 cell fusion, whereas CNN3 gene knockdown promoted skeletal myosin expression and fusion. CNN3 was phosphorylated at Ser293/296 in the C-terminal region. The basal inhibitory property of CNN3 against myoblast differentiation was enhanced by Ser293/296Ala mutation or deletion of the C-terminal region, and this inhibition was reversed by Ser293/296Asp mutation. Ser293/296 phosphorylation was required for CNN3 to bind actin and was dependent on Rho-associated kinases 1/2 (ROCK 1/2). Gene knockdown of ROCK1/2 suppressed CNN3 phosphorylation and impaired myoblast fusion, and these effects were partially attenuated by additional CNN3 overexpression of Ser293/296Asp CNN3. These findings indicated that CNN3 phosphorylation by ROCK blunts CNN3's inhibitory effects on muscle cell differentiation and fusion. In muscle tissues, satellite cells, but not mature myofibrils, expressed CNN3. CNN3 was also expressed and phosphorylated during myotube induction in isolated muscle satellite cells. Taken together, these results indicate that CNN3 is a downstream regulator of the ROCK signaling pathway for myogenesis. PMID:23276748

  14. Phosphorylation Barcoding as a Mechanism of Directing GPCR Signaling

    NSDL National Science Digital Library

    Stephen B. Liggett (University of Maryland School of Medicine; Departments of Medicine and Physiology REV)

    2011-08-09

    A unifying mechanism by which G protein–coupled receptors (GPCRs) signal in cell type–dependent and G protein–independent ways has developed over the past decade. GPCR kinases (GRKs) are mediators of homologous desensitization: GRK phosphorylation of the receptors leads to the subsequent binding of ?-arrestins, which partially quenches receptor coupling to G proteins. For some receptors, this GRK-mediated phosphorylation stimulates additional signaling through the scaffolding action of ?-arrestin. These downstream signals are configured by ?-arrestin conformation, which is dictated by the GRK phosphoacceptors on the receptors in a barcode-like fashion. Furthermore, each of the GRKs can potentially phosphorylate different serine and threonine residues on a given receptor, and the phosphorylation pattern can be biased by the receptor conformation established by bound ligand. Finally, the arrangement of potential GRK phosphorylation sites—and thus the conformation of ?-arrestin and its effect on downstream signaling—can differ substantially between even closely related GPCRs stimulated by the same agonist. The diversity of the barcoding to flexible ?-arrestin explains the multidimensional nature of signaling in the superfamily and represents new opportunities for drug discovery.

  15. Phosphorylation of the human full-length protein kinase Ciota.

    PubMed

    Macek, Boris; Benda, Christian; Jestel, Anja; Maskos, Klaus; Mann, Matthias; Messerschmidt, Albrecht

    2008-07-01

    Atypical protein kinases C, including protein kinase Ciota (PKCiota), play critical roles in signaling pathways that control cell growth, differentiation and survival. This qualifies them as attractive targets for development of novel therapeutics for the treatment of various human diseases. In this study, the full-length PKCiota was expressed in Sf9 insect cells, purified, and digested with trypsin and endoproteinase Asp-N, and its phosphorylation analyzed by liquid chromatography-high accuracy mass spectrometry. This strategy mapped 97% of the PKCiota protein sequence and revealed seven new Ser/Thr phosphorylation sites, in addition to the two previously known, pThr403 in the activation loop and pThr555 in the turn motif of the kinase domain. Most of the newly identified phosphorylation sites had low estimated occupancies (below 2%). Two phosphorylation sites were located in domain connecting amino acid sequence stretches (pSer217 and pSer237/pSer238) and may contribute to an improved stability and solubility of the protein. The most interesting new phosphorylation site was detected in a well-accessible loop of the PB1 domain (pSer35/pSer37) and may be involved in the interactions of the PB1 domain with different partners in the relevant signaling pathways. PMID:18489133

  16. Ferredoxin:NADP+ oxidoreductase in junction with CdSe/ZnS quantum dots: characteristics of an enzymatically active nanohybrid

    NASA Astrophysics Data System (ADS)

    Szczepaniak, Krzysztof; Worch, Remigiusz; Grzyb, Joanna

    2013-05-01

    Ferredoxin:NADP+ oxidoreductase (FNR) is a plant and cyanobacterial photosynthetic enzyme, also found in non-photosynthetic tissues, where it is involved in redox reactions of biosynthetic pathways. In vivo it transfers electrons to nicotinamide adenine dinucleotide phosphate (NADP+), forming its reduced version, NADPH, while in vitro it can also use NADPH to reduce several substrates, such as ferricyanide, various quinones and nitriles. As an oxidoreductase catalyzing reaction of a broad range of substrates, FNR may be used in biotechnological processes. Quantum dots are semiconductor nanocrystals of a few to several nanometers diameter, having very useful luminescent properties. We present the spectroscopic and functional characteristics of a covalent conjugation of FNR and CdSe/ZnS quantum dots. Two types of quantum dots, of different diameter and emission maximum (550 and 650 nm), were used for comparison. Steady-state fluorescence and gel electrophoresis confirmed efficient conjugation, while fluorescence correlation spectroscopy (FCS) allowed for determination of the conjugates’ radii. The nanohybrids sustained enzymatic activity; however, changes in maximal reaction rates and Michaelis constant were found. Detailed analysis of the kinetic parameters showed that the changes in the enzyme activity depend on the substrate used for activity measurement but also on the size of the quantum dots. The presented nanohybrids, as the first example using plant and photosynthetic enzyme as a protein partner, may became a tool to study photosynthesis as well as other biosynthetic and biotechnological processes, involving enzymatically catalyzed electron transfer.

  17. An enzymatic model of the growth hormone-releasing hormone oscillator incorporating neuronal synchronization.

    PubMed

    Caplan, S Roy; Tannenbaum, Gloria S; Johnstone, Rose M

    2010-06-01

    Models of growth hormone (GH) rhythmogenesis which we and others have presented suggest that the GH pulses in the circulation are generated by a GH-releasing hormone (GHRH) oscillator with a 1h periodicity. Here we examine the possibility that this is an intrinsic neuronal rhythm resulting from enzymatic reactions occurring in the axon terminals. A "Baselator" feedback reaction sequence can generate an hourly chemical burst of a primer (presumably a low molecular weight peptide) regulating Ca(2+)-triggered exocytosis of GHRH-loaded vesicles. Accordingly we propose that the priming species is largely immobilized by binding within the terminals. Free unbound primer is able to diffuse and is alternately phosphorylated and dephosphorylated by a kinase and a phosphatase (or undergoes a similar pair of complementary reactions). Under appropriate conditions involving feedback control of one or other of the enzymes the levels of both unreacted and reacted free primer peak sharply at hourly intervals. It is self-evident that synchronization between the packed terminals of the GHRH neurons at the median eminence is necessary to generate highly ordered in vivo pulses of GH release. Gap junctions provide a means of interterminal communication for the primer. Simulations of clusters of 4 adjacent axon terminals in a linear array were performed to assess whether and when synchrony can occur. With gap junctions closed the axons were set to be 90 degrees out of phase, i.e. their chemical bursts were separated by 15 min. Opening the gap junctions, assuming either that only the unphosphorylated species permeates, or that both species permeate, resulted in rapid overall synchronization. The oscillatory systems undergo mutual entrainment and all peaks appeared simultaneously at an intermediate hourly interval. This result was independent of the mode of chemical feedback, whether positive or negative. Closing the gap junctions led to a gradual, but not immediate, loss of synchrony. PMID:20211631

  18. Conformational Changes in Protein Loops and Helices Induced by Post-Translational Phosphorylation

    PubMed Central

    Jacobson, Matthew P

    2006-01-01

    Post-translational phosphorylation is a ubiquitous mechanism for modulating protein activity and protein-protein interactions. In this work, we examine how phosphorylation can modulate the conformation of a protein by changing the energy landscape. We present a molecular mechanics method in which we phosphorylate proteins in silico and then predict how the conformation of the protein will change in response to phosphorylation. We apply this method to a test set comprised of proteins with both phosphorylated and non-phosphorylated crystal structures, and demonstrate that it is possible to predict localized phosphorylation-induced conformational changes, or the absence of conformational changes, with near-atomic accuracy in most cases. Examples of proteins used for testing our methods include kinases and prokaryotic response regulators. Through a detailed case study of cyclin-dependent kinase 2, we also illustrate how the computational methods can be used to provide new understanding of how phosphorylation drives conformational change, why substituting Glu or Asp for a phosphorylated amino acid does not always mimic the effects of phosphorylation, and how a phosphatase can “capture” a phosphorylated amino acid. This work illustrates how computational methods can be used to elucidate principles and mechanisms of post-translational phosphorylation, which can ultimately help to bridge the gap between the number of known sites of phosphorylation and the number of structures of phosphorylated proteins. PMID:16628247

  19. Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro.

    PubMed Central

    Connell-Crowley, L; Solomon, M J; Wei, N; Harper, J W

    1993-01-01

    p33cdk2 is a serine-threonine protein kinase that associates with cyclins A, D, and E and has been implicated in the control of the G1/S transition in mammalian cells. Recent evidence indicates that cyclin-dependent kinase 2 (Cdk2), like its homolog Cdc2, requires cyclin binding and phosphorylation (of threonine-160) for activation in vivo. However, the extent to which mechanistic details of the activation process are conserved between Cdc2 and Cdk2 is unknown. We have developed bacterial expression and purification systems for Cdk2 and cyclin A that allow mechanistic studies of the activation process to be performed in the absence of cell extracts. Recombinant Cdk2 is essentially inactive as a histone H1 kinase (< 4 x 10(-5) pmol phosphate transferred.min-1 x microgram-1 Cdk2). However, in the presence of equimolar cyclin A, the specific activity is approximately 16 pmol.mon-1 x microgram-1, 4 x 10(5)-fold higher than Cdk2 alone. Mutation of T160 in Cdk2 to either alanine or glutamic acid had little impact on the specific activity of the Cdk2/cyclin A complex: the activity of Cdk2T160E was indistinguishable from Cdk2, whereas that of Cdk2T160A was reduced by five-fold. To determine if the Cdk2/cyclin A complex could be activated further by phosphorylation of T160, complexes were treated with Cdc2 activating kinase (CAK), purified approximately 12,000-fold from Xenopus eggs. This treatment resulted in an 80-fold increase in specific activity. This specific activity is comparable with that of the Cdc2/cyclin B complex after complete activation by CAK (approximately 1600 pmol.mon-1 x microgram-1). Neither Cdk2T160A/cyclin A nor Cdk2T160E/cyclin A complexes were activated further by treatment with CAK. In striking contrast with cyclin A, cyclin B did not directly activate Cdk2. However, both Cdk2/cyclin A and Cdk2/cyclin B complexes display similar activity after activation by CAK. For the Cdk2/cyclin A complex, both cyclin binding and phosphorylation contribute significantly to activation, although the energetic contribution of cyclin A binding is greater than that of T160 phosphorylation by approximately 5 kcal/mol. The potential significance of direct activation of Cdk2 by cyclins with respect to regulation of cell cycle progression is discussed. Images PMID:8443411

  20. Heat Transfer

    NSDL National Science Digital Library

    Integrated Teaching and Learning Program,

    Students explore heat transfer and energy efficiency using the context of energy efficient houses. They gain a solid understanding of the three types of heat transfer: radiation, convection and conduction, which are explained in detail and related to the real world. They learn about the many ways solar energy is used as a renewable energy source to reduce the emission of greenhouse gasses and operating costs. Students also explore ways in which a device can capitalize on the methods of heat transfer to produce a beneficial result. They are given the tools to calculate the heat transferred between a system and its surroundings.

  1. Enzymatic modifications of exopolysaccharides enhance bacterial persistence

    PubMed Central

    Whitfield, Gregory B.; Marmont, Lindsey S.; Howell, P. Lynne

    2015-01-01

    Biofilms are surface-attached communities of bacterial cells embedded in a self-produced matrix that are found ubiquitously in nature. The biofilm matrix is composed of various extracellular polymeric substances, which confer advantages to the encapsulated bacteria by protecting them from eradication. The matrix composition varies between species and is dependent on the environmental niche that the bacteria inhabit. Exopolysaccharides (EPS) play a variety of important roles in biofilm formation in numerous bacterial species. The ability of bacteria to thrive in a broad range of environmental settings is reflected in part by the structural diversity of the EPS produced both within individual bacterial strains as well as by different species. This variability is achieved through polymerization of distinct sugar moieties into homo- or hetero-polymers, as well as post-polymerization modification of the polysaccharide. Specific enzymes that are unique to the production of each polymer can transfer or remove non-carbohydrate moieties, or in other cases, epimerize the sugar units. These modifications alter the physicochemical properties of the polymer, which in turn can affect bacterial pathogenicity, virulence, and environmental adaptability. Herein, we review the diversity of modifications that the EPS alginate, the Pel polysaccharide, Vibrio polysaccharide, cepacian, glycosaminoglycans, and poly-N-acetyl-glucosamine undergo during biosynthesis. These are EPS produced by human pathogenic bacteria for which studies have begun to unravel the effect modifications have on their physicochemical and biological properties. The biological advantages these polymer modifications confer to the bacteria that produce them will be discussed. The expanding list of identified modifications will allow future efforts to focus on linking these modifications to specific biosynthetic genes and biofilm phenotypes. PMID:26029200

  2. Crystal structure of a phosphorylation-coupled saccharide transporter

    PubMed Central

    Cao, Yu; Jin, Xiangshu; Levin, Elena J.; Huang, Hua; Zong, Yinong; Quick, Matthias; Weng, Jun; Pan, Yaping; Love, James; Punta, Marco; Rost, Burkhard; Hendrickson, Wayne A.; Javitch, Jonathan A.; Rajashankar, Kanagalaghatta R.; Zhou, Ming

    2011-01-01

    Saccharides play a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the N-terminal halves of the two protomers. The C-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation. PMID:21471968

  3. Crystal Structure of a Phosphorylation-coupled Saccharide Transporter

    SciTech Connect

    Y Cao; X Jin; E Levin; H Huang; Y Zong; W Hendrickson; J Javitch; K Rajashankar; M Zhou; et al.

    2011-12-31

    Saccharides have a central role in the nutrition of all living organisms. Whereas several saccharide uptake systems are shared between the different phylogenetic kingdoms, the phosphoenolpyruvate-dependent phosphotransferase system exists almost exclusively in bacteria. This multi-component system includes an integral membrane protein EIIC that transports saccharides and assists in their phosphorylation. Here we present the crystal structure of an EIIC from Bacillus cereus that transports diacetylchitobiose. The EIIC is a homodimer, with an expansive interface formed between the amino-terminal halves of the two protomers. The carboxy-terminal half of each protomer has a large binding pocket that contains a diacetylchitobiose, which is occluded from both sides of the membrane with its site of phosphorylation near the conserved His250 and Glu334 residues. The structure shows the architecture of this important class of transporters, identifies the determinants of substrate binding and phosphorylation, and provides a framework for understanding the mechanism of sugar translocation.

  4. Odor-induced phosphorylation of olfactory cilia proteins.

    PubMed

    Boekhoff, I; Schleicher, S; Strotmann, J; Breer, H

    1992-12-15

    Stimulation of isolated rat olfactory cilia in the presence of [gamma-32P]ATP leads to a significantly enhanced incorporation of [32P]phosphate. Depending on the type of odorants applied, the induced phosphorylation is completely blocked by specific inhibitors of either protein kinase A or protein kinase C. Time-course experiments indicate that the odor-induced modification of ciliary proteins is transient; the intensity of labeling decayed over time (1-10 sec). Separation of ciliary proteins by SDS/polyacrylamide gel electrophoresis followed by autoradiography demonstrated that upon stimulation with lilial, a single polypeptide (50,000 Da) was phosphorylated; the size of the modified protein is in line with the hypothesis that odorant receptors are phosphorylated subsequent to activation by specific odors. PMID:1334554

  5. Odor-induced phosphorylation of olfactory cilia proteins.

    PubMed Central

    Boekhoff, I; Schleicher, S; Strotmann, J; Breer, H

    1992-01-01

    Stimulation of isolated rat olfactory cilia in the presence of [gamma-32P]ATP leads to a significantly enhanced incorporation of [32P]phosphate. Depending on the type of odorants applied, the induced phosphorylation is completely blocked by specific inhibitors of either protein kinase A or protein kinase C. Time-course experiments indicate that the odor-induced modification of ciliary proteins is transient; the intensity of labeling decayed over time (1-10 sec). Separation of ciliary proteins by SDS/polyacrylamide gel electrophoresis followed by autoradiography demonstrated that upon stimulation with lilial, a single polypeptide (50,000 Da) was phosphorylated; the size of the modified protein is in line with the hypothesis that odorant receptors are phosphorylated subsequent to activation by specific odors. Images PMID:1334554

  6. Reversible Phosphorylation of Histidine Residues in Proteins from Vertebrates

    NSDL National Science Digital Library

    Susanne Klumpp (Munster; Westfalische Wilhelms-Universitat REV)

    2009-03-10

    Signaling by kinases and phosphatases that act on serine, threonine, and tyrosine residues of proteins is among the most extensively studied regulatory mechanisms in mammalian cells, and research focused in this area is ongoing. We are just beginning to appreciate that such signaling mechanisms are extended and enriched by the reversible phosphorylation of histidine residues. The most exciting developments in this field to date come from studies on the ? subunit of heterotrimeric guanosine triphosphate–binding proteins (G proteins), the enzyme adenosine 5?-triphosphate-citrate lyase, and now the Ca2+-activated K+ channel KCa3.1, all of which are targeted by nucleoside diphosphate kinase (which phosphorylates histidines) and protein histidine phosphatase (which dephosphorylates phosphorylated histidines).

  7. Molecular mechanism of phosphorylation-dependent arrestin activation.

    PubMed

    Ostermaier, Martin K; Schertler, Gebhard F X; Standfuss, Joerg

    2014-12-01

    The past years have seen tremendous progress towards understanding how arrestins recognize phosphorylated G protein-coupled receptors (GPCRs). Two arrestin crystal structures, one of a pre-activated splice variant and one bound to a GPCR phosphopeptide, provided insights into the conformational changes upon phosphate recognition. Scanning mutagenesis and spectroscopic studies complete the picture of arrestin activation and receptor binding. Most perspicuous is the C-tail exchange mechanism, by which the C-tail of arrestin is released from its basal conformation and replaced by the phosphorylated GPCR C-terminus. Three positively charged clusters could act as conserved arrestin phosphosensors. Variations in the pattern of phosphorylation in a GPCR and variations within the C-terminus of different GPCRs may encode specificity to arrestin subtypes and particular physiological responses. PMID:25484000

  8. Bioinformatics Study of Cancer-Related Mutations within p53 Phosphorylation Site Motifs

    PubMed Central

    Ji, Xiaona; Huang, Qiang; Yu, Long; Nussinov, Ruth; Ma, Buyong

    2014-01-01

    p53 protein has about thirty phosphorylation sites located at the N- and C-termini and in the core domain. The phosphorylation sites are relatively less mutated than other residues in p53. To understand why and how p53 phosphorylation sites are rarely mutated in human cancer, using a bioinformatics approaches, we examined the phosphorylation site and its nearby flanking residues, focusing on the consensus phosphorylation motif pattern, amino-acid correlations within the phosphorylation motifs, the propensity of structural disorder of the phosphorylation motifs, and cancer mutations observed within the phosphorylation motifs. Many p53 phosphorylation sites are targets for several kinases. The phosphorylation sites match 17 consensus sequence motifs out of the 29 classified. In addition to proline, which is common in kinase specificity-determining sites, we found high propensity of acidic residues to be adjacent to phosphorylation sites. Analysis of human cancer mutations in the phosphorylation motifs revealed that motifs with adjacent acidic residues generally have fewer mutations, in contrast to phosphorylation sites near proline residues. p53 phosphorylation motifs are mostly disordered. However, human cancer mutations within phosphorylation motifs tend to decrease the disorder propensity. Our results suggest that combination of acidic residues Asp and Glu with phosphorylation sites provide charge redundancy which may safe guard against loss-of-function mutations, and that the natively disordered nature of p53 phosphorylation motifs may help reduce mutational damage. Our results further suggest that engineering acidic amino acids adjacent to potential phosphorylation sites could be a p53 gene therapy strategy. PMID:25075982

  9. GABAB receptor phosphorylation regulates KCTD12-induced K+ current desensitization

    PubMed Central

    Adelfinger, Lisa; Turecek, Rostislav; Ivankova, Klara; Jensen, Anders A.; Moss, Stephen J.; Gassmann, Martin; Bettler, Bernhard

    2015-01-01

    GABAB receptors assemble from GABAB1 and GABAB2 subunits. GABAB2 additionally associates with auxiliary KCTD subunits (named after their K+ channel tetramerization-domain). GABAB receptors couple to heterotrimeric G–proteins and activate inwardly-rectifying K+ channels through the ?? subunits released from the G-protein. Receptor-activated K+ currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity. Desensitization of K+ currents integrates distinct mechanistic underpinnings. GABAB receptor activity reduces protein kinase-A activity, which reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G-protein ?? subunits. Here we show that the two mechanisms of desensitization influence each other. Serine-892 phosphorylation in heterologous cells rearranges KCTD12 at the receptor and slows KCTD12-induced desensitization. Likewise, protein kinase-A activation in hippocampal neurons slows fast desensitization of GABAB receptor-activated K+ currents while protein kinase-A inhibition accelerates fast desensitization. Protein kinase-A fails to regulate fast desensitization in KCTD12 knock-out mice or knock-in mice with a serine-892 to alanine mutation, thus demonstrating that serine-892 phosphorylation regulates KCTD12-induced desensitization in vivo. Fast current desensitization is accelerated in hippocampal neurons carrying the serine-892 to alanine mutation, showing that tonic serine-892 phosphorylation normally limits KCTD12-induced desensitization. Tonic serine-892 phosphorylation is in turn promoted by assembly of receptors with KCTD12. This cross-regulation of serine-892 phosphorylation and KCTD12 activity sharpens the response during repeated receptor activation. PMID:25065880

  10. ERK phosphorylation is required for retention of trace fear memory.

    PubMed

    Villarreal, Julissa S; Barea-Rodriguez, Edwin J

    2006-01-01

    The extracellular signal-regulated kinase (ERK) has been previously associated with long-term memory formation. Earlier studies have demonstrated a role for phospho-ERK in delay fear conditioning and it has been shown to disrupt trace fear memory when inhibited after training. cAMP response element binding protein (CREB) is a key transcription factor that has been implicated in long-term memory formation across different species. It has also been shown to be modulated by ERK. In our study, we used the drug SL327 to prevent ERK phosphorylation. Two groups of Fischer 344 male rats (2-4 months) were injected intraperitoneally with 100% DMSO (2 ml/kg) or SL327 (100 mg/kg/2 ml dissolved in DMSO) 45 min before 10 trials of trace fear conditioning. Each trial consisted of a tone paired with a footshock with a 30-s interval separating the stimuli. Twenty-four hours later, rats were tested for fear to the tone. Our results showed that SL327-treated rats displayed memory deficits 24 h after training. Western blot analyses of total hippocampal protein revealed a significant increase in phosphorylated ERK immediately after training. There were also decreases in phosphorylated ERK at 45 and 90 min post-injection of SL327-treated rats as compared to DMSO-treated rats, but levels of phosphorylated CREB remained the same. These findings indicate that ERK phosphorylation is increased immediately after trace fear conditioning and inhibiting this increase is correlated with memory deficits in trace fear conditioning 24 h later. These findings support a role for ERK phosphorylation in the formation of trace fear memories. PMID:16182574

  11. Global consequences of activation loop phosphorylation on protein kinase A.

    PubMed

    Steichen, Jon M; Iyer, Ganesh H; Li, Sheng; Saldanha, S Adrian; Deal, Michael S; Woods, Virgil L; Taylor, Susan S

    2010-02-01

    Phosphorylation of the activation loop is one of the most common mechanisms for regulating protein kinase activity. The catalytic subunit of cAMP-dependent protein kinase autophosphorylates Thr(197) in the activation loop when expressed in Escherichia coli. Although mutation of Arg(194) to Ala prevents autophosphorylation, phosphorylation of Thr(197) can still be achieved by a heterologous protein kinase, phosphoinositide-dependent protein kinase (PDK1), in vitro. In this study, we examined the structural and functional consequences of adding a single phosphate to the activation loop of cAMP-dependent protein kinase by comparing the wild type C-subunit to the R194A mutant either in the presence or the absence of activation loop phosphorylation. Phosphorylation of Thr(197) decreased the K(m) by approximately 15- and 7-fold for kemptide and ATP, respectively, increased the stability of the enzyme as measured by fluorescence and circular dichroism, and enhanced the binding between the C-subunit and IP20, a protein kinase inhibitor peptide. Additionally, deuterium exchange coupled to mass spectrometry was used to compare the structural dynamics of these proteins. All of the regions of the C-subunit analyzed underwent amide hydrogen exchange at a higher or equal rate in the unphosphorylated enzyme compared with the phosphorylated enzyme. The largest changes occurred at the C terminus of the activation segment in the p + 1 loop/APE regions and the alphaH-alphaI loop motifs and leads to the prediction of a coordinated phosphorylation-induced salt bridge between two conserved residues, Glu(208) and Arg(280). PMID:19965870

  12. Spatiotemporal Dynamics of Phosphorylation in Lipid Second Messenger Signaling*

    PubMed Central

    Antal, Corina E.; Newton, Alexandra C.

    2013-01-01

    The plasma membrane serves as a dynamic interface that relays information received at the cell surface into the cell. Lipid second messengers coordinate signaling on this platform by recruiting and activating kinases and phosphatases. Specifically, diacylglycerol and phosphatidylinositol 3,4,5-trisphosphate activate protein kinase C and Akt, respectively, which then phosphorylate target proteins to transduce downstream signaling. This review addresses how the spatiotemporal dynamics of protein kinase C and Akt signaling can be monitored using genetically encoded reporters and provides information on how the coordination of signaling at protein scaffolds or membrane microdomains affords fidelity and specificity in phosphorylation events. PMID:23788531

  13. Atg1 kinase organizes autophagosome formation by phosphorylating Atg9

    PubMed Central

    Papinski, Daniel; Kraft, Claudine

    2014-01-01

    The conserved Ser/Thr kinase Atg1/ULK1 plays a crucial role in the regulation of autophagy. However, only very few Atg1 targets have been identified, impeding elucidation of the mechanisms by which Atg1 regulates autophagy. In our study, we determined the Saccharomyces cerevisiae Atg1 consensus phosphorylation sequence using a peptide array-based approach. Among proteins containing this sequence we identified Atg9, another essential component of the autophagic machinery. We showed that phosphorylation of Atg9 by Atg1 is required for phagophore elongation, shedding light on the mechanism by which Atg1 regulates early steps of autophagy. PMID:24905091

  14. Atg1 kinase organizes autophagosome formation by phosphorylating Atg9.

    PubMed

    Papinski, Daniel; Kraft, Claudine

    2014-07-01

    The conserved Ser/Thr kinase Atg1/ULK1 plays a crucial role in the regulation of autophagy. However, only very few Atg1 targets have been identified, impeding elucidation of the mechanisms by which Atg1 regulates autophagy. In our study, we determined the Saccharomyces cerevisiae Atg1 consensus phosphorylation sequence using a peptide array-based approach. Among proteins containing this sequence we identified Atg9, another essential component of the autophagic machinery. We showed that phosphorylation of Atg9 by Atg1 is required for phagophore elongation, shedding light on the mechanism by which Atg1 regulates early steps of autophagy. PMID:24905091

  15. Human Cytomegalovirus UL97 Phosphorylates the Viral Nuclear Egress Complex

    PubMed Central

    Sharma, Mayuri; Bender, Brian J.; Kamil, Jeremy P.; Lye, Ming F.; Pesola, Jean M.; Reim, Natalia I.; Hogle, James M.

    2014-01-01

    ABSTRACT Herpesvirus nucleocapsids exit the host cell nucleus in an unusual process known as nuclear egress. The human cytomegalovirus (HCMV) UL97 protein kinase is required for efficient nuclear egress, which can be explained by its phosphorylation of the nuclear lamina component lamin A/C, which disrupts the nuclear lamina. We found that a dominant negative lamin A/C mutant complemented the replication defect of a virus lacking UL97 in dividing cells, validating this explanation. However, as complementation was incomplete, we investigated whether the HCMV nuclear egress complex (NEC) subunits UL50 and UL53, which are required for nuclear egress and recruit UL97 to the nuclear rim, are UL97 substrates. Using mass spectrometry, we detected UL97-dependent phosphorylation of UL50 residue S216 (UL50-S216) and UL53-S19 in infected cells. Moreover, UL53-S19 was specifically phosphorylated by UL97 in vitro. Notably, treatment of infected cells with the UL97 inhibitor maribavir or infection with a UL97 mutant led to a punctate rather than a continuous distribution of the NEC at the nuclear rim. Alanine substitutions in both UL50-S216 and UL53-S19 resulted in a punctate distribution of the NEC in infected cells and also decreased virus production and nuclear egress in the absence of maribavir. These results indicate that UL97 phosphorylates the NEC and suggest that this phosphorylation modulates nuclear egress. Thus, the UL97-NEC interaction appears to recruit UL97 to the nuclear rim both for disruption of the nuclear lamina and phosphorylation of the NEC. IMPORTANCE Human cytomegalovirus (HCMV) causes birth defects and it can cause life-threatening diseases in immunocompromised patients. HCMV assembles in the nucleus and then translocates to the cytoplasm in an unusual process termed nuclear egress, an attractive target for antiviral therapy. A viral enzyme, UL97, is important for nuclear egress. It has been proposed that this is due to its role in disruption of the nuclear lamina, which would otherwise impede nuclear egress. In validating this proposal, we showed that independent disruption of the lamina can overcome a loss of UL97, but only partly, suggesting additional roles for UL97 during nuclear egress. We then found that UL97 phosphorylates the viral nuclear egress complex (NEC), which is essential for nuclear egress, and we obtained evidence that this phosphorylation modulates this process. Our results highlight a new role for UL97, the mutual dependence of the viral NEC and UL97 during nuclear egress, and differences among herpesviruses. PMID:25339763

  16. Theoretical Studies of Interactions between O-Phosphorylated and Standard Amino-Acid Side-Chain Models in Water.

    PubMed

    Wi?niewska, Marta; Sobolewski, Emil; O?dziej, Stanis?aw; Liwo, Adam; Scheraga, Harold A; Makowski, Mariusz

    2015-07-01

    Phosphorylation is a common post-translational modification of the amino-acid side chains (serine, tyrosine, and threonine) that contain hydroxyl groups. The transfer of the negatively charged phosphate group from an ATP molecule to such amino-acid side chains leads to changes in the local conformations of proteins and the pattern of interactions with other amino-acid side-chains. A convenient characteristic of the side chain-side chain interactions in the context of an aqueous environment is the potential of mean force (PMF) in water. A series of umbrella-sampling molecular dynamic (MD) simulations with the AMBER force field were carried out for pairs of O-phosphorylated serine (pSer), threonine (pThr), and tyrosine, (pTyr) with natural amino acids in a TIP3P water model as a solvent at 298 K. The weighted-histogram analysis method was used to calculate the four-dimensional potentials of mean force. The results demonstrate that the positions and depths of the contact minima and the positions and heights of the desolvation maxima, including their dependence on the relative orientation depend on the character of the interacting pairs. More distinct minima are observed for oppositely charged pairs such as, e.g., O-phosphorylated side-chains and positively charged ones, such as the side-chains of lysine and arginine. PMID:26100791

  17. Enzymatic Amplification of DNA/RNA Hybrid Molecular Beacon Signaling in Nucleic Acid Detection

    PubMed Central

    Jacroux, Thomas; Rieck, Daniel C.; Cui, Rong; Ouyang, Yexin; Dong, Wen-Ji

    2012-01-01

    A rapid assay operable under isothermal or non-isothermal conditions is described wherein the sensitivity of a typical molecular beacon (MB) system is improved by utilizing thermostable RNase H to enzymatically cleave an MB comprised of a DNA stem and RNA loop (R/D-MB). Upon hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7x above background) due to an opening of the probe and concomitant reduction in the Förster resonance energy transfer efficiency. Addition of thermostable RNase H resulted in the cleavage of the RNA loop which eliminated energy transfer. The cleavage step also released bound target DNA, enabling it to bind to another R/D-MB probe and rendering the approach a cyclic amplification scheme. Full processing of R/D-MBs maximized the fluorescence signal to the fullest extent possible (12.9x above background), resulting in a ~2–2.8 fold increase in the signal-to-noise ratio observed isothermally at 50 °C following the addition of RNase H. The probe was also used to monitor real-time PCR reactions by measuring enhancement of donor fluorescence upon R/D-MB binding to amplified pUC19 template dilutions. Hence, the R/D-MB-RNase H scheme can be applied to a broad range of nucleic acid amplification methods. PMID:23000602

  18. Scale-up and evaluation of high solid ionic liquid pretreatment and enzymatic hydrolysis of switchgrass

    PubMed Central

    2013-01-01

    Background Ionic liquid (IL) pretreatment is receiving significant attention as a potential process that enables fractionation of lignocellulosic biomass and produces high yields of fermentable sugars suitable for the production of renewable fuels. However, successful optimization and scale up of IL pretreatment involves challenges, such as high solids loading, biomass handling and transfer, washing of pretreated solids and formation of inhibitors, which are not addressed during the development stages at the small scale in a laboratory environment. As a first in the research community, the Joint BioEnergy Institute, in collaboration with the Advanced Biofuels Process Demonstration Unit, a Department of Energy funded facility that supports academic and industrial entities in scaling their novel biofuels enabling technologies, have performed benchmark studies to identify key challenges associated with IL pretreatment using 1-ethyl-3-methylimidazolium acetate and subsequent enzymatic saccharification beyond bench scale. Results Using switchgrass as the model feedstock, we have successfully executed 600-fold, relative to the bench scale (6 L vs 0.01 L), scale-up of IL pretreatment at 15% (w/w) biomass loading. Results show that IL pretreatment at 15% biomass generates a product containing 87.5% of glucan, 42.6% of xylan and only 22.8% of lignin relative to the starting material. The pretreated biomass is efficiently converted into monosaccharides during subsequent enzymatic hydrolysis at 10% loading over a 150-fold scale of operations (1.5 L vs 0.01 L) with 99.8% fermentable sugar conversion. The yield of glucose and xylose in the liquid streams were 94.8% and 62.2%, respectively, and the hydrolysate generated contains high titers of fermentable sugars (62.1 g/L of glucose and 5.4 g/L cellobiose). The overall glucan and xylan balance from pretreatment and saccharification were 95.0% and 77.1%, respectively. Enzymatic inhibition by [C2mim][OAc] at high solids loadings requires further process optimization to obtain higher yields of fermentable sugars. Conclusion Results from this initial scale up evaluation indicate that the IL-based conversion technology can be effectively scaled to larger operations and the current study establishes the first scaling parameters for this conversion pathway but several issues must be addressed before a commercially viable technology can be realized, most notably reduction in water consumption and efficient IL recycle. PMID:24160440

  19. Phosphorylation of connexin43 on S279/282 may contribute to laminopathy-associated conduction defects

    SciTech Connect

    Chen, Steven C., E-mail: bug@uw.edu [Fred Hutchinson Cancer Research Center (FHCRC), Public Health Sciences Division, 1100 Fairview Ave. N., Seattle, WA 98109 (United States); University of Washington Department of Biochemistry, 1959 NE Pacific St., Seattle, WA 98195 (United States); Kennedy, Brian K., E-mail: bkennedy@buckinstitute.org [University of Washington Department of Biochemistry, 1959 NE Pacific St., Seattle, WA 98195 (United States); Buck Institute for Research on Aging, 8001 Redwood Boulevard, Novato, CA 94945 (United States); Lampe, Paul D., E-mail: plampe@fhcrc.org [Fred Hutchinson Cancer Research Center (FHCRC), Public Health Sciences Division, 1100 Fairview Ave. N., Seattle, WA 98109 (United States)

    2013-04-01

    An understanding of the molecular mechanism behind the arrhythmic phenotype associated with laminopathies has yet to emerge. A-type lamins have been shown to interact and sequester activated phospho-ERK1/2(pERK1/2) at the nucleus. The gap junction protein connexin43 (Cx43) can be phosphorylated by pERK1/2 on S279/282 (pS279/282), inhibiting intercellular communication. We hypothesized that without A-type lamins, pS279/282 Cx43 will increase due to inappropriate phosphorylation by pERK1/2, resulting in decreased gap junction function. We observed a 1.6-fold increase in pS279/282 Cx43 levels in Lmna{sup ?/?} mouse embryonic fibroblasts (MEFs) compared to Lmna{sup +/+}, and 1.8-fold more pERK1/2 co-precipitated from Lmna{sup ?/?} MEFs with Cx43 antibodies. We found a 3-fold increase in the fraction of non-nuclear pERK1/2 and a concomitant 2-fold increase in the fraction of pS279/282 Cx43 in Lmna{sup ?/?} MEFs by immunofluorescence. In an assay of gap junctional function, Lmna{sup ?/?} MEFs transferred dye to 60% fewer partners compared to Lmna{sup +/+} controls. These results are mirrored in 5–6 week-old Lmna{sup ?/?} mice compared to their Lmna{sup +/+} littermates as we detect increased pS279/282 Cx43 in gap junctions by immunofluorescence and 1.7-fold increased levels by immunoblot. We conclude that increased pS279/282 Cx43 in the Lmna{sup ?/?} background results in decreased cell communication and may contribute to the arrhythmic pathology in vivo. - Highlights: ? Connexin43 phosphorylation plays a role in laminopathy-associated conduction defects. ? Loss of A-type lamin activity results in release of pERK1/2 from the nucleus. ? Increased cytoplasmic localization of pERK1/2 acts to phosphorylate S279/282 of Cx43. ? Phosphorylation of S279/282 on Cx43 decreases gap junction activity in cell culture. ? Mice lacking A-type lamins have increased phosphorylation on S279/282 of Cx43.

  20. Enzymatic degradation of chitins and partially deacetylated chitins.

    PubMed

    Shigemasa, Y; Saito, K; Sashiwa, H; Saimoto, H

    1994-02-01

    The enzymatic (lysozyme, chitinase etc.) digestibility of chitins obtained from squid pen and shrimp shell, and of partially deacetylated chitins (DA-chitins) was investigated. The digestibility of various chitins by the chitinase from Bacillus sp. PI-7S was much higher than that by lysozyme, and beta-chitin was digested more smoothly than alpha-chitin. DA-chitin deacetylated under homogeneous conditions (DAC) was hydrolysed by lysozyme more rapidly than that deacetylated under heterogeneous conditions (DAC). DACs from shrimp shell and squid pen showed the same degree of digestibility by lysozyme in spite of a difference in the crystal structure of the original chitins. The crystal structure of chitin and the degree of N-acetyl group aggregation among DA-chitin molecules affect the enzymatic digestibility of chitin and DA-chitin, respectively. PMID:8180144

  1. Enhancement of enzymatic hydrolysis by simultaneous attrition of cellulosic substrates

    SciTech Connect

    Neilson, M.J.; Kelsey, R.G.; Shafizadeh, F.

    1982-02-01

    It has been shown that simultaneous attrition of cellulose in an attritor containing stainless-steel beads results in a substantial enhancement of the enzymatic hydrolysis. The attrition exerts two opposing effects, continuous delamination and comminution of the substrate with formation of new reactive sites and a gradual denaturation and inactivation of the enzyme. Consequently, the hydrolysis proceeds very rapidly at first and levels off at about 70% saccharification of the substrate. Accumulation of hydrolysis products is also responsible for inhibition of the enzyme. The attrition method is effective for the saccharification of wood, lignocellulose, holocellulose, and cellulose with simultaneous attrition showed that the lignin component provided more hindrance toward the saccharification process than hemicelluloses, which are themselves subject to enzymatic hydrolysis.

  2. Enzymatic methods in food analysis: determination of ascorbic acid.

    PubMed

    Shekhovtsova, Tatyana N; Muginova, Svetlana V; Luchinina, Julia A; Galimova, Anna Z

    2006-07-28

    The feasibility and expediency of enzymatic methods application in food analysis is demonstrated by the example of ascorbic acid (AsA) determination in foods. Enzymatic determination of ascorbic acid is based on its action as a second substrate of horseradish (HRP) and peanut (PNP) peroxidases in the reactions of o-dianisidine (OD) and 3,3',5,5'-tetramethylbenzidine (TMB) oxidation with hydrogen peroxide. The rates of the reactions are monitored spectrophotometrically by measuring the duration of the induction period on kinetic curves plotted in coordinates absorption-time. The proposed procedures are sensitive (c(L)=0.1 microM), simple, and rapid. The procedure using horseradish peroxidase and the reaction of TMB oxidation was used to determine ascorbic acid in fruit juices, milk and sour-milk products for babies' nutrition. PMID:17723515

  3. Bioremediation of uranium contamination with enzymatic uranium reduction

    USGS Publications Warehouse

    Lovley, D.R.; Phillips, E.J.P.

    1992-01-01

    Enzymatic uranium reduction by Desulfovibrio desulfuricans readily removed uranium from solution in a batch system or when D. desulfuricans was separated from the bulk of the uranium-containing water by a semipermeable membrane. Uranium reduction continued at concentrations as high as 24 mM. Of a variety of potentially inhibiting anions and metals evaluated, only high concentrations of copper inhibited uranium reduction. Freeze-dried cells, stored aerobically, reduced uranium as fast as fresh cells. D. desulfuricans reduced uranium in pH 4 and pH 7.4 mine drainage waters and in uraniumcontaining groundwaters from a contaminated Department of Energy site. Enzymatic uranium reduction has several potential advantages over other bioprocessing techniques for uranium removal, the most important of which are as follows: the ability to precipitate uranium that is in the form of a uranyl carbonate complex; high capacity for uranium removal per cell; the formation of a compact, relatively pure, uranium precipitate.

  4. Enzymatic hydrolysis of cellulosic materials: a kinetic study

    SciTech Connect

    Beltrame, P.L.; Carniti, P.; Focher, B.; Marzetti, A.; Sarto, V.

    1984-01-01

    A kinetic study of the enzymatic hydrolysis of two celluloses with different structural features was performed at various temperatures (26-50/sup 0/C). The enzymatic system consisted of three types of enzymes: E/sub 1/-..beta..-1,4-glucan glucanohydrolase; E/sub 2/-..beta..-1,4-glucan cellobiohydrolase; and E/sub 3/-..beta..-glucosidase. A mathematical model for the mechanism of the hydrolysis of cellulosic materials catalyzed by a multienzymatic system was checked and a good rationalization of the experimental results was achieved. Uncompetitive and competitive glucose inhibition on E/sub 1/ and E/sub 2/, respectively, appeared to occur for both substrates. Inhibition by cellobiose was checked at 34/sup 0/C on one substrate. The V/sub max/, K/sub m/, and glucose inhibition constants were optimized and their dependence on temperature determined.

  5. 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

  6. Enzymatic cybernetics: An unpublished work by Jacques Monod.

    PubMed

    Gayon, Jean

    2015-06-01

    In 1959, Jacques Monod wrote a manuscript entitled Cybernétique enzymatique [Enzymatic cybernetics]. Never published, this unpublished manuscript presents a synthesis of how Monod interpreted enzymatic adaptation just before the publication of the famous papers of the 1960s on the operon. In addition, Monod offers an example of a philosophy of biology immersed in scientific investigation. Monod's philosophical thoughts are classified into two categories, methodological and ontological. On the methodological side, Monod explicitly hints at his preferences regarding the scientific method in general: hypothetical-deductive method, and use of theoretical models. He also makes heuristic proposals regarding molecular biology: the need to analyse the phenomena in question at the level of individual cells, and the dual aspect of all biological explanation, functional and evolutionary. Ontological issues deal with the notions of information and genetic determinism, "cellular memory", the irrelevance of the notion of "living matter", and the usefulness of a cybernetic comprehension of molecular biology. PMID:26003505

  7. Pretreatment of Agave americana stalk for enzymatic saccharification.

    PubMed

    Yang, Qiang; Pan, Xuejun

    2012-12-01

    Agave americana is one of commonly grown agave species but currently less valuable because its large flower stalk cannot be used for producing alcoholic beverage. In the present study, the stalk was pretreated with dilute acid (DA), sulfite (SPORL), and sodium hydroxide (NaOH) to preliminarily assess its potential as feedstock for bioethanol production. The changes of cell wall components during the pretreatments, enzymatic digestibility of the pretreated stalks, and the adsorption of cellulases on the substrates were investigated. Results indicated that the pretreatments significantly improved the enzymatic digestibility of the agave stalk. SPORL pretreatment gave higher substrate and sugar yields, while NaOH pretreated stalk had better digestibility under the investigated conditions. The better hydrolysability of NaOH-pretreated stalk was attributed to low lignin and hemicellulose content and high affinity to cellulases. PMID:23122484

  8. Phytochrome Induces Rapid PIF5 Phosphorylation and Degradation in Response to Red-Light Activation1[W][OA

    PubMed Central

    Shen, Yu; Khanna, Rajnish; Carle, Christine M.; Quail, Peter H.

    2007-01-01

    The phytochrome (phy) family of sensory photoreceptors (phyA–phyE in Arabidopsis thaliana) induces changes in target-gene expression upon light-induced translocation to the nucleus, where certain members interact with selected members of the constitutively nuclear basic helix-loop-helix transcription factor family, such as PHYTOCHROME-INTERACTING FACTOR3 (PIF3). Previous evidence indicates that the binding of the photoactivated photoreceptor molecule to PIF3 induces rapid phosphorylation of the transcription factor in the cell prior to its degradation via the ubiqitin-proteosome system. To investigate whether this apparent primary signaling mechanism can be generalized to other phy-interacting partners, we have examined the molecular behavior of a second related phy-interacting member of the basic helix-loop-helix family, PIF5, during early deetiolation, immediately following initial exposure of dark-grown seedlings to light. The data show that red light induces very rapid phosphorylation and subsequent degradation (t1/2 < 5 min) of PIF5 via the proteosome system upon irradiation. Photobiological and genetic evidence indicates that the photoactivated phy molecule acts within 60 s to induce this phosphorylation of PIF5, and that phyA and phyB redundantly dominate this process, with phyD playing an apparently minor role. Collectively, the data support the proposal that the rapid phy-induced phosphorylation of PIF3 and PIF5 may represent the biochemical mechanism of primary signal transfer from photoactivated photoreceptor to binding partner, and that phyA and phyB (and possibly phyD) may signal to multiple, shared partners utilizing this common mechanism. PMID:17827270

  9. Enzymatic activity of allergenic house dust and storage mite extracts.

    PubMed

    Morales, Maria; Iraola, Víctor; Leonor, Jose R; Carnés, Jerónimo

    2013-01-01

    Proteases are involved in the pathogenicity of allergy, increasing epithelial permeability and acting as adjuvants. Enzymatic activity is therefore important for the allergenicity of an extract and also affects its stability and safety. However, the enzymatic activity of extracts is not usually evaluated. The objective of this study was to evaluate the enzymatic activity of the most allergenic mite extracts and to investigate their allergenic properties. Extracts from nine allergenic mite species (Dermatophagoides pteronyssinus, Dermatophagoides farinae Hughes, Euroglyphus maynei, Lepidoglyphus destructor, Tyrophagus putrescentiae (Schrank), Glycyphagus domesticus (DeGeer), Acarus siro L., Chortoglyphus arcuatus, and Blomia tropicalis) were characterized. Protein and allergen profiles were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western-blot, respectively. Gelatinolytic activity was evaluated with a zymogram and the activity of other enzymes (cysteine, serine proteases, and esterases) was evaluated individually or with the API-ZYM system. The main differences in protease activity were found between house dust mites and storage mites. House dust mites presented higher cysteine protease activity while storage mites presented higher serine protease activity. These differences are in line with their trophic specialization. A wide range of different activities was found in all the extracts analyzed, reflecting the fact that the extracts preserve the activity of many enzymes, this being necessary for a correct diagnosis. However, enzymes may act as adjuvants and, therefore, could lead to undesirable effects in immunotherapies, making this activity not suitable for treatment products. Modified extracts with lower enzymatic activity could be more appropriate for immunotherapy. PMID:23427664

  10. Detection of infant faecal bifidobacteria by enzymatic methods

    Microsoft Academic Search

    E. Vlková; J. Nevoral; B. Jencikova; J. Kopecÿny; J. Godefrooij; I. Trojanová; V. Rada

    2005-01-01

    An enzyme-based assay was developed for the detection of bifidobacteria in infant faeces. Ninety-five samples from 51 breast-fed infants in the age between 3 and 276 days were investigated. Bifidobacteria and other bacterial groups were determined by cultivation and fluorescence in situ hybridisation (FISH). Faecal samples were examined for the activity of fructoso-6-phosphate phosphoketolase (F6PPK) and for other enzymatic reactions

  11. CO(2) fixation through hydrogenation by chemical or enzymatic methods.

    PubMed

    Beller, Matthias; Bornscheuer, Uwe T

    2014-04-25

    Two birds with one stone: The simulaneous fixation of the greenhouse gas carbon dioxide and storage of the alternative fuel hydrogen can be accomplished with the formation of formic acid. In principle, this is now possible either with an enzymatic system based on a newly discovered bacterial hydrogen-dependent carbon dioxide reductase or by using organometallic catalysts at room temperature and ambient pressure. PMID:24706361

  12. Activity-based proteomics: enzymatic activity profiling in complex proteomes

    Microsoft Academic Search

    H. Schmidinger; A. Hermetter; R. Birner-Gruenberger

    2006-01-01

    Summary.  In the postgenomic era new technologies are emerging for global analysis of protein function. The introduction of active site-directed\\u000a chemical probes for enzymatic activity profiling in complex mixtures, known as activity-based proteomics has greatly accelerated\\u000a functional annotation of proteins. Here we review probe design for different enzyme classes including serine hydrolases, cysteine\\u000a proteases, tyrosine phosphatases, glycosidases, and others. These probes

  13. Influences of Different Enzymatic Treatment on Denim Garment

    Microsoft Academic Search

    Majid Montazer; Ali Sadeghian Maryan

    2010-01-01

    In current study, the effects of different enzymatic treatment including acid cellulases, neutral cellulases, and combination\\u000a of laccases with cellulases on denim garment were investigated. The color changes of different samples were compared by colorimetric\\u000a indices for the garment surface and back and also white pocket. Abrasion resistance, tensile strength, and crease recovery\\u000a angle of the samples were measured within

  14. Electronic detection of the enzymatic degradation of starch.

    PubMed

    Star, Alexander; Joshi, Vikram; Han, Tzong-Ru; Altoé, M Virginia P; Grüner, George; Stoddart, J Fraser

    2004-06-24

    [reaction: see text] The enzymatic degradation of starch can be monitored electronically using single-walled carbon nanotubes (SWNTs) as semiconducting probes in field-effect transistors (FETs). Incubation of these devices in aqueous buffer solutions of amyloglucosidase (AMG) results in the removal of the starch from both the silicon surfaces and the side walls of the SWNTs in the FETs, as evidenced by direct imaging and electronic measurements. PMID:15200292

  15. MFR PAPER 1034 An enzymatic FPC process could

    E-print Network

    - tured in such a way that they act as catalysts in the breakage of peptide bonds by a process call edMFR PAPER 1034 An enzymatic FPC process could make profitable use of some of our underutilized/lles TO solubili::.e prOTein and release lipids. All en- ::'YIiUlTic FPC process could /IIake profiTable use oj s

  16. Enzymatic liberation of lycotetraose from the Solanum glycoalkaloid ?-tomatine

    Microsoft Academic Search

    Katherine Woods; Chris J. Hamilton; Robert A. Field

    2004-01-01

    The branched tetrasaccharide, O-?-d-glucopyranosyl-(1?2)-O-[?-d-xylopyranosyl-(1?3)]-O-?-d-glucopyranosyl-(1?4)-d-galactose (lycotetraose) is a key constituent of many biologically interesting natural products. Described herein is a convenient enzymatic preparation of lycotetraose from the readily available Solanum glycoalkaloid ?-tomatine. The preparation makes use of the recombinant endo-glycosidase, tomatinase, from the plant pathogen Fusarium oxysporum f. sp. lycopersici.

  17. Composite aromatic boxes for enzymatic transformations of quaternary ammonium substrates.

    PubMed

    Nagy, Gergely N; Marton, Lívia; Contet, Alicia; Ozohanics, Olivér; Ardelean, Laura-Mihaela; Révész, Agnes; Vékey, Károly; Irimie, Florin Dan; Vial, Henri; Cerdan, Rachel; Vértessy, Beáta G

    2014-12-01

    Cation-? interactions to cognate ligands in enzymes have key roles in ligand binding and enzymatic catalysis. We have deciphered the key functional role of both charged and aromatic residues within the choline binding subsite of CTP:phosphocholine cytidylyltransferase and choline kinase from Plasmodium falciparum. Comparison of quaternary ammonium binding site structures revealed a general composite aromatic box pattern of enzyme recognition sites, well distinguished from the aromatic box recognition site of receptors. PMID:25283789

  18. Electrode adsorption method for determination of enzymatic activity

    Microsoft Academic Search

    H. Arwin; I. Lundström; A. Palmqvist

    1982-01-01

    The paper describes an electrical method for the determination of enzymatic activity. It is based on the fact that certain\\u000a peptides, which are hydrolysed by enzymes, adsorb on a metal electrode in such a way that the double-layer capacitance of\\u000a the electrode is changed. The two parts of the hydrolysed peptide do not have this property. The capacitance change depends

  19. Enzymatic Microreactor Using Si, Glass and EPON SU8

    Microsoft Academic Search

    D. J. Strike; G.-C. Fiaccabrino; M. Koudelka-Hep; N. F. de Rooij

    2000-01-01

    This article describes the design, fabrication and initial testing of an enzymatic microreactor using Si, glass, epoxy and Perspex components. The microreactor geometry was defined by a photostructured EPON SU-8 spacer. This gave two chambers, an upstream chamber (vol. ~2.7 µl) which was packed with control pore glass (CPG) and a downstream chamber (vol. ~1.5 µl) which contained the detectors.

  20. Organosolv pretreatment of lignocellulosic biomass for enzymatic hydrolysis

    Microsoft Academic Search

    Xuebing Zhao; Keke Cheng; Dehua Liu

    2009-01-01

    Production of ethanol by bioconversion of lignocellulosic biomass has attracted much interest in recent years. However, the\\u000a pretreatment process for increasing the enzymatic digestibility of cellulose has become a key step in commercialized production\\u000a of cellulosic ethanol. During the last decades, many pretreatment processes have been developed for decreasing the biomass\\u000a recalcitrance, but only a few of them seem to

  1. Biological pretreatment of lignocellulose for enzymatic hydrolysis of cellulose

    Microsoft Academic Search

    A. I. Hatakka

    1984-01-01

    Pretreatment of lignocellulosic materials is considered as the rate-limiting step in an economically feasible process for\\u000a enzymatic hydrolysis of cellulose. Biological delignification techniques have not been developed as intensively as physical\\u000a and chemical methods. However, white-rot fungi are effective degraders of lignin, and some of them even preferentially remove\\u000a lignin from wood compared with carbohydrates, and therefore might be suitable

  2. Fast Enzymatic Saccharification of Switchgrass After Pretreatment with Ionic Liquids

    SciTech Connect

    Zhou, Hua [Argonne National Laboratory (ANL); Baker, Gary A [ORNL; Cowins, Janet V. [Savannah State University

    2010-01-01

    The pretreatment of cellulose using ionic liquids (ILs) has been shown to be an effective method for improving the enzymatic hydrolysis of cellulose; this technique affords a fast and complete saccharification of cellulose into reducing sugars (Dadi et al., Biotechnol Bioeng. 2006; 95:904 910; Liu and Chen, Chinese Sci Bull. 2006; 51:2432 2436; Zhao et al., J Biotechnol. 2009; 139:47 54). Motivated by these advances, this study examines the effect of IL-pretreatment on the enzymatic hydrolysis of purified xylan (as a model system of hemicellulose) and switchgrass (as a real lignocellulose). The IL-pretreatment resulted in no improvement in the hydrolysis of xylan. The likely reason is that pure xylan has a low degree of polymerization (DP), and is readily biodegraded even without any pretreatment. However, in real cellulosic materials (such as switchgrass), xylan is entrapped within the cellulosic matrix, and cannot be conveniently accessed by enzymes. Our data demonstrate that the IL-pretreatment of switchgrass significantly improved the enzymatic saccharification of both cellulose (96% D-glucose yield in 24 h) and xylan (63% D-xylose yield in 24 h). The compositional analysis of switchgrass suggests a lower lignin content after IL-pretreatment. In addition, the infrared spectrum of regenerated switchgrass indicates a lower substrate crystallinity, whereas the enzyme adsorption isotherm further implies that the regenerated substrate is more accessible to enzymes. This study has further confirmed that ILpretreatment is an effective tool in enhancing the enzymatic hydrolysis of cellulosic biomass, and allowing a more complete saccharification.

  3. Stereospecificity in the enzymatic hydrolysis of cyclosarin (GF)

    Microsoft Academic Search

    Steven P. Harvey; Jan E. Kolakowski; Tu-Chen Cheng; Vipin K. Rastogi; Louis P. Reiff; Joseph J. DeFrank; Frank M. Raushel; Craig Hill

    2005-01-01

    Enzymatic catalysis is one means of accelerating the rate of hydrolysis of G-type organophosphorus nerve agents. Here, the stereospecificity of the catalysis of cyclosarin (GF, O-cyclohexyl methylphosphonofluoridate) hydrolysis by several enzymes was investigated. Stereospecificity was not evident at 3mM GF but was evident at 0.5mM GF. The differential effect was apparently due to fluoride-catalyzed racemization of the substrate. Alteromonas sp.

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

    Microsoft Academic Search

    Charlotte Tengborg; Mats Galbe; Guido Zacchi

    2001-01-01

    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

  5. Effects of manganese on tyrosine hydroxylase (TH) activity and TH-phosphorylation in a dopaminergic neural cell line

    SciTech Connect

    Zhang Danhui; Kanthasamy, Arthi; Anantharam, Vellareddy; Kanthasamy, Anumantha, E-mail: akanthas@iastate.edu

    2011-07-15

    Manganese (Mn) exposure causes manganism, a neurological disorder similar to Parkinson's disease. However, the cellular mechanism by which Mn impairs the dopaminergic neurotransmitter system remains unclear. We previously demonstrated that caspase-3-dependent proteolytic activation of protein kinase C delta (PKC{delta}) plays a key role in Mn-induced apoptotic cell death in dopaminergic neurons. Recently, we showed that PKC{delta} negatively regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, by enhancing protein phosphatase-2A activity in dopaminergic neurons. Here, we report that Mn exposure can affect the enzymatic activity of TH, the rate-limiting enzyme in dopamine synthesis, by activating PKC{delta}-PP2A signaling pathway in a dopaminergic cell model. Low dose Mn (3-10 {mu}M) exposure to differentiated mesencephalic dopaminergic neuronal cells for 3 h induced a significant increase in TH activity and phosphorylation of TH-Ser40. The PKC{delta} specific inhibitor rottlerin did not prevent Mn-induced TH activity or TH-Ser40 phosphorylation. On the contrary, chronic exposure to 0.1-1 {mu}M Mn for 24 h induced a dose-dependent decrease in TH activity. Interestingly, chronic Mn treatment significantly increased PKC{delta} kinase activity and protein phosphatase 2A (PP2A) enzyme activity. Treatment with the PKC{delta} inhibitor rottlerin almost completely prevented chronic Mn-induced reduction in TH activity, as well as increased PP2A activity. Neither acute nor chronic Mn exposures induced any cytotoxic cell death or altered TH protein levels. Collectively, these results demonstrate that low dose Mn exposure impairs TH activity in dopaminergic cells through activation of PKC{delta} and PP2A activity.

  6. Phosphorylation of the Peptidoglycan Synthase PonA1 Governs the Rate of Polar Elongation in Mycobacteria

    PubMed Central

    Kieser, Karen J.; Baer, Christina E.; Barczak, Amy K.; Meniche, Xavier; Chao, Michael C.; Rego, E. Hesper; Sassetti, Christopher M.; Fortune, Sarah M.; Rubin, Eric J.

    2015-01-01

    Cell growth and division are required for the progression of bacterial infections. Most rod-shaped bacteria grow by inserting new cell wall along their mid-section. However, mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce new cell wall material at their poles. How mycobacteria control this different mode of growth is incompletely understood. Here we find that PonA1, a penicillin binding protein (PBP) capable of transglycosylation and transpeptidation of cell wall peptidoglycan (PG), is a major governor of polar growth in mycobacteria. PonA1 is required for growth of Mycobacterium smegmatis and is critical for M. tuberculosis during infection. In both cases, PonA1’s catalytic activities are both required for normal cell length, though loss of transglycosylase activity has a more pronounced effect than transpeptidation. Mutations that alter the amount or the activity of PonA1 result in abnormal formation of cell poles and changes in cell length. Moreover, altered PonA1 activity results in dramatic differences in antibiotic susceptibility, suggesting that a balance between the two enzymatic activities of PonA1 is critical for survival. We also find that phosphorylation of a cytoplasmic region of PonA1 is required for normal activity. Mutations in a critical phosphorylated residue affect transglycosylase activity and result in abnormal rates of cell elongation. Together, our data indicate that PonA1 is a central determinant of polar growth in mycobacteria, and its governance of cell elongation is required for robust cell fitness during both host-induced and antibiotic stress. PMID:26114871

  7. Sialidase NEU3 dynamically associates to different membrane domains specifically modifying their ganglioside pattern and triggering Akt phosphorylation.

    PubMed

    Bonardi, Dario; Papini, Nadia; Pasini, Mario; Dileo, Loredana; Orizio, Flavia; Monti, Eugenio; Caimi, Luigi; Venerando, Bruno; Bresciani, Roberto

    2014-01-01

    Lipid rafts are known to regulate several membrane functions such as signaling, trafficking and cellular adhesion. The local enrichment in sphingolipids and cholesterol together with the low protein content allows their separation by density gradient flotation after extraction with non-ionic detergent at low temperature. These structures are also referred to as detergent resistant membranes (DRM). Among sphingolipids, gangliosides play important roles in different biological events, including signal transduction and tumorigenesis. Sialidase NEU3 shows high enzymatic specificity toward gangliosides. Moreover, the enzyme is present both at the cell surface and in endosomal structures and cofractionates with caveolin. Although changes in the expression level of NEU3 have been correlated to different tumors, little is known about the precise distribution of the protein and its ability in modifying the ganglioside composition of DRM and non-DRM, thus regulating intracellular events. By means of inducible expression cell system we found that i) newly synthesized NEU3 is initially associated to non-DRM; ii) at steady state the protein is equally distributed between the two membrane subcompartments, i.e., DRM and non-DRM; iii) NEU3 is degraded via the proteasomal pathway; iv) the enzyme specifically modifies the ganglioside composition of the membrane areas where it resides; and v) NEU3 triggers phosphorylation of Akt, even in absence of exogenously administered EGF. Taken together our data demonstrate that NEU3 regulates the DRM ganglioside content and it can be considered as a modulator of Akt phosphorylation, further supporting the role of this enzyme in cancer and tumorigenesis. PMID:24925219

  8. Neonatal Exendin-4 Leads to Protection from Reperfusion Injury and Reduced Rates of Oxidative Phosphorylation in the Adult Rat Heart

    PubMed Central

    Brown, Suzanne B.; Libonati, Joseph R.; Selak, Mary A.; Shannon, Richard P.

    2015-01-01

    Purpose Glucagon like peptide-1 (7-36) amide (GLP-1) is an incretin hormone with multiple salutary cardiovascular effects. A short course of the GLP-1 analogue Exendin-4 (Ex-4) in the neonatal period prevents the development of mitochondrial dysfunction and oxidative stress in a rat prone to obesity and diabetes. We sought to evaluate whether neonatal Ex-4 can exert the same effect in the normal rat heart, as well as whether Ex-4 could affect susceptibility to cardiac reperfusion injury. Methods After birth, Sprague Dawley rat pups were given either Ex-4 (1 nmole/kg body weight) or vehicle (1% BSA in 0.9% saline) subcutaneously for 6 days. Animals were studied at juvenile (4–6 weeks) and adult (8–9 months) ages. Using the Langendorff isolated perfused heart, cardiovascular function was assessed at baseline and following ischemia-reperfusion. Mitochondria were isolated from fresh heart tissue, and oxidative phosphorylation and calcium sequestration were analyzed. TBARS, MnSOD activity, and non-enzymatic anti-oxidant capacity were measured to assess the degree of oxidative stress present in the two groups. Results Both at the juvenile and adult age, Ex-4 treated rats demonstrated improved recovery from an ischemic insult. Rates of oxidative phosphorylation were globally reduced in adult, but not juvenile Ex-4 treated animals. Furthermore, mitochondria isolated from adult Ex-4 treated rats sequestered less calcium before undergoing the mitochondrial permeability transition. Oxidative stress did not differ between groups at any time point. Conclusion A short course of Exendin-4 in the neonatal period leads to protection from ischemic injury and a preconditioned mitochondrial phenotype in the adult rat. PMID:20582459

  9. Effects of Manganese on Tyrosine Hydroxylase (TH) Activity and TH-phosphorylation in a Dopaminergic Neural Cell Line

    PubMed Central

    Zhang, Danhui; Kanthasamy, Arthi; Anantharam, Vellareddy; Kanthasamy, Anumantha

    2011-01-01

    Manganese (Mn) exposure causes manganism, a neurological disorder similar to Parkinson’s disease. However, the cellular mechanism by which Mn impairs the dopaminergic neurotransmitter system remains unclear. We previously demonstrated that caspase-3-dependent proteolytic activation of protein kinase C delta (PKC?) plays a key role in Mn-induced apoptotic cell death in dopaminergic neurons. Recently, we showed that PKC? negatively regulates tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, by enhancing protein phosphatase-2A activity in dopaminergic neurons. Here we report that Mn exposure can affect the enzymatic activity of TH, the rate-limiting enzyme in dopamine synthesis, by activating PKC? - PP2A signaling pathway in a dopaminergic cell model. Low dose Mn (3–10 ?M) exposure to differentiated mesencephalic dopaminergic neuronal cells for 3 h induced a significant increase in TH activity and phosphorylation of TH-Ser40. The PKC? specific inhibitor rottlerin did not prevent Mn-induced TH activity or TH-Ser40 phosphorylation. On the contrary, chronic exposure to 0.1–1 ?M Mn for 24 h induced a dose-dependent decrease in TH activity. Interestingly, chronic Mn treatment significantly increased PKC? kinase activity and protein phosphatase 2A (PP2A) enzyme activity. Treatment with the PKC? inhibitor rottlerin almost completely prevented chronic Mn-induced reduction in TH activity, as well as increased PP2A activity. Neither acute nor chronic Mn exposures induced any cytotoxic cell death or altered TH protein levels. Collectively, these results demonstrate that low dose Mn exposure impairs TH activity in dopaminergic cells through activation of PKC? and PP2A activity. PMID:21310168

  10. Enzymatic measurement of phosphatidic acid in cultured cells.

    PubMed

    Morita, Shin-ya; Ueda, Kazumitsu; Kitagawa, Shuji

    2009-09-01

    In this work, we developed a novel enzymatic method for measuring phosphatidic acid (PA) in cultured cells. The enzymatic reaction sequence of the method involves hydrolysis of PA to produce glycerol-3-phosphate (G3P), which is then oxidized by G3P oxidase to generate hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacted with Amplex Red to produce highly fluorescent resorufin. We found that lipase from Pseudomonas sp. can completely hydrolyze PA to G3P and FAs. The calibration curve for PA measurement was linear between 20 and 250 microM, and the detection limit was 5 microM (50 pmol in the reaction mixture). We also modified the method for the enzymatic measurement of lysophosphatidic acid. By this new method, we determined the PA content in the lipid extract from HEK293 cells. The cellular content of PA was decreased with increasing cell density but not correlated with the proliferation rate. The diacylglycerol kinase inhibitor R59949 markedly reduced the cellular PA content, suggesting the diacylglycerol kinase activity was involved in a large part of the PA production in HEK293 cells. This novel method for PA quantification is simple, rapid, specific, sensitive, and high-throughput and will help to study the biological functions of PA and its related enzymes. PMID:19369695

  11. Enzymatic measurement of phosphatidic acid in cultured cells

    PubMed Central

    Morita, Shin-ya; Ueda, Kazumitsu; Kitagawa, Shuji

    2009-01-01

    In this work, we developed a novel enzymatic method for measuring phosphatidic acid (PA) in cultured cells. The enzymatic reaction sequence of the method involves hydrolysis of PA to produce glycerol-3-phosphate (G3P), which is then oxidized by G3P oxidase to generate hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide reacted with Amplex Red to produce highly fluorescent resorufin. We found that lipase from Pseudomonas sp. can completely hydrolyze PA to G3P and FAs. The calibration curve for PA measurement was linear between 20 and 250 µM, and the detection limit was 5 µM (50 pmol in the reaction mixture). We also modified the method for the enzymatic measurement of lysophosphatidic acid. By this new method, we determined the PA content in the lipid extract from HEK293 cells. The cellular content of PA was decreased with increasing cell density but not correlated with the proliferation rate. The diacylglycerol kinase inhibitor R59949 markedly reduced the cellular PA content, suggesting the diacylglycerol kinase activity was involved in a large part of the PA production in HEK293 cells. This novel method for PA quantification is simple, rapid, specific, sensitive, and high-throughput and will help to study the biological functions of PA and its related enzymes. PMID:19369695

  12. Enzymatic degradation of PLLA-PEOz-PLLA triblock copolymers.

    PubMed

    Wang, Chau-Hui; Fan, Kuo-Rong; Hsiue, Ging-Ho

    2005-06-01

    The enzymatic degradation of poly(L-lactide)-block- poly(2-ethyl-2-oxazoline)-block-poly(L-lactide) triblock copolymer (PLLA-PEOz-PLLA) was investigated using efficient enzyme proteinase K. PLLA-PEOz-PLLA solution-cast film lost a considerable amount of hydrophilic copolymers in the first 2 h, and the degradation after 2 h proceeded predominantly by surface erosion. The two faces of the hydrolyzed film exhibited different morphologies following enzymatic degradation. The lower face showed many spherulites, which are the superstructural morphology of polymer crystals. Porous spheres based on crystalline PLLA were observed on the upper face, because they were more resistant to enzymatic attack. The crystallinity of the films increased monotonously with the hydrolysis time, thus, the absorption of water gradually decreased. The analysis of degradation residues revealed that many colloids of poly(2-ethyl-2-oxazoline)-co-polyethylenimine (PEOz-co-PEI) copolymers were dispersed in the buffer solution. The average diameter, 1 microm, of the colloids was reduced to 200 nm by advanced degradation. The proteinase K exhibited remarkable hydrolysis not only at the ester bond but also the amide bond. PMID:15603776

  13. 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.)

  14. Elucidation of Factors Effecting Enzymatic Saccharification using Transgenic Hardwoods

    NASA Astrophysics Data System (ADS)

    Min, Douyong

    Three groups of transgenic wood samples were used as starting materials to elucidate the recalcitrance of enzymatic saccharification with/without pretreatments. The first group of transgenic wood samples is low lignin P. trichocarpa. The second group is low xylan P. trichocarpa. The third one is 12 hybrid poplars which have different levels of S/V ratio and lignin content. Four pretreatments were carried out in this research including dilute sulfuric acid, green liquor, auto hydrolysis and ozone delignification. The behavior among pretreatments as a function of removal of lignin appears to be different. Lignin is the major factor of recalcitrance of the lignocellulosic material to ethanol conversion process. Xylan also plays key role in this process. In addition, the crude milled wood lignin was isolated from these three groups of transgenic samples. Lignin carbohydrate complexes was characterized by 1H-13C HMQC and 13C NMR. Thus the effect of LCCs on enzymatic saccharification was elucidated. High S/V ratio propels the lignin removal during pretreatments however; high S/V ratio retards the enzymatic saccharification on the lignocellulosic material without pretreatments. The level of LCCs linkages accounts for additional recalcitrance of the lignocellulosic material to ethanol conversion process. The amount of LCCs linkages is affected by xylan content, lignin content and S/V ratio.

  15. Creating Advanced Multifunctional Biosensors with Surface Enzymatic Transformations

    PubMed Central

    Lee, Hye Jin; Wark, Alastair W.; Corn, Robert M.

    2008-01-01

    This article summarizes our recent work on the coupling of surface enzyme chemistry and bioaffinity interactions on biopolymer microarrays for the creation of multiplexed biosensors with enhanced selectivity and sensitivity. The surface sensitive techniques of surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS) are used to detect the surface enzymatic transformations in real time. Three specific examples of novel coupled surface bioaffinity/surface enzymatic processes are demonstrated: (i) a surface enzymatic amplification method utilizing the enzyme ribonuclease H (RNase H) in conjunction with RNA microarrays that permits the ultrasensitive direct detection of genomic DNA at a concentration of 1 fM without labeling or PCR amplification, (ii) the use of RNA-DNA ligation chemistry to create renewable RNA microarrays from single stranded DNA microarrays, and (iii) the application of T7 RNA polymerase for the on-chip replication of RNA from double stranded DNA microarray elements. In addition, a simple yet powerful theoretical framework that includes the contributions of both enzyme adsorption and surface enzyme kinetics is used to quantitate surface enzyme reactivity. This model is successfully applied to SPRI and SPFS measurements of surface hydrolysis reactions of RNase H and Exonuclease III (Exo III) on oligonucleotide microarrays. PMID:16732647

  16. Immobilization and enzymatic properties of Bacillus megaterium glucose dehydrogenase

    SciTech Connect

    Baron, M.; Fontana, J.D.; Guimaraes, M.F. [Federal Univ. of Parana, Curitiba, PR (Brazil); Woodward, J. [Oak Ridge National Lab., TN (United States)

    1996-12-31

    The enzymatic production of hydrogen gas from renewable sources of energy; e.g., cellulose, starch, lactose, can be obtained by coupling the reactions catalyzed by glucose dehydrogenase (GDH) and hydrogenase. In order to enhance the thermostability of GDH from Bacillus megaterium, the enzyme was immobilized by ionic adsorption using the polycationic polymer DEAE-(dextran)Sephadex. The effect of enzyme concentration on immobilization showed a tendency to increase the activity of the immobilized enzyme with the increase of the amount of added GDH. When the enzyme: support ratio was 15.97 U: 100 mg, the immobilization yield was 84.76%. The enzymatic profiles for the immobilized GDH were a little different when compared to those for free enzyme with respect to the effects of pH and temperature. Concerning the effect of incubation time carried at pH 7.5 and at 40{degrees}C, the maximum production of reduced coenzyme by the immobilized enzyme was reached within 4 h and it was maintained up to 16 h without loss of enzymatic activity. The coupling of the immobilized GDH activity with that for free alkaline cellulose (Novozym. 342) demonstrated the possibility for obtaining reduced coenzyme from the cellulose hydrolysis and the immobilized GDH could be reassayed 10 times maintaining its enzyme activity.

  17. Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan

    E-print Network

    Campbell, Kevin P.

    receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with highCell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan Marie-Laurence Moraz,1. Introduction The Old World arenavirus Lassa virus (LASV) is the causative agent of a severe viral haemorrhagic

  18. On the importance of protein phosphorylation in cell cycle control

    Microsoft Academic Search

    James L. Maller

    1993-01-01

    This chapter is written as a contribution to a volume commemorating the work of Krebs and Fischer that led to awarding of the Nobel Prize in 1992. This award was made because of their fundamental discovery in the mid-1950s that protein phosphorylation was the underlying mechanism that accounted for the reversible modification of activity of glycogen phosphorylase in mammalian skeletal

  19. Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved

    E-print Network

    Hirt, Heribert

    Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved in RNA a small and therefore easier analyzable component of the com- plex phosphoproteome. In contrast to the wealth of phosphoproteomic investi- gations performed on yeast and animal systems, only a few studies

  20. Spatial Separation of Plk1 Phosphorylation and Activity

    PubMed Central

    Bruinsma, Wytse; Aprelia, Melinda; Kool, Jolanda; Macurek, Libor; Lindqvist, Arne; Medema, René H.

    2015-01-01

    Polo-like kinase 1 (Plk1) is one of the major kinases controlling mitosis and cell division. Plk1 is first recruited to the centrosome in S phase, then appears on the kinetochores in late G2, and at the end of mitosis, it translocates to the central spindle. Activation of Plk1 requires phosphorylation of T210 by Aurora A, an event that critically depends on the co-factor Bora. However, conflicting reports exist as to where Plk1 is first activated. Phosphorylation of T210 is first observed at the centrosomes, but kinase activity seems to be restricted to the nucleus in the earlier phases of G2. Here, we demonstrate that Plk1 activity manifests itself first in the nucleus using a nuclear FRET-based biosensor for Plk1 activity. However, we find that Bora is restricted to the cytoplasm and that Plk1 is phosphorylated on T210 at the centrosomes. Our data demonstrate that while Plk1 activation occurs on centrosomes, downstream target phosphorylation by Plk1 first occurs in the nucleus. We discuss several explanations for this surprising separation of activation and function.

  1. Anxiolytic action of pterostilbene: involvement of hippocampal ERK phosphorylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pterostilbene, a natural analog of resveratrol, has diverse health-beneficial properties. However, the neurological activities of this compound are largely unexplored. Here we report that pterostilbene shows anxiolytic action by downregulating phosphorylated levels of ERKs in the hippocampus of mice...

  2. Role of tyrosine phosphorylation in sperm capacitation \\/ acrosome reaction

    Microsoft Academic Search

    Rajesh K Naz; Preeti B Rajesh

    2004-01-01

    Capacitation is an important physiological pre-requisite before the sperm cell can acrosome react and fertilize the oocyte. Recent reports from several laboratories have amply documented that the protein phosphorylation especially at tyrosine residues is one of the most important events that occur during capacitation. In this article, we have reviewed the data from our and other laboratories, and have constructed

  3. METHIONINE OXIDATION AND PROTEIN PHOSPHORYLATION: INTERACTIVE PARTNERS IN SIGNALING?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein phosphorylation can affect the activity, stability or localization of a protein and as result plays a broad role in regulation of processes ranging from metabolism to control of plant growth and development. One aspect of current interest in our lab is how protein kinases target their substr...

  4. Quantitation, networking, and function of protein phosphorylation in plant cell

    PubMed Central

    Zhu, Lin; Li, Ning

    2013-01-01

    Protein phosphorylation is one of the most important post-translational modifications (PTMs) as it participates in regulating various cellular processes and biological functions. It is therefore crucial to identify phosphorylated proteins to construct a phosphor-relay network, and eventually to understand the underlying molecular regulatory mechanism in response to both internal and external stimuli. The changes in phosphorylation status at these novel phosphosites can be accurately measured using a 15N-stable isotopic labeling in Arabidopsis (SILIA) quantitative proteomic approach in a high-throughput manner. One of the unique characteristics of the SILIA quantitative phosphoproteomic approach is the preservation of native PTM status on protein during the entire peptide preparation procedure. Evolved from SILIA is another quantitative PTM proteomic approach, AQUIP (absolute quantitation of isoforms of post-translationally modified proteins), which was developed by combining the advantages of targeted proteomics with SILIA. Bioinformatics-based phosphorylation site prediction coupled with an MS-based in vitro kinase assay is an additional way to extend the capability of phosphosite identification from the total cellular protein. The combined use of SILIA and AQUIP provides a novel strategy for molecular systems biological study and for investigation of in vivo biological functions of these phosphoprotein isoforms and combinatorial codes of PTMs. PMID:23316209

  5. Phosphorylation at Tyrosine 262 Promotes GADD34 Protein Turnover*

    PubMed Central

    Zhou, Wei; Jeyaraman, Krishna; Yusoff, Permeen; Shenolikar, Shirish

    2013-01-01

    In mammalian cells, metabolic and environmental stress increases the phosphorylation of the eukaryotic translational initiation factor, eIF2?, and attenuates global protein synthesis. Subsequent transcriptional activation of GADD34 assembles an eIF2? phosphatase that feeds back to restore mRNA translation. Active proteasomal degradation of GADD34 protein then reestablishes the sensitivity of cells to subsequent bouts of stress. Mass spectrometry established GADD34 phosphorylation on multiple serines, threonines, and tyrosines. Phosphorylation at tyrosine 262 enhanced the rate of the GADD34 protein turnover. Substrate-trapping studies identified TC-PTP (PTPN2) as a potential GADD34 phosphatase, recognizing phosphotyrosine 262. Reduced GADD34 protein levels in TC-PTP-null MEFs following ER stress emphasized the importance of TC-PTP in determining the cellular levels of GADD34 protein. The susceptibility of TC-PTP-null MEFs to ER stress-induced apoptosis was significantly ameliorated by ectopic expression of GADD34. The data suggested that GADD34 phosphorylation on tyrosine 262 modulates endoplasmic reticulum stress signaling and cell fate. PMID:24092754

  6. Histone H1 Phosphorylation in Breast Cancer Sean W. Harshman,,,

    E-print Network

    Wysocki, Vicki H.

    types including kidney, prostate, lung, pancreatic, and breast cancers.3-6 While there is a deepHistone H1 Phosphorylation in Breast Cancer Sean W. Harshman,,,¶ Michael E. Hoover,,,¶ Chengsi and Medical Genetics, Comprehensive Cancer Center, § Department of Chemistry & Biochemistry, Veterinary

  7. RESEARCH ARTICLE CSN1 inhibits c-Jun phosphorylation and

    E-print Network

    Deng, Xing-Wang

    phosphorylation of proto-oncogene c-Jun product and repress c-Jun depen- dent transcription. Further, CSN1-terminal kinase 1 (JNK1) INTRODUCTION The c-Jun proto-oncogene encodes a major component of activator protein 1

  8. Observation of Phosphorylation Site-Specific Dissociation of Singly

    E-print Network

    Kim, Myung Soo

    Young Sik Shin,a Jeong Hee Moon,b and Myung Soo Kima a Department of Chemistry, Seoul National-terminus, intense an 97 peaks were observed. These ions were formed by cleavage at phosphorylated residues only/D exchange studies showed that an 97 was formed by H3PO4 loss from an 1 radical cations. The site

  9. Phosphorylation of histone H3 correlates with transcriptionally active loci

    E-print Network

    Corces, Victor G.

    ) is a well-characterized model for the in- duction of gene expression in response to environmental stress in the induction of transcription during the heat shock response. [Key Words: Histone phosphorylation; histone October 16, 2000. The eukaryotic genome is highly compacted within the nucleus into the chromatin fiber

  10. Activation of purified calcium channels by stoichiometric protein phosphorylation

    SciTech Connect

    Nunoki, K.; Florio, V.; Catterall, W.A. (Univ. of Washington, Seattle (USA))

    1989-09-01

    Purified dihydropyridine-sensitive calcium channels from rabbit skeletal muscle were reconstituted into phosphatidylcholine vesicles to evaluate the effect of phosphorylation by cyclic AMP-dependent protein kinase (PK-A) on their function. Both the rate and extent of {sup 45}Ca{sup 2+} uptake into vesicles containing reconstituted calcium channels were increased severalfold after incubation with ATP and PK-A. The degree of stimulation of {sup 45}Ca{sup 2+} uptake was linearly proportional to the extent of phosphorylation of the alpha 1 and beta subunits of the calcium channel up to a stoichiometry of approximately 1 mol of phosphate incorporated into each subunit. The calcium channels activated by phosphorylation were determined to be incorporated into the reconstituted vesicles in the inside-out orientation and were completely inhibited by low concentrations of dihydropyridines, phenylalkylamines, Cd{sup 2+}, Ni{sup 2+}, and Mg{sup 2+}. The results demonstrate a direct relationship between PK-A-catalyzed phosphorylation of the alpha 1 and beta subunits of the purified calcium channel and activation of the ion conductance activity of the dihydropyridine-sensitive calcium channels.

  11. Tyrosine Phosphorylation Allows Integration of Multiple Signaling Inputs by IKK?

    PubMed Central

    Meyer, April N.; Drafahl, Kristine A.; McAndrew, Christopher W.; Gilda, Jennifer E.; Gallo, Leandro H.; Haas, Martin; Brill, Laurence M.; Donoghue, Daniel J.

    2013-01-01

    Signaling regulated by NF?B and related transcription factors is centrally important to many inflammatory and autoimmune diseases, cancer, and stress responses. The kinase that directly regulates the canonical NF?B transcriptional pathway, Inhibitor of ?B kinase ? (IKK?), undergoes activation by Ser phosphorylation mediated by NIK or TAK1 in response to inflammatory signals. Using titanium dioxide-based phosphopeptide enrichment (TiO2)-liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS), we analyzed IKK? phosphorylation in human HEK293 cells expressing IKK? and FGFR2, a Receptor tyrosine kinase (RTK) essential for embryonic differentiation and dysregulated in several cancers. We attained unusually high coverage of IKK?, identifying an abundant site of Tyr phosphorylation at Tyr169 within the Activation Loop. The phosphomimic at this site confers a level of kinase activation and NF?B nuclear localization exceeding the iconic mutant S177E/S181E, demonstrating that RTK-mediated Tyr phosphorylation of IKK? has the potential to directly regulate NF?B transcriptional activation. PMID:24386391

  12. Coregulation of Ion Channels by Neurosteroids and Phosphorylation

    NSDL National Science Digital Library

    Jeffrey Tasker (Tulane University; Department of Cell and Molecular Biology REV)

    2000-11-21

    This Perspective by Tasker is one of two discussing the regulation of ion channels by neurosteroids. Neurosteroids are steroids synthesized in the brain that act as allosteric modulators of ion-channel activity. The dual regulation of ?-aminobutyric acid type A (GABAA) receptors and voltage-gated calcium channels by phosphorylation and neurosteroids is the focus of this Perspective.

  13. Resolvin E1 receptor activation signals phosphorylation and phagocytosis.

    PubMed

    Ohira, Taisuke; Arita, Makoto; Omori, Kazuhiro; Recchiuti, Antonio; Van Dyke, Thomas E; Serhan, Charles N

    2010-01-29

    Resolvins are endogenous lipid mediators that actively regulate the resolution of acute inflammation. Resolvin E1 (RvE1; (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is an endogenous anti-inflammatory and pro-resolving mediator derived from eicosapentaenoic acid that regulates leukocyte migration and enhances macrophage phagocytosis of apoptotic neutrophils to resolve inflammation. In the inflammatory milieu, RvE1 mediates counter-regulatory actions initiated via specific G protein-coupled receptors. Here, we have identified RvE1-specific signaling pathways initiated by the RvE1 receptor ChemR23. RvE1 stimulated phosphorylation of Akt that was both ligand- and receptor-dependent. RvE1 regulated Akt phosphorylation in a time (0-15 min)- and dose-dependent (0.01-100 nm) manner in human ChemR23-transfected Chinese hamster ovary cells. RvE1 stimulated phosphorylation of both Akt and a 30-kDa protein, a downstream target of Akt, identified using a phospho-Akt substrate antibody. The 30-kDa protein was identified as ribosomal protein S6, a translational regulator, and its phosphorylation was inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin) and an ERK inhibitor (PD98059) but not by a p38-MAPK inhibitor (SB203580). Ribosomal protein S6 is a downstream target of the PI3K/Akt signaling pathway as well as the Raf/ERK pathway. In ChemR23-expressing differentiated HL60 cells, RvE1 also stimulated the phosphorylation of ribosomal protein S6. In addition, RvE1 enhanced phagocytosis of zymosan A by human macrophages, which are inhibited by PD98059 and rapamycin (mTOR inhibitor). These results indicate that RvE1 initiates direct activation of ChemR23 and signals receptor-dependent phosphorylation. These phosphorylation-signaling pathways identified for RvE1 receptor-ligand interactions underscore the importance of endogenous pro-resolving agonists in resolving acute inflammation. PMID:19906641

  14. Canine pulmonary adenocarcinoma tyrosine kinase receptor expression and phosphorylation

    PubMed Central

    2014-01-01

    Background This study evaluated tyrosine kinase receptor (TKR) expression and activation in canine pulmonary adenocarcinoma (cpAC) biospecimens. As histological similarities exist between human and cpAC, we hypothesized that cpACs will have increased TKR mRNA and protein expression as well as TKR phosphorylation. The molecular profile of cpAC has not been well characterized making the selection of therapeutic targets that would potentially have relevant biological activity impossible. Therefore, the objectives of this study were to define TKR expression and their phosphorylation state in cpAC as well as to evaluate the tumors for the presence of potential epidermal growth factor receptor (EGFR) tyrosine kinase activating mutations in exons 18–21. Immunohistochemistry (IHC) for TKR expression was performed using a tissue microarray (TMA) constructed from twelve canine tumors and companion normal lung samples. Staining intensities of the IHC were quantified by a veterinary pathologist as well as by two different digitalized algorithm image analyses software programs. An antibody array was used to evaluate TKR phosphorylation of the tumor relative to the TKR phosphorylation of normal tissues with the resulting spot intensities quantified using array analysis software. Each EGFR exon PCR product from all of the tumors and non-affected lung tissues were sequenced using sequencing chemistry and the sequencing reactions were run on automated sequencer. Sequence alignments were made to the National Center for Biotechnology Information canine EGFR reference sequence. Results The pro-angiogenic growth factor receptor, PDGFR?, had increased cpAC tumor mRNA, protein expression and phosphorylation when compared to the normal lung tissue biospecimens. Similar to human pulmonary adenocarcinoma, significant increases in cpAC tumor mRNA expression and receptor phosphorylation of the anaplastic lymphoma kinase (ALK) tyrosine receptor were present when compared to the corresponding normal lung tissue. The EGFR mRNA, protein expression and phosphorylation were not increased compared to the normal lung and no activating mutations were identified in exons 18–21. Conclusions Canine pulmonary adenocarcinoma TKRs are detected at both the mRNA and protein levels and are activated. Further investigation into the contribution of TKR activation in cpAC tumorigenesis is warranted. PMID:24423144

  15. Caged phosphopeptides and phosphoproteins : probes to dissect the role of phosphorylation in complex signaling pathways

    E-print Network

    Vogel, Elizabeth Maura

    2007-01-01

    Protein phosphorylation is a central regulatory mechanism in signal transduction pathways and cellular migration. Current genetic strategies for the study of phosphorylation, including gene knockout and point mutation, are ...

  16. Correlating Protein Phosphorylation With Genomic Alterations in Cancer - Jianjiong Gao, TCGA Scientific Symposium 2011

    Cancer.gov

    Home News and Events Multimedia Library Videos Correlating Protein Phosphorylation With Genomic Alterations in Cancer - Jianjiong Gao Correlating Protein Phosphorylation With Genomic Alterations in Cancer - Jianjiong Gao, TCGA Scientific Symposium

  17. Environmental Effects on Phosphoryl Group Bonding Probed by Vibrational Spectroscopy: Implications for Understanding

    E-print Network

    Herschlag, Dan

    Environmental Effects on Phosphoryl Group Bonding Probed by Vibrational Spectroscopy: Implications spectroscopy to study bonding in monosubstituted dianionic phosphates, both to learn more about basic group. We examined the bonding properties of the phosphoryl group via vibrational spectroscopy

  18. Ligand binding promotes CDK-dependent phosphorylation of ER-alpha on hinge serine 294 but inhibits ligand-independent phosphorylation of serine 305.

    PubMed

    Held, Jason M; Britton, David J; Scott, Gary K; Lee, Elbert L; Schilling, Birgit; Baldwin, Michael A; Gibson, Bradford W; Benz, Christopher C

    2012-08-01

    Phosphorylation of estrogen receptor-? (ER?) is critical for its transcription factor activity and may determine its predictive and therapeutic value as a biomarker for ER?-positive breast cancers. Recent attention has turned to the poorly understood ER? hinge domain, as phosphorylation at serine 305 (Ser305) associates with poor clinical outcome and endocrine resistance. We show that phosphorylation of a neighboring hinge domain site, Ser294, analyzed by multiple reaction monitoring mass spectrometry of ER? immunoprecipitates from human breast cancer cells is robustly phosphorylated exclusively by ligand (estradiol and tamoxifen) activation of ER? and not by growth factor stimulation (EGF, insulin, heregulin-?). In a reciprocal fashion, Ser305 phosphorylation is induced by growth factors but not ligand activation of ER?. Phosphorylation at Ser294 and Ser305 is suppressed upon co-stimulation by EGF and ligand, respectively, unlike the N-terminal (AF-1) domain Ser118 and Ser167 sites of ER? where phosphorylation is enhanced by ligand and growth factor co-stimulation. Inhibition of cyclin-dependent kinases (CDK) by roscovitine or SNS-032 suppresses ligand-activated Ser294 phosphorylation without affecting Ser118 or Ser104/Ser106 phosphorylation. Likewise, cell-free studies using recombinant ER? and specific cyclin-CDK complexes suggest that Ser294 phosphorylation is primarily induced by the transcription-regulating and cell-cycle-independent kinase CDK7. Thus, CDK-dependent phosphorylation at Ser294 differentiates ligand-dependent from ligand-independent activation of Ser305 phosphorylation, showing that hinge domain phosphorylation patterns uniquely inform on the various ER? activation mechanisms thought to underlie the biologic and clinical diversity of hormone-dependent breast cancers. PMID:22669764

  19. Thyroid hormone regulation of phospholamban phosphorylation in the rat heart.

    PubMed

    Ojamaa, K; Kenessey, A; Klein, I

    2000-06-01

    Thyroid hormone exerts predictable effects on the contractile performance of the heart in part by regulating the transcription of genes encoding specific calcium transporter proteins. In a rat model of hypothyroidism, left ventricular (LV) contractile function as measured by ejection fraction was decreased by 22% (P < 0.05), and this was returned to control values with T3 treatment. In confirmation of prior studies, LV phospholamban (PLB) protein content was significantly decreased by 25% and 40% compared with hypothyroid LV when the animals were treated with T3 at two doses, 2.5 and 7.0 microg/day, respectively. The ratio of sarcoplasmic reticulum calcium adenosine triphosphatase (SERCA2) to PLB protein content was thus increased by 171% and 207%, respectively (P < 0.01). Resolution of the phosphorylated PLB pentamers by SDS-PAGE showed that T3 infusion at 2.5 and 7.0 microg/day decreased (P < 0.001) the amount nonphosphorylated pentamers by 82% and 95%, respectively, in a dose-dependent manner. T3 treatment produced an increase in the proportion of highly phosphorylated PLB pentamers (more than five phosphates) when expressed as a fraction of total pentameric molecules (P < 0.05). Site-specific antibodies showed that the T3-induced increase in phosphorylated PLB pentamers was the result of an increase in both serine 16 and threonine 17 phosphorylation. We conclude that thyroid hormone, in addition to regulating the expression of cardiac PLB, is able to alter the degree of PLB phosphorylation, which correlates with enhancement of LV contractile function. These studies suggest that T3 may augment myocyte calcium cycling via changes in both cAMP- and calcium/calmodulin-dependent protein kinase activities. PMID:10830301

  20. Cell Density Regulates Tyrosine Phosphorylation and Localization of Focal Adhesion Kinase

    Microsoft Academic Search

    Fengping Xu; Zhizhuang Joe Zhao

    2001-01-01

    We have investigated tyrosine phosphorylation of cellular proteins at different cell densities. A tyrosine-phosphorylated protein of 120 kDa was detected when cells were plated sparsely. The phosphorylation level of the protein gradually declined as the cells were plated at higher densities or when the sparsely plated cells approached confluence. This density-dependent phosphorylation was also associated with cell attachment since it

  1. Stainless steel electrospray probe: A dead end for phosphorylated organic compounds?

    Microsoft Academic Search

    R. Tuytten; F. Lemière; E. Witters; W. Van Dongen; H. Slegers; R. P. Newton; H. Van Onckelen; E. L. Esmans

    2006-01-01

    A study of the interaction of phosphorylated organic compounds with the stainless components of a liquid chromatography–electrospray ionisation–mass spectrometry system (LC–ESI–MS) was carried out to disclose a (forgotten?) likely pitfall in the LC–ESI–MS analysis of phosphorylated compounds. The retention behaviour of some representative compounds of different important classes of phosphorylated biomolecules such as nucleotides, oligonucleotides, phosphopeptides, phospholipids and phosphorylated sugars

  2. Scale-up of enzymatic production of lactobionic acid using the rotary jet head system.

    PubMed

    Hua, Ling; Nordkvist, Mikkel; Nielsen, Per Munk; Villadsen, John

    2007-07-01

    Enzymatic oxidation of lactose to lactobionic acid (LBA) by a carbohydrate oxidase from Microdochium nivale was studied in a pilot-scale batch reactor of 600 L working volume using a rotary jet head (RJH) for mixing and mass transfer (Nordkvist et al., 2003, Chem Eng Sci 58:3877-3890). Both lactose and whey permeate were used as substrate, air was used as oxygen source, and catalase was added to eliminate the byproduct hydrogen peroxide. More than 98% conversion to LBA was achieved. Neither enzyme deactivation nor enzyme inhibition was observed under the experimental conditions. The dissolved oxygen tension (DOT) was constant throughout the tank for a given set of operating conditions, indicating that liquid mixing was sufficiently good to avoid oxygen gradients in the tank. However, at a given oxygen tension measured in the tank, the specific rate of reaction found in the RJH system was somewhat higher than previously obtained in a 1 L mechanically stirred tank reactor (Nordkvist et al., 2007, in this issue, pp. 694-707). This can be ascribed to a higher pressure in the recirculation loop which is part of the RJH system. Compared to mechanically stirred systems, high values of the volumetric mass transfer coefficient, k(L)a, were obtained when lactose was used as substrate, especially at low values of the specific power input and the superficial gas velocity. k(L)a was lower for experiments with whey permeate than with lactose due to addition of antifoam. The importance of mass transfer and of the saturation concentration of oxygen on the volumetric rate of reaction was demonstrated by simulations. PMID:17154315

  3. Phosphorylation of Vesicular Stomatitis Virus Proteins as a Possible Contributing Factor in Virion Uncoating

    Microsoft Academic Search

    DONALD J. WITT; CLAYTON W. NAEVE; DONALD F. SUMMERS

    1981-01-01

    SUMMARY The relationship between the in vitro phosphorylation of vesicular stomatitis virus (VSV) proteins and virion uncoating was examined. Activation of the VSV virion kinase with low concentrations of melittin, the active peptide component of bee venom, in the presence of y-(32p)ATP resulted in the phosphorylation of virion proteins. Following the in vitro phosphorylation of VSV proteins in the presence

  4. Phosphorylation of Ser3 of cofilin regulates its essential function on actin

    Microsoft Academic Search

    Kenji Moriyama; Kazuko Iida; Ichiro Yahara

    1996-01-01

    Background: Cofilin is a low-molecular weight actin- modulating protein, and is structurally and function- ally conserved in eucaryotes from yeast to mammals. The functions of cofilin appear to be regulated by phosphorylation and dephosphorylation. Results: A proteolytic study of phosphorylated por- cine cofilin and expression of a mutated cofilin in cultured cells revealed that Ser-3 is the unique phosphorylation site.

  5. Identification of the Phosphorylation Sites of Cytosolic Phospholipase A2 in Agonist-stimulated Human Platelets

    E-print Network

    Gelb, Michael

    -stimulated Human Platelets and HeLa Cells* (Received for publication, July 25, 1997, and in revised form, OctoberPLA2) in hu- man platelets and HeLa cells. Tryptic digests of 32 P- phosphorylated phosphorylation of platelet cPLA2 at two sites, Ser-505 and Ser-727. Exclu- sive phosphorylation on these two

  6. Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage

    PubMed Central

    Ebhardt, H Alexander; Nan, Jie; Chaulk, Steven G; Fahlman, Richard P; Aebersold, Ruedi

    2014-01-01

    RATIONALE Tandem mass (MS/MS) spectra generated by collision-induced dissociation (CID) typically lack redundant peptide sequence information in the form of e.g. b- and y-ion series due to frequent use of sequence-specific endopeptidases cleaving C- or N-terminal to Arg or Lys residues. METHODS Here we introduce arginyl-tRNA protein transferase (ATE, EC 2.3.2.8) for proteomics. ATE recognizes acidic amino acids or oxidized Cys at the N-terminus of a substrate peptide and conjugates an arginine from an aminoacylated tRNAArg onto the N-terminus of the substrate peptide. This enzymatic reaction is carried out under physiological conditions and, in combination with Lys-C/Asp-N double digest, results in arginylated peptides with basic amino acids on both termini. RESULTS We demonstrate that in vitro arginylation of peptides using yeast arginyl tRNA protein transferase 1 (yATE1) is a robust enzymatic reaction, specific to only modifying N-terminal acidic amino acids. Precursors originating from arginylated peptides generally have an increased protonation state compared with their non-arginylated forms. Furthermore, the product ion spectra of arginylated peptides show near complete 2× fragment ladders within the same MS/MS spectrum using commonly available electrospray ionization peptide fragmentation modes. Unexpectedly, arginylated peptides generate complete y- and c-ion series using electron transfer dissociation (ETD) despite having an internal proline residue. CONCLUSIONS We introduce a rapid enzymatic method to generate peptides flanked on either terminus by basic amino acids, resulting in a rich, redundant MS/MS fragment pattern. © 2014 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd. PMID:25380496

  7. Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

    PubMed

    Cheng, Rui; Tao, Mangjuan; Shi, Zhilu; Zhang, Xiafei; Jin, Yan; Li, Baoxin

    2015-11-15

    Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (? exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4)U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research. PMID:26057733

  8. Haloperidol Regulates the State of Phosphorylation of Ribosomal Protein S6 via Activation of PKA and Phosphorylation of DARPP-32

    PubMed Central

    Valjent, Emmanuel; Bertran-Gonzalez, Jesus; Bowling, Heather; Lopez, Sébastien; Santini, Emanuela; Matamales, Miriam; Bonito-Oliva, Alessandra; Hervé, Denis; Hoeffer, Charles; Klann, Eric; Girault, Jean-Antoine; Fisone, Gilberto

    2011-01-01

    Administration of typical antipsychotic drugs, such as haloperidol, promotes cAMP-dependent signaling in the medium spiny neurons (MSNs) of the striatum. In this study, we have examined the effect of haloperidol on the state of phosphorylation of the ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit. We found that haloperidol increases the phosphorylation of rpS6 at the dual site Ser235/236, which is involved in the regulation of mRNA translation. This effect was exerted in the MSNs of the indirect pathway, which express specifically dopamine D2 receptors (D2Rs) and adenosine A2 receptors (A2ARs). The effect of haloperidol was decreased by blockade of A2ARs or by genetic attenuation of the G?olf protein, which couples A2ARs to activation of adenylyl cyclase. Moreover, stimulation of cAMP-dependent protein kinase A (PKA) increased Ser235/236 phosphorylation in cultured striatal neurons. The ability of haloperidol to promote rpS6 phosphorylation was abolished in knock-in mice deficient for PKA activation of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32?kDa. In contrast, pharmacological or genetic inactivation of p70 rpS6 kinase 1, or extracellular signal-regulated kinases did not affect haloperidol-induced rpS6 phosphorylation. These results identify PKA as a major rpS6 kinase in neuronal cells and suggest that regulation of protein synthesis through rpS6 may be a potential target of antipsychotic drugs. PMID:21814187

  9. Serine 34 Phosphorylation of Rho Guanine Dissociation Inhibitor (RhoGDI?) Links Signaling from Conventional Protein Kinase C to RhoGTPase in Cell Adhesion*

    PubMed Central

    Dovas, Athanassios; Choi, Youngsil; Yoneda, Atsuko; Multhaupt, Hinke A. B.; Kwon, Seung-Hae; Kang, Dongmin; Oh, Eok-Soo; Couchman, John R.

    2010-01-01

    Conventional protein kinase C (PKC) isoforms are essential serine/threonine kinases regulating many signaling networks. At cell adhesion sites, PKC? can impact the actin cytoskeleton through its influence on RhoGTPases, but the intermediate steps are not well known. One important regulator of RhoGTPase function is the multifunctional guanine nucleotide dissociation inhibitor RhoGDI? that sequesters several related RhoGTPases in an inactive form, but it may also target them through interactions with actin-associated proteins. Here, it is demonstrated that conventional PKC phosphorylates RhoGDI? on serine 34, resulting in a specific decrease in affinity for RhoA but not Rac1 or Cdc42. The mechanism of RhoGDI? phosphorylation is distinct, requiring the kinase and phosphatidylinositol 4,5-bisphosphate, consistent with recent evidence that the inositide can activate, localize, and orient PKC? in membranes. Phosphospecific antibodies reveal endogenous phosphorylation in several cell types that is sensitive to adhesion events triggered, for example, by hepatocyte growth factor. Phosphorylation is also sensitive to PKC inhibition. Together with fluorescence resonance energy transfer microscopy sensing GTP-RhoA levels, the data reveal a common pathway in cell adhesion linking two essential mediators, conventional PKC and RhoA. PMID:20472934

  10. OXIDATIVE PHOSPHORYLATION: Kinetic and Thermodynamic Correlation between Electron Flow, Proton Translocation, Oxygen Consumption and ATP Synthesis under Close to In Vivo Concentrations of Oxygen

    PubMed Central

    Reynafarje, Baltazar D.; Ferreira, Jorge

    2008-01-01

    For the fist time the mitochondrial process of oxidative phosphorylation has been studied by determining the extent and initial rates of electron flow, H+ translocation, O2 uptake and ATP synthesis under close to in vivo concentrations of oxygen. The following novel results were obtained. 1) The real rates of O2 uptake and ATP synthesis are orders of magnitude higher than those observed under state-3 metabolic conditions. 2) The phosphorylative process of ATP synthesis is neither kinetically nor thermodynamically related to the respiratory process of H+ ejection. 3) The ATP/O stoichiometry is not constant but varies depending on all, the redox potential (?Eh), the degree of reduction of the membrane and the relative concentrations of O2, ADP, and protein. 4) The free energy of electron flow is not only used for the enzymatic binding and release of substrates and products but fundamentally for the actual synthesis of ATP from ADP and Pi. 5) The concentration of ADP that produces half-maximal responses of ATP synthesis (EC50) is not constant but varies depending on both ?Eh and O2 concentration. 6) The process of ATP synthesis exhibits strong positive catalytic cooperativity with a Hill coefficient, n, of ~3.0. It is concluded that the most important factor in determining the extent and rates of ATP synthesis is not the level of ADP or the proton gradient but the concentration of O2 and the state of reduction and/or protonation of the membrane. PMID:18566675

  11. Regulation of Protein Tyrosine Phosphorylation in Human Sperm by a Calcium\\/Calmodulin-Dependent Mechanism: Identification of A Kinase Anchor Proteins as Major Substrates for Tyrosine Phosphorylation

    Microsoft Academic Search

    Alfonso Carrera; Jiri Moos; Xiao Ping Ning; George L. Gerton; Jan Tesarik; Gregory S. Kopf; Stuart B. Moss

    1996-01-01

    Signal transduction pathways regulate various aspects of mammalian sperm function. When human sperm were incubated in a medium supporting capacitation, proteins became tyrosine-phosphorylated in a time-dependent manner. This phosphorylation was inhibited by genistein, a protein tyrosine kinase inhibitor. Phosphorylation was also reduced when sperm were incubated either in the presence of increasing concentrations of extracellular Ca2+or in a medium containing

  12. Regulatory phosphorylation of C 4 phospho enol pyruvate carboxylase from Sorghum : An immunological study using specific anti-phosphorylation site-antibodies

    Microsoft Academic Search

    Valérie Pacquit; Nathalie Giglioli; Claude Crétin; Jean Noel Pierre; Jean Vidal; Cristina Echevarria

    1995-01-01

    A peptide containing the N-terminal phosphorylation site (Ser-8) of Sorghum C4-phospho enolpyruvate carboxylase (PEPC) was synthesized, purified and used to raise an antiserum in rabbits. Affinity-purified IgGs prevented PEPC phosphorylation in a reconstituted in vitro assay and reacted with both the phosphorylated and dephosphorylated forms of either native or denatured PEPC in immunoblotting experiments. Saturation of dephospho-PEPC with these specific

  13. Negative regulation of Raf-1 by phosphorylation of serine 621.

    PubMed Central

    Mischak, H; Seitz, T; Janosch, P; Eulitz, M; Steen, H; Schellerer, M; Philipp, A; Kolch, W

    1996-01-01

    The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation. PMID:8816453

  14. Ground State Destabilization by Anionic Nucleophiles Contributes to the Activity of Phosphoryl Transfer

    E-print Network

    Herschlag, Dan

    . Alkaline Phosphatase (AP) and other nonspecific phosphatases are some of Nature's most impressive catalysts that no competing interests exist. Abbreviations: AP, alkaline phosphatase; Pi, inorganic phosphate; PTP, protein binding measurements with a protein tyrosine phosphatase suggest the generality of this ground state

  15. Enzymatic Polymer Functionalisation: Advances in Laccase and Peroxidase Derived Lignocellulose Functional Polymers

    Microsoft Academic Search

    Gibson S. Nyanhongo; Tukayi Kudanga; Endry Nugroho Prasetyo; Georg M. Guebitz

    \\u000a \\u000a Abstract  Enzymatic polymer functionalisation has entered its most fascinating period with development in this field largely at the\\u000a basic research level and pilot scale applications. Development of enzymatic processes for the development of lignocellulose-based\\u000a functional polymers has not been spared, ranging from textile fibres with novel properties (antimicrobials properties, hydrophobic\\u000a properties, attractive shed colours, etc.) to fibreboards. Enzymatic processes are also

  16. Regioselective enzymatic acylation of multi-hydroxyl compounds in organic synthesis

    Microsoft Academic Search

    Hyun Gyu Park; Jin Hwan Do; Ho Nam Chang

    2003-01-01

    With current developments in enzyme-catalyzed reactions and techniques available for rational redesign of natural biocatalysts,\\u000a the enzymatic biosynthesis can become one of the most valuable synthetic methods. Enzymatic regioselective catalysis in organic\\u000a media has played a key role in pursuing asymmetric synthesis for active chiral compounds. Here, we shortly describe some historical\\u000a issues of the rapidly growing area, enzymatic catalysis

  17. Enzymatic and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs)

    Microsoft Academic Search

    Shann S Yu; Randy L Scherer; Ryan A Ortega; Charleson S Bell; Conlin P O’Neil; Jeffrey A Hubbell; Todd D Giorgio

    2011-01-01

    Background  Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming\\u000a increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis.\\u000a Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for\\u000a applications in magnetic resonance imaging (MRI). To achieve

  18. A novel phosphorylation-independent interaction between SMG6 and UPF1 is essential for human NMD

    PubMed Central

    Nicholson, Pamela; Josi, Christoph; Kurosawa, Hitomi; Yamashita, Akio; Mühlemann, Oliver

    2014-01-01

    Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD substrates can be degraded by different routes that all require phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of the three known NMD factors thought to be recruited to nonsense mRNAs via an interaction with P-UPF1, leading to eventual mRNA degradation. By artificial tethering of SMG6 and mutants thereof to a reporter mRNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 to reduce reporter mRNA levels. Using in vivo and in vitro approaches, we further document that SMG6 and the unique stalk region of the UPF1 helicase domain, along with a contribution from the SQ domain, form a novel interaction and we also show that this region of the UPF1 helicase domain is critical for SMG6 function and NMD. Our results show that this interaction is required for NMD and for the capability of tethered SMG6 to degrade its bound RNA, suggesting that it contributes to the intricate regulation of UPF1 and SMG6 enzymatic activities. PMID:25053839

  19. Microbial enzymatic production and applications of short-chain fructooligosaccharides and inulooligosaccharides: recent advances and current perspectives.

    PubMed

    Mutanda, T; Mokoena, M P; Olaniran, A O; Wilhelmi, B S; Whiteley, C G

    2014-06-01

    The industrial production of short-chain fructooligosaccharides (FOS) and inulooligosaccharides is expanding rapidly due to the pharmaceutical importance of these compounds. These compounds, concisely termed prebiotics, have biofunctional properties and hence health benefits if consumed in recommended dosages. Prebiotics can be produced enzymatically from sucrose elongation or via enzymatic hydrolysis of inulin by exoinulinases and endoinulinases acting alone or synergistically. Exoinulinases cleave the non-reducing ?-(2, 1) end of inulin-releasing fructose while endoinulinases act on the internal linkages randomly to release inulotrioses (F3), inulotetraoses (F4) and inulopentaoses (F5) as major products. Fructosyltransferases act by cleaving a sucrose molecule and then transferring the liberated fructose molecule to an acceptor molecule such as sucrose or another oligosaccharide to elongate the short-chain fructooligosaccharide. The FOS produced by the action of fructosyltransferases are 1-kestose (GF2), nystose (GF3) and fructofuranosyl nystose (GF4). The production of high yields of oligosaccharides of specific chain length from simple raw materials such as inulin and sucrose is a technical challenge. This paper critically explores recent research trends in the production and application of short-chain oligosaccharides. Inulin and enzyme sources for the production of prebiotics are discussed. The mechanism of FOS chain elongation and also the health benefits associated with prebiotics consumption are discussed in detail. PMID:24793124

  20. An enzymatic biosensor for hydrogen peroxide based on CeO2 nanostructure electrodeposited on ITO surface.

    PubMed

    Yagati, Ajay Kumar; Lee, Taek; Min, Junhong; Choi, Jeong-Woo

    2013-09-15

    In this study, an enzymatic biosensor for amperometric detection of hydrogen peroxide was developed based on the direct electrochemistry of myoglobin (Mb) on a porous cerium dioxide (CeO2) nanostructured film. The developed film accomplished with large surface area was electrodeposited on an indium tin oxide (ITO) substrate. Surface morphological studies revealed that the formed CeO2 film has a large specific surface area with a unique nanostructure on the ITO surface. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were employed to demonstrate the electrochemical behavior of Mb immobilized on the fabricated film, which exhibited facile, direct electrochemistry and good electrocatalytic performance without any electron mediator. The electrode displayed a pair of quasi-reversible reduction-oxidation peaks at -0.3 and -0.2V, respectively, due to the Mb [Fe(3+)/Fe(2+)] redox couple, which is a surface-controlled electrochemical process with one electron transfer. This reagent-less biosensor showed good stability and high sensitivity for detecting H2O2 without any influence of intermediate compounds. This protein-based biosensor was capable of detecting H2O2 as low as 0.6?M with linearity up to 3mM and a response time of ~8s, compared to those of other modified electrodes. Hence, porous CeO2 is a possible candidate material for fabricating enzymatic sensors or devices. PMID:23608540

  1. Validation of doubled haploid plants by enzymatic mismatch cleavage

    PubMed Central

    2013-01-01

    Background Doubled haploidy is a fundamental tool in plant breeding as it provides the fastest way to generate populations of meiotic recombinants in a genetically fixed state. A wide range of methods has been developed to produce doubled haploid (DH) plants and recent advances promise efficient DH production in otherwise recalcitrant species. Since the cellular origin of the plants produced is not always certain, rapid screening techniques are needed to validate that the produced individuals are indeed homozygous and genetically distinct from each other. Ideal methods are easily implemented across species and in crops where whole genome sequence and marker resources are limited. Results We have adapted enzymatic mismatch cleavage techniques commonly used for TILLING (Targeting Induced Local Lesions IN Genomes) for the evaluation of heterozygosity in parental, F1 and putative DH plants. We used barley as a model crop and tested 26 amplicons previously developed for TILLING. Experiments were performed using self-extracted single-strand-specific nuclease and standard native agarose gels. Eleven of the twenty-six tested primers allowed unambiguous assignment of heterozygosity in material from F1 crosses and loss of heterozygosity in the DH plants. Through parallel testing of previously developed Simple Sequence Repeat (SSR) markers, we show that 3/32 SSR markers were suitable for screening. This suggests that enzymatic mismatch cleavage approaches can be more efficient than SSR based screening, even in species with well-developed markers. Conclusions Enzymatic mismatch cleavage has been applied for mutation discovery in many plant species, including those with little or no available genomic DNA sequence information. Here, we show that the same methods provide an efficient system to screen for the production of DH material without the need of specialized equipment. This gene target based approach further allows discovery of novel nucleotide polymorphisms in candidate genes in the parental lines. PMID:24220637

  2. Detection of infant faecal bifidobacteria by enzymatic methods.

    PubMed

    Vlková, E; Nevoral, J; Jencikova, B; Kopecný, J; Godefrooij, J; Trojanová, I; Rada, V

    2005-03-01

    An enzyme-based assay was developed for the detection of bifidobacteria in infant faeces. Ninety-five samples from 51 breast-fed infants in the age between 3 and 276 days were investigated. Bifidobacteria and other bacterial groups were determined by cultivation and fluorescence in situ hybridisation (FISH). Faecal samples were examined for the activity of fructoso-6-phosphate phosphoketolase (F6PPK) and for other enzymatic reactions using the API-ZYM kit. Twenty-nine infants had high numbers of bifidobacteria (usually higher than 9 log CFU/g) in their faeces. Seventeen infants (35%) did not contain detectable amounts of bifidobacteria in their faecal samples. The remaining five individuals had low counts of bifidobacteria (3-6 log CFU/g). Most negative infants possessed major amounts of clostridia in their faecal flora. There were no significant differences among bifidobacterial counts obtained by cultivation and FISH, detection of F6PPK, alpha-galactosidase and alpha-glucosidase activities could routinely be used for the rapid and simple detection of bifidobacteria in infant faecal samples. Bifidobacterial colonies were identified using enzymatic tests and PCR procedure based on 16S rRNA gene sequences species-specific primers. In 14 samples, the identifications of individual isolates were compared with direct analyses of faeces using the nested PCR-denaturing gradient gel electrophoresis (nested DGGE) procedure. The results obtained in several cases are not identical. Bifidobacterium longum and Bifidobacterium breve were most frequently identified. Bifidobacteria-positive samples had high activities of alpha-galactosidase and alpha-glucosidase. On the contrary, negative samples missed either one or both of these enzymatic activities. While all positive samples tested showed distinctive fructose-6-phosphate phosphoketolase activity (F6PPK), none of the negative samples expressed F6PPK activity. PMID:15649538

  3. Understanding of alkaline pretreatment parameters for corn stover enzymatic saccharification

    PubMed Central

    2013-01-01

    Background Previous research on alkaline pretreatment has mainly focused on optimization of the process parameters to improve substrate digestibility. To achieve satisfactory sugar yield, extremely high chemical loading and enzyme dosages were typically used. Relatively little attention has been paid to reduction of chemical consumption and process waste management, which has proven to be an indispensable component of the bio-refineries. To indicate alkali strength, both alkali concentration in pretreatment solution (g alkali/g pretreatment liquor or g alkali/L pretreatment liquor) and alkali loading based on biomass solids (g alkali/g dry biomass) have been widely used. The dual approaches make it difficult to compare the chemical consumption in different process scenarios while evaluating the cost effectiveness of this pretreatment technology. The current work addresses these issues through pretreatment of corn stover at various combinations of pretreatment conditions. Enzymatic hydrolysis with different enzyme blends was subsequently performed to identify the effects of pretreatment parameters on substrate digestibility as well as process operational and capital costs. Results The results showed that sodium hydroxide loading is the most dominant variable for enzymatic digestibility. To reach 70% glucan conversion while avoiding extensive degradation of hemicellulose, approximately 0.08 g NaOH/g corn stover was required. It was also concluded that alkali loading based on total solids (g NaOH/g dry biomass) governs the pretreatment efficiency. Supplementing cellulase with accessory enzymes such as ?-arabinofuranosidase and ?-xylosidase significantly improved the conversion of the hemicellulose by 6–17%. Conclusions The current work presents the impact of alkaline pretreatment parameters on the enzymatic hydrolysis of corn stover as well as the process operational and capital investment costs. The high chemical consumption for alkaline pretreatment technology indicates that the main challenge for commercialization is chemical recovery. However, repurposing or co-locating a biorefinery with a paper mill would be advantageous from an economic point of view. PMID:23356733

  4. Non-contact measurement technique for enzymatic reaction of glucokinase

    NASA Astrophysics Data System (ADS)

    Staforelli, Juan P.; Gallardo, María. J.; Meza, Pablo; Torres, Sergio; Mella, Héctor; Reyes, Claudio

    2014-03-01

    A non-contact infrared imaging-based measurement technique is applied to quantify the enzymatic reaction of glucokinase. The method is implemented by a long-wave (8-12 [?m]) infrared microbolometer imaging array and a germanium-based infrared optical vision system adjusted to the size of a small biological sample. The enzymatic reaction is carried out by the glucokinase enzyme, which is representative of the internal dynamics of the cell. Such reactions produce a spontaneous exothermal release of energy detected by the infrared imaging system as a non-contact measurement technique. It is shown by stoichiometry computations and infrared thermal resolution metrics that the infrared imaging system can detect the energy release at the [mK] range. This allows to quantify the spontaneity of the enzymatic reaction in a three dimensional (surface and time) single and noncontact real- time measurement. The camera is characterized for disclosing its sensibility, and the fixed pattern noise is compensated by a two point calibration method. On the other hand, the glucokinase enzyme is isolated from Pyrococcus furiosus. Therefore, the experiment is carried out by manual injection with graduated micropipettes using 40 [?l] of glucokinase at the surface of the substrate contained in an eppendorf tube. For recording, the infrared camera is adjusted in-focus at 25.4 [mm] from the superficial level of the substrate. The obtained values of energy release are 139 +/- 22 [mK] at room temperature and 274 +/- 22 [mK] for a bath temperature of 334 [K].

  5. Neurosteroids promote phosphorylation and membrane insertion of extrasynaptic GABAA receptors

    PubMed Central

    Abramian, Armen M.; Comenencia-Ortiz, Eydith; Modgil, Amit; Vien, Thuy N.; Nakamura, Yasuko; Moore, Yvonne E.; Maguire, Jamie L.; Terunuma, Miho; Davies, Paul A.; Moss, Stephen J.

    2014-01-01

    Neurosteroids are synthesized within the brain and act as endogenous anxiolytic, anticonvulsant, hypnotic, and sedative agents, actions that are principally mediated via their ability to potentiate phasic and tonic inhibitory neurotransmission mediated by ?-aminobutyric acid type A receptors (GABAARs). Although neurosteroids are accepted allosteric modulators of GABAARs, here we reveal they exert sustained effects on GABAergic inhibition by selectively enhancing the trafficking of GABAARs that mediate tonic inhibition. We demonstrate that neurosteroids potentiate the protein kinase C-dependent phosphorylation of S443 within ?4 subunits, a component of GABAAR subtypes that mediate tonic inhibition in many brain regions. This process enhances insertion of ?4 subunit-containing GABAAR subtypes into the membrane, resulting in a selective and sustained elevation in the efficacy of tonic inhibition. Therefore, the ability of neurosteroids to modulate the phosphorylation and membrane insertion of ?4 subunit-containing GABAARs may underlie the profound effects these endogenous signaling molecules have on neuronal excitability and behavior. PMID:24778259

  6. Integration of troponin I phosphorylation with cardiac regulatory networks.

    PubMed

    Solaro, R John; Henze, Marcus; Kobayashi, Tomoyoshi

    2013-01-18

    We focus here on the modulation of thin filament activity by cardiac troponin I phosphorylation as an integral and adaptive mechanism in cardiac homeostasis and as a mechanism vulnerable to maladaptive response to stress. We discuss a current concept of cardiac troponin I function in the A-band region of the sarcomere and potential signaling to cardiac troponin I in a network involving the ends of the thin filaments at the Z-disk and the M-band regions. The cardiac sarcomere represents a remarkable set of interacting proteins that functions not only as a molecular machine generating the heartbeat but also as a hub of signaling. We review how phosphorylation signaling to cardiac troponin I is integrated, with parallel signals controlling excitation-contraction coupling, hypertrophy, and metabolism. PMID:23329791

  7. Cell entry of Lassa virus induces tyrosine phosphorylation of dystroglycan.

    PubMed

    Moraz, Marie-Laurence; Pythoud, Christelle; Turk, Rolf; Rothenberger, Sylvia; Pasquato, Antonella; Campbell, Kevin P; Kunz, Stefan

    2013-05-01

    The extracellular matrix (ECM) receptor dystroglycan (DG) serves as a cellular receptor for the highly pathogenic arenavirus Lassa virus (LASV) that causes a haemorrhagic fever with high mortality in human. In the host cell, DG provides a molecular link between the ECM and the actin cytoskeleton via the adapter proteins utrophin or dystrophin. Here we investigated post-translational modifications of DG in the context of LASV cell entry. Using the tyrosine kinase inhibitor genistein, we found that tyrosine kinases are required for efficient internalization of virus particles, but not virus-receptor binding. Engagement of cellular DG by LASV envelope glycoprotein (LASV GP) in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. This virus-induced dissociation of utrophin was affected by genistein treatment, suggesting a role of receptor tyrosine phosphorylation in the process. PMID:23279385

  8. Ser/Thr phosphorylation as a regulatory mechanism in bacteria.

    PubMed

    Dworkin, Jonathan

    2015-04-01

    This review will discuss some recent work describing the role of Ser/Thr phosphorylation as a post-translational mechanism of regulation in bacteria. I will discuss the interaction between bacterial eukaryotic-like Ser/Thr kinases (eSTKs) and two-component systems as well as hints as to physiological function of eSTKs and their cognate eukaryotic-like phosphatases (eSTPs). In particular, I will highlight the role of eSTKs and eSTPs in the regulation of peptidoglycan metabolism and protein synthesis. In addition, I will discuss how data from phosphoproteomic surveys suggest that Ser/Thr phosphorylation plays a much more significant physiological role than would be predicted simply based on in vivo and in vitro analyses of individual kinases. PMID:25625314

  9. Thermophysical and flammability characterization of phosphorylated epoxy adhesives

    NASA Technical Reports Server (NTRS)

    Kourtides, D. A.; Parker, J. A.; Giants, T. W.; Bilow, N.; Hsu, M.-T.

    1980-01-01

    Some of the thermophysical and flammability properties of a phosphorylated epoxy adhesive, which has potential applications in aircraft interior panels, are described. The adhesive consists of stoichiometric ratios of bis(3-glycidyloxphenyl)methylphosphine oxide and bis(3-aminophenyl)methylphosphine oxide containing approximately 7.5% phosphorus. Preliminary data are presented from adhesive bonding studies conducted utilizing this adhesive with polyvinyl fluoride (PVF) film and phenolic-glass laminates. Limiting oxygen index and smoke density data are presented and compared with those of the tetraglycidyl methylene dianiline epoxy resin-adhesive system currently used in aircraft interiors. Initial results indicate that the phosphorylated epoxy compound has excellent adhesive properties when used with PVF film and that desirable fire-resistant properties are maintained.

  10. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    PubMed Central

    Shi, Lei; Pigeonneau, Nathalie; Ravikumar, Vaishnavi; Dobrinic, Paula; Macek, Boris; Franjevic, Damjan; Noirot-Gros, Marie-Francoise; Mijakovic, Ivan

    2014-01-01

    Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD, and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells. PMID:25278935

  11. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation

    PubMed Central

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-01-01

    Linker histones are involved in chromatin higher-order structure and gene regulation. We have successfully achieved partial phosphorylation of linker histones in chicken erythrocyte soluble chromatin with CDK2, as indicated by HPCE, MALDI-TOF and Tandem MS. We have studied the effects of linker histone partial phosphorylation on secondary structure and chromatin condensation. Infrared spectroscopy analysis showed a gradual increase of ?-structure in the phosphorylated samples, concomitant to a decrease in ?-helix/turns, with increasing linker histone phosphorylation. This conformational change could act as the first step in the phosphorylation-induced effects on chromatin condensation. A decrease of the sedimentation rate through sucrose gradients of the phosphorylated samples was observed, indicating a global relaxation of the 30-nm fiber following linker histone phosphorylation. Analysis of specific genes, combining nuclease digestion and qPCR, showed that phosphorylated samples were more accessible than unphosphorylated samples, suggesting local chromatin relaxation. Chromatin aggregation was induced by MgCl2 and analyzed by dynamic light scattering (DLS). Phosphorylated chromatin had lower percentages in volume of aggregated molecules and the aggregates had smaller hydrodynamic diameter than unphosphorylated chromatin, indicating that linker histone phosphorylation impaired chromatin aggregation. These findings provide new insights into the effects of linker histone phosphorylation in chromatin condensation. PMID:25870416

  12. Phosphorylation of C-protein, troponin I and phospholamban in isolated rabbit hearts.

    PubMed Central

    Garvey, J L; Kranias, E G; Solaro, R J

    1988-01-01

    Phosphorylation of myofibrillar and sacroplasmic-reticulum (SR) proteins was studied in Langendorff-perfused rabbit hearts subjected to various inotropic interventions. Stimulation of hearts with isoprenaline resulted in the phosphorylation of both troponin I (TnI) and C-protein in myofibrils and phospholamban in SR. Phosphorylation of phospholamban could be reversed by a 15 min perfusion with drug-free buffer, after a 1 minute pulse perfusion with isoprenaline, at which time the mechanical effects of isoprenaline stimulation had also been reversed. However, both TnI and C-protein remained phosphorylated at this time. Moreover, the inhibition of Ca2+ activation of the Mg2+-dependent ATPase (Mg-ATPase) activity associated with myofibrillar phosphorylation persisted in myofibrils prepared from hearts frozen after 15 min of washout of isoprenaline. To assess the contribution of C-protein phosphorylation in the decrease of Ca2+ activation of the myofibrillar Mg-ATPase activity, we reconstituted a regulated actomyosin system in which only C-protein was phosphorylated. In this system, C-protein phosphorylation did not contribute to the decrease in Ca2+ activation of Mg-ATPase activity, indicating that TnI phosphorylation is responsible for the diminished sensitivity of the myofibrils to Ca2+. These observations support the hypothesis that phospholamban phosphorylation plays a more dominant role than TnI or C-protein phosphorylation in the mechanical response of the mammalian heart to beta-adrenergic stimulation. Images Fig. 1. Fig. 3. PMID:2895634

  13. Three siderophores from one bacterial enzymatic assembly line.

    PubMed

    Wuest, William M; Sattely, Elizabeth S; Walsh, Christopher T

    2009-04-15

    Siderophores play a vital role in the survival of bacteria, as they facilitate the transport of iron in low-concentration environments. Nature employs a variety of coordinating functional groups in siderophore scaffolds as a way of creating structural diversity. We have successfully shown that the pseudomonine synthetase can produce three distinct siderophore natural products and five siderophore-like compounds. The in vitro enzymatic production of acinetobactin has prompted a revision of the reported structure from an oxazoline to an isoxazolidinone. Our results reveal the inherent flexibility of the pseudomonine synthetase and thus provide insight into the evolution of siderophore biosynthetic gene clusters in bacteria. PMID:19320483

  14. Detecting platform for phenolic compounds-characteristic of enzymatic electrode

    NASA Astrophysics Data System (ADS)

    Cabaj, Joanna; Chyla, Antoni; J?drychowska, Agnieszka; Olech, Kamila; So?oducho, Jadwiga

    2012-08-01

    We report here on simple and universal method for the highly efficient, electrolytic immobilization of tyrosinase (from Agaricus bisporus), for amperometric biosensing purposes. Tyrosinase has been successfully deposited on the surface of thin, ordered films of copolymerized derivative of thiophene (3-methylthiophene/3-thiopheneacetic acid/bis(ethylenedioxythiophene)diphenylamine). The tyrosinase retains well its activity well within the fabricated copolymer matrix. Reduction peaks, observed in cyclic voltammetry at 0.125 to +0.07 V, were attributed to the reduction of enzymatically liberated quinone species. Considering the fact, that immobilization strategy showed high efficiency, obtained results suggest that the method for phenoloxidase immobilization has a great potential for fabrication of bioelectronics' devices.

  15. Enzymatic synthesis of polyphenols with longer conjugation lengths

    Microsoft Academic Search

    Yushuang Cui; Xueqin Han; Yuanju Ding; Zaijun Lu

    2010-01-01

    Polyphenols containing Schiff base pendent groups, poly(4-[benzylidene-amino]-phenol) (PBP), and poly(4-[(anthracen-9-ylmethylene)-amino]-phenol)\\u000a (PAP) have been synthesized through the combination of Schiff base reaction and enzymatic polymerization using horseradish\\u000a peroxidase (HRP) as catalyst. The polymers were characterized by GPC, FTIR, 1H NMR, and UV spectroscopy. It shows that PBP and PAP are composed of polyphenol main chains bearing Schiff base pendent side\\u000a groups.

  16. Influences of different enzymatic treatment on denim garment.

    PubMed

    Montazer, Majid; Sadeghian Maryan, Ali

    2010-04-01

    In current study, the effects of different enzymatic treatment including acid cellulases, neutral cellulases, and combination of laccases with cellulases on denim garment were investigated. The color changes of different samples were compared by colorimetric indices for the garment surface and back and also white pocket. Abrasion resistance, tensile strength, and crease recovery angle of the samples were measured within the standard methods. Also, surfaces of the treated samples were observed by SEM. The results showed that the combination of laccases with cellulases help to improve the lightness and decrease staining on both back of garment and on white pocket. PMID:19728171

  17. Non-enzymatic glucose detection using magnetic nanoemulsions

    SciTech Connect

    Mahendran, V.; Philip, John, E-mail: philip@igcar.gov.in [SMARTS, Metallurgy and Materials Group, Indira Gandhi Centre for Atomic Research, Kalpakkam 603 102, Tamil Nadu (India)

    2014-09-22

    We probe the optical properties and intermolecular interactions in magnetically responsive nanoemulsions in the presence of glucose. The equilibrium interdroplet distance between the emulsion droplets in an one-dimensional array increases by several nanometers in the presence of glucose because of intermolecular hydrogen bonding with sodium dodecyl sulphate molecules at the oil-water interface that gives rise to stretched lamellae-like structure. The observed large red shift in the diffracted Bragg peak (?50–100?nm) and the linear response in the glucose concentration range of 0.25–25?mM offer a simple, fast, and cost effective non-enzymatic approach for glucose detection.

  18. Stereochemistry of enzymatic water addition to C=C bonds.

    PubMed

    Chen, Bi-Shuang; Otten, Linda G; Hanefeld, Ulf

    2015-01-01

    Water addition to carbon-carbon double bonds using hydratases is attracting great interest in biochemistry. Most of the known hydratases are involved in primary metabolism and to a lesser extent in secondary metabolism. New hydratases have recently been added to the toolbox, both from natural sources or artificial metalloenzymes. In order to comprehensively understand how the hydratases are able to catalyse the water addition to carbon-carbon double bonds, this review will highlight the mechanistic and stereochemical studies of the enzymatic water addition to carbon-carbon double bonds, focusing on the syn/anti-addition and stereochemistry of the reaction. PMID:25640045

  19. Optimization of Enzymatic Clarification of Sapodilla Juice: A Statistical Perspective

    Microsoft Academic Search

    Nicemol Jacob; R. K. Sukumaran; P. Prema

    2008-01-01

    Response surface methodology (RSM) was employed to establish optimum conditions for enzymatic clarification of sapodilla juice.\\u000a Polygalacturonase obtained from Streptomyces lydicus had been purified to homogeneity and was used for the treatment. The independent variables were temperature (30–45 °C), enzyme\\u000a concentration (0.5–1.5 U), and treatment time (30–90 min), whose effects on viscosity and clarity of the juice were evaluated\\u000a using a Box–Behnken design.

  20. Rheological Properties of Enzymatically Isolated Tomato Fruit Cuticle.

    PubMed Central

    Petracek, P. D.; Bukovac, M. J.

    1995-01-01

    Rheological properties were determined for cuticular membranes (CMs) enzymatically isolated from mature tomato (Lycopersicon esculentum Mill. cv Pik Red) fruit. The cuticle responded as a viscoelastic polymer in stress-strain studies. Both CM and dewaxed CM expanded and became more elastic and susceptible to fracture when hydrated, suggesting that water plasticized the cuticle. Dewaxing of the CM caused similar changes in elasticity and fracturing, indicating that wax may serve as a supporting filler in the cutin matrix. Exposure of the cuticle to the surfactant Triton X-100 did not significantly affect its rheological properties. PMID:12228622

  1. 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

  2. N-[Bis(benzylamino)phosphoryl]-2,2,2-trichloroacetamide

    PubMed Central

    Ovchynnikov, Vladimir A.

    2010-01-01

    In the title compound, C16H17Cl3N3O2P, the P atom has a slightly distorted tetra­hedral configuration. The conformations of the carbonyl and phosphoryl groups are anti to each other. In the crystal, inter­molecular N—H?O hydrogen bonds link the mol­ecules into infinite chains parallel to the b axis. PMID:21579501

  3. PROTEIN PHOSPHORYLATION AND THE REGULATION OF CATION COTRANSPORT

    Microsoft Academic Search

    H. CLIVE PALFREY; SETH L. ALPER; PAUL GREENGARD

    1980-01-01

    The turkey erythrocyte is a useful system in which to investigate the relationship between protein phosphorylation and membrane transport. This cell possesses a catecholamine- and cyclic AMP-stimulated Na+-K+ 'co- transport ' system in its plasma membrane, and the physiological properties of this system have been partially elucidated. It is distinct from the Na+-K+ 'pump' and is insensitive to ouabain, but

  4. Extraction of rare-earth nitrates by phosphoryl podands

    Microsoft Academic Search

    A. N. Turanov; V. K. Karandashev; V. E. Baulin

    2006-01-01

    The distribution of trace amounts of rare-earth nitrates between aqueous solutions of NH4NO3 and organic solutions of phosphoryl podands is studied for Ln = La, Ce, Pr, Nd, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and Y.\\u000a The stoichiometry of the extraction complexes is determined. The effect of the structure of the extractant and the nature\\u000a of the

  5. Intrinsic Protein Kinase Activity in Mitochondrial Oxidative Phosphorylation Complexes

    PubMed Central

    Phillips, Darci; Aponte, Angel M.; Covian, Raul Garcia; Balaban, Robert S.

    2012-01-01

    Mitochondrial protein phosphorylation is a well-recognized metabolic control mechanism, with the classical example of pyruvate dehydrogenase (PDH) regulation by specific kinases and phosphatases of bacterial origin. However, despite the growing number of reported mitochondrial phosphoproteins, identity of the protein kinases mediating these phosphorylation events remains largely unknown. The detection of mitochondrial protein kinases is complicated by the low concentration of kinase relative to target protein, lack of specific antibodies and contamination from associated, but non-matrix, proteins. In this study we used blue native gel electrophoresis (BN-PAGE) to isolate rat and porcine heart mitochondrial complexes for screening of protein kinase activity. To detect kinase activity, 1D BN-PAGE gels were exposed to ?-32P-ATP and then followed by SDS gel electrophoresis (2D BN/SDS-PAGE). 32P-labeled dozen of mitochondrial proteins in this setting, including all five complexes of oxidative phosphorylation and several citric acid cycle enzymes. The near ubiquitous 32P protein labeling demonstrates protein kinase activity within each mitochondrial protein complex. The validity of this 2D-BN-PAGE method was demonstrated by detecting the known PDH kinases and phosphatases within the PDH complex band using Western blots and mass spectrometry. Surprisingly, these same approaches detected only a few additional conventional protein kinases suggesting a major role for autophosphorylation in mitochondrial proteins. Studies on purified Complex V and creatine kinase confirmed that these proteins undergo autophosphorylation, and, to a lesser degree, tenacious 32P-metabolite association. In-gel Complex IV activity was shown to be inhibited by ATP, and partially reversed by phosphatase activity, consistent with an inhibitory role for protein phosphorylation in this complex. Collectively, this study proposes that many of the mitochondrial complexes contain an autophosphorylation mechanism, which may play a functional role in the regulation of these multi-protein units. PMID:21329348

  6. Apoptotic Membrane Blebbing Is Regulated by Myosin Light Chain Phosphorylation

    PubMed Central

    Mills, Jason C.; Stone, Nicole L.; Erhardt, Joseph; Pittman, Randall N.

    1998-01-01

    The evolutionarily conserved execution phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the execution phase of apoptosis. PMID:9456322

  7. Cellular Adaptations to Oxidative Phosphorylation Defects in Cancer

    Microsoft Academic Search

    Sarika Srivastava; Carlos T. Moraes

    \\u000a Mitochondrial DNA (mtDNA) somatic mutations or mutations in nuclear genes encoding mitochondrial proteins important for the\\u000a assembly, activity, or maintenance of the individual oxidative phosphorylation (OXPHOS) complexes have been observed in tumors.\\u000a Although the functional consequence of such mutations is unclear at the moment, retrograde signaling in response to OXPHOS\\u000a defects can activate various nuclear genes and signaling pathways that

  8. Phosphorylation characteristics of brain clathrin-coated vesicle endogenous proteins.

    PubMed

    Schook, W J; Parker, C; Silva, W I; Kohtz, D S; Puszkin, S

    1987-08-01

    Clathrin-coated vesicles purified from bovine brain express protein kinase activity on two principal endogenous vesicle-associated substrates: a 50,000-Mr polypeptide (pp50) and clathrin-associated protein2 (CAP2; the faster-migrating clathrin light chain). Various exogenous substrates, e.g., casein, phosvitin, histone II, and histone III, also are phosphorylated. The pp50 protein kinase activity of clathrin-coated vesicles is not modulated by Ca2+, calmodulin, phosphatidylserine, or cyclic AMP. On the other hand, phosphorylation of the other endogenous substrates requires certain activators, including histone, polylysine, polyarginine, or polyethylenimine. Phosphate incorporation into pp50 was sensitive to divalent cations that inhibit sulfhydryl-dependent enzymes in the following order of potency: Zn2+ greater than Hg2+ greater than Cd2+, Cu2+, and Pb2+. Phosphate incorporation into CAP2 with polylysine present was insensitive to divalent cations. The alkylating agents dithiodinitrobenzene, phenacyl bromide, and N-ethylmaleimide inhibited phosphate incorporation into pp50 up to 90% without affecting incorporation into the other substrates. Vanadium pentoxide inhibited phosphorylation of CAP2 but had a minimal effect on pp50. CAP2 kinase activity was separated from the coated vesicle membrane and from dis-assembled clathrin triskelions, coeluting with the assembly polypeptide complex on a Sepharose 4B column. It retained phosphorylation properties similar to those of intact vesicles. These data imply that clathrin-coated vesicle kinases are elements of the coat proteins and may be involved in the assembly/disassembly of clathrin triskelions or interactions of coated vesicles with other cellular components. PMID:2885397

  9. Odor-Induced Phosphorylation of Olfactory Cilia Proteins

    Microsoft Academic Search

    Ingrid Boekhoff; Sabine Schleicher; Jorg Strotmann; Heinz Breer

    1992-01-01

    Stimulation of isolated rat olfactory cilia in the presence of [gamma-32P]ATP leads to a significantly enhanced incorporation of [32P]phosphate. Depending on the type of odorants applied, the induced phosphorylation is completely blocked by specific inhibitors of either protein kinase A or protein kinase C. Time-course experiments indicate that the odor-induced modification of ciliary proteins is transient; the intensity of labeling

  10. Phosphorylation of Ribose-Borate Complexes at Convergent Margins?

    Microsoft Academic Search

    N. G. Holm

    2008-01-01

    The potential of pyrophosphate formation upon heating of hydrogenated orthophosphates like whitlockite ((Ca18Mg2H2(PO4)14) to a few hundred °C in geological environments with low water to rock ratio has probably been underestimated. Once pyrophosphate is available, phosphorylation of pentoses, ribose in particular, may occur. Experiments involving heating of sodium dihydrogen phosphate have even shown high yields of trimetaphosphate. This compound is

  11. Identification of phosphorylation sites in the mouse oestrogen receptor

    Microsoft Academic Search

    H. Lahooti; R. White; S. A. Hoare; D. Rahman; D. J. C. Pappin; M. G. Parker

    1995-01-01

    Phosphorylation sites in the mouse oestrogen receptor, expressed in COS-1 cells in the presence of 17?-oestradiol, have been mapped by solid phase microsequencing. The receptor was first radiolabelled with [32P]orthophosphate and a number of3H- or14C-labelled amino acids, immunopurified and then tryptic peptides were separated by thin layer chromatography or high performance liquid chromatography. Amino acid sequence analysis indicated that Ser-122,

  12. Basal phosphorylation of mu opioid receptor is agonist modulated and Ca2+-dependent.

    PubMed

    Wang, Z; Arden, J; Sadée, W

    1996-05-27

    The mu opioid receptor was shown to be phosphorylated at a basal rate in the absence of agonist, measured in permeabilized HEK293 cells transfected with an epitope tagged mu receptor (EE-mu) [Arden, J., Segredo, V., Wang, Z., Lameh, J. and Sadee, W. (1995) J. Neurochem. 65, 1636-1645]. In the present study, basal phosphorylation was found to be Ca2+ dependent; however, several inhibitors of protein kinase C and Ca2+-calmodulin dependent kinases failed to affect basal mu receptor phosphorylation. Thus, the basal mu receptor phosphorylating activity differed from the main kinases involved in receptor regulation. The general kinase inhibitor H7 (100 microM) suppressed basal mu receptor phosphorylation. Pretreatment with the agonist morphine, followed by drug removal, resulted in a sustained increase of basal mu receptor phosphorylation. The gradual agonist dependent modulation of basal mu receptor phosphorylation suggests a novel regulatory mechanism which may play a role in narcotic tolerance and dependence. PMID:8654566

  13. Sequential phosphorylation analysis using dye-tethered peptides and microfluidic isoelectric focusing electrophoresis.

    PubMed

    Choi, Hoseok; Choi, Nakchul; Lim, Butaek; Kim, Tae-Wuk; Song, Simon; Kim, Young-Pil

    2015-11-15

    We report a simple method for analyzing sequential phosphorylation by protein kinases using fluorescent peptide substrates and microfluidic isoelectric focusing (?IEF) electrophoresis. When a dye-labeled peptide substrate was sequentially phosphorylated by two consecutive protein kinases (mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3)), its differently phosphorylated forms were easily separated and visualized by fluorescent focusing zones in the ?IEF channel based on a change in the isoelectric point (pI) by phosphorylation. As a result, ratiometric and quantitative analysis of the fluorescent focusing regions shifted by phosphorylation enabled the analysis of phosphorylation efficiency and the relevant inhibition of protein kinases (MAPK and GSK3) with high simplicity and selectivity. Furthermore, the GSK3 activity in the cell lysates was elucidated by ?IEF electrophoresis in combination with immunoprecipitation. Our results suggest that this method has great potential for analyzing the sequential phosphorylation of multiple protein kinases that are implicated in cellular signaling pathways. PMID:26050965

  14. Endothelial Akt1 mediates angiogenesis by phosphorylating multiple angiogenic substrates

    PubMed Central

    Lee, Monica Y.; Luciano, Amelia K.; Ackah, Eric; Rodriguez-Vita, Juan; Bancroft, Tara A.; Eichmann, Anne; Simons, Michael; Kyriakides, Themis R.; Morales-Ruiz, Manuel; Sessa, William C.

    2014-01-01

    The PI3K/Akt pathway is necessary for several key endothelial cell (EC) functions, including cell growth, migration, survival, and vascular tone. However, existing literature supports the idea that Akt can be either pro- or antiangiogenic, possibly due to compensation by multiple isoforms in the EC when a single isoform is deleted. Thus, biochemical, genetic, and proteomic studies were conducted to examine isoform-substrate specificity for Akt1 vs. Akt2. In vitro, Akt1 preferentially phosphorylates endothelial nitric oxide synthase (eNOS) and promotes NO release, whereas nonphysiological overexpression of Akt2 can bypass the loss of Akt1. Conditional deletion of Akt1 in the EC, in the absence or presence of Akt2, retards retinal angiogenesis, implying that Akt1 exerts a nonredundant function during physiological angiogenesis. Finally, proteomic analysis of Akt substrates isolated from Akt1- or Akt2-deficient ECs documents that phosphorylation of multiple Akt substrates regulating angiogenic signaling is reduced in Akt1-deficient, but not Akt2-deficient, ECs, including eNOS and Forkhead box proteins. Therefore, Akt1 promotes angiogenesis largely due to phosphorylation and regulation of important downstream effectors that promote aspects of angiogenic signaling. PMID:25136137

  15. TTBK2: A Tau Protein Kinase beyond Tau Phosphorylation

    PubMed Central

    Liao, Jung-Chi; Yang, T. Tony; Weng, Rueyhung Roc; Kuo, Ching-Te; Chang, Chih-Wei

    2015-01-01

    Tau tubulin kinase 2 (TTBK2) is a kinase known to phosphorylate tau and tubulin. It has recently drawn much attention due to its involvement in multiple important cellular processes. Here, we review the current understanding of TTBK2, including its sequence, structure, binding sites, phosphorylation substrates, and cellular processes involved. TTBK2 possesses a casein kinase 1 (CK1) kinase domain followed by a ~900 amino acid segment, potentially responsible for its localization and substrate recruitment. It is known to bind to CEP164, a centriolar protein, and EB1, a microtubule plus-end tracking protein. In addition to autophosphorylation, known phosphorylation substrates of TTBK2 include tau, tubulin, CEP164, CEP97, and TDP-43, a neurodegeneration-associated protein. Mutations of TTBK2 are associated with spinocerebellar ataxia type 11. In addition, TTBK2 is essential for regulating the growth of axonemal microtubules in ciliogenesis. It also plays roles in resistance of cancer target therapies and in regulating glucose and GABA transport. Reported sites of TTBK2 localization include the centriole/basal body, the midbody, and possibly the mitotic spindles. Together, TTBK2 is a multifunctional kinase involved in important cellular processes and demands augmented efforts in investigating its functions. PMID:25950000

  16. Monte Carlo Simulations of Tau Proteins: Effect of Phosphorylation

    PubMed Central

    Jho, Y.S.; Zhulina, E.B.; Kim, M.W.; Pincus, P.A.

    2010-01-01

    We perform Monte Carlo simulations of tau proteins bound to a cylinder that mimics a microtubule (MT), and then study them in solution. Tau protein binds to a highly anionic MT surface to stabilize the cylindrical structure of MT. The negatively charged tail domain floats away from the anionic MT surface while positively charged tau segments localize near the MT surface. Monte Carlo simulations demonstrate that, in 3RS tau isoform (which has three imperfect repeats (R) short (S) isoform), amino acids are more condensed near a highly charged interface compared to 4RL isoform (which has four imperfect repeats (R) long (L) isoform). In 4RL isoform, amino acids in tail domain stay mostly apart from the MT surface. In the bulk solution, dephosphorylated taus are separated due to Coulomb repulsion between similarly charged isoforms. Moderate phosphorylation of 3RS isoform decreases average intermolecular distance between dephosphorylated and phosphorylated taus and lead to their overlap. Further phosphorylation does not change noticeably the intermolecular distances. PMID:20959078

  17. Src tyrosyl phosphorylates cortactin in response to prolactin.

    PubMed

    Hammer, Alan; Laghate, Sneha; Diakonova, Maria

    2015-08-01

    The hormone/cytokine prolactin (PRL) is implicated in breast cancer cell invasion and metastasis. PRL-induced pathways are mediated by two non-receptor tyrosine kinases, JAK2 and Src. We previously demonstrated that prolactin stimulates invasion of breast cancer cells TMX2-28 through JAK2 and its target serine/threonine kinase PAK1. We hypothesize herein that the actin-binding protein cortactin, a protein involved in invadopodia formation and cell invasion, is activated by PRL. We demonstrate that TMX2-28 cells are more invasive than T47D breast cancer cells in response to PRL. We determine that cortactin is tyrosyl phosphorylated in response to PRL in a time and dose-dependent manner in TMX2-28 cells, but not in T47D cells. Furthermore, we show that PRL mediates cortactin tyrosyl phosphorylation via Src, but not JAK2. Finally, we demonstrate that maximal PRL-mediated TMX2-28 cell invasion requires both Src and JAK2 kinase activity, while T47D cell invasion is JAK2- but not Src-dependent. Thus PRL may induce cell invasion via two pathways: through a JAK2/PAK1 mediated pathway that we have previously demonstrated, and Src-dependent activation and tyrosyl phosphorylation of cortactin. PMID:26043691

  18. Occurrence of phosphorylated castasterone in Arabidopsis thaliana and Lycopersicum esculentum.

    PubMed

    Kim, Min Kyun; Jang, Mun-Seok; Youn, Ji-Hyun; Son, Seung-Hyun; Lee, Ji Eun; Kim, Tae-Wuk; Kim, Seong-Ki

    2015-01-01

    An in vitro enzyme assay using radioisotope-labeled (3) H-castasterone ((3) H-CS) or (32) P-ATP showed that CS can be phosphorylated by ATP in Arabidopsis and tomato plants. Gas chromatography-mass spectrometry (GC-MS) analysis using non-isotope-labeled CS and ATP revealed that the phosphorylation of CS occurs at the side chain, most likely at the C-23 hydroxyl. The polar fractions than free brassinosteroids (BRs) obtained from extracts of Arabidopsis and tomato showed almost no BRs activity in a rice lamina inclination bioassay. However, the fractions showed increased bioactivity after treatment with wheat germ acidic phosphatase (WGAP). Additionally, CS was identified from the hydrolysate by WGAP using GC-MS analysis in both plants. In contrast, the polar fractions obtained from BR-deficient mutants, Arabidopsis cyp85a2 and tomato d(x) , did not show an increase in biological activity with WGAP treatment, and no free BRs, including CS, were detected in the hydrolysate. This suggests that CS phosphate is a naturally occurring biologically inactive conjugate that is generated when CS is normally synthesized in Arabidopsis and tomato plants. Taken together, these results suggest that phosphorylation of CS is an important conjugation process for the maintenance of the homeostatic level of an active BR and thus the regulation of the growth and development of plants. PMID:24939035

  19. Does prothymosin alpha affect the phosphorylation of elongation factor 2?

    PubMed

    Enkemann, S A; Pavur, K S; Ryazanov, A G; Berger, S L

    1999-06-25

    Prothymosin alpha is a small, acidic, essential nuclear protein that plays a poorly defined role in the proliferation and survival of mammalian cells. Recently, Vega et al. proposed that exogenous prothymosin alpha can specifically increase the phosphorylation of eukaryotic elongation factor 2 (eEF-2) in extracts of NIH3T3 cells (Vega, F. V., Vidal, A., Hellman, U., Wernstedt, C., and Domínguez, F. (1998) J. Biol. Chem. 273, 10147-10152). Using similar lysates prepared by four methods (detergent lysis, Dounce homogenization, digitonin permeabilization, and sonication) and three preparations of prothymosin alpha, one of which was purified by gentle means (the native protein, and a histidine-tagged recombinant prothymosin alpha expressed either in bacteria or in COS cells), we failed to find a response. A reconstituted system composed of eEF-2, recombinant eEF-2 kinase, calmodulin, and calcium was also unaffected by prothymosin alpha. However, unlike our optimized buffer, Vega's system included a phosphatase inhibitor, 50 mM fluoride, which when evaluated in our laboratories severely reduced phosphorylation of all species. Under these conditions, any procedure that decreases the effective fluoride concentration will relieve the inhibition and appear to activate. Our data do not support a direct relationship between the function of prothymosin alpha and the phosphorylation of eEF-2. PMID:10373476

  20. Hydrogen Peroxide-Induced Akt Phosphorylation Regulates Bax Activation

    PubMed Central

    Sadidi, Mahdieh; Lentz, Stephen I.; Feldman, Eva L.

    2009-01-01

    Reactive oxygen species such as hydrogen peroxide (H2O2) are involved in many cellular processes that positively and negatively regulate cell fate. H2O2, acting as an intracellular messenger, activates phosphatidylinositol-3 kinase (PI3K) and its downstream target Akt, and promotes cell survival. The aim of the current study was to understand the mechanism by which PI3K/Akt signaling promotes survival in SH-SY5Y neuroblastoma cells. We demonstrate that PI3K/Akt mediates phosphorylation of the pro-apoptotic Bcl-2 family member Bax. This phosphorylation suppresses apoptosis and promotes cell survival. Increased survival in the presence of H2O2 was blocked by LY294002, an inhibitor of PI3K activation. LY294002 prevented Bax phosphorylation and resulted in Bax translocation to the mitochondria, cytochrome c release, caspase-3 activation, and cell death. Collectively, these findings reveal a mechanism by which H2O2-induced activation of PI3K/Akt influences posttranslational modification of Bax and inactivate a key component of the cell death machinery. PMID:19278624

  1. Regulation of Monoamine Transporters: Role of Transporter Phosphorylation

    PubMed Central

    Ramamoorthy, Sammanda; Shippenberg, Toni S.; Jayanthi, Lankupalle D.

    2010-01-01

    Presynaptic biogenic amine transporters mediate reuptake of released amines from the synapse, thus regulating serotonin, dopamine and norepinephrine neurotransmission. Medications utilized in the treatment of depression, attention deficit-hyperactivity disorder and other psychiatric disorders possess high affinity for amine transporters. In addition, amine transporters are targets for psychostimulants. Altered expression of biogenic amine transporters has long been implicated in several psychiatric and degenerative disorders. Therefore, appropriate regulation and maintenance of biogenic amine transporter activity is critical for the maintenance of normal amine homoeostasis. Accumulating evidence suggests that cellular protein kinases and phosphatases regulate amine transporter expression, activity, trafficking and degradation. Amine transporters are phosphoproteins that undergo dynamic control under the influence of various kinase and phosphatase activities. This review presents a brief overview of the role of amine transporter phosphorylation in the regulation of amine transport in the normal and diseased brain. Understanding the molecular mechanisms by which phosphorylation events affect amine transporter activity is essential for understanding the contribution of transporter phosphorylation to the regulation of monoamine neurotransmission and for identifying potential new targets for the treatment of various brain diseases. PMID:20951731

  2. Aquaporin 9 phosphorylation mediates membrane localization and neutrophil polarization.

    PubMed

    Karlsson, Thommie; Glogauer, Michael; Ellen, Richard P; Loitto, Vesa-Matti; Magnusson, Karl-Eric; Magalhães, Marco A O

    2011-11-01

    Neutrophils are of prime importance in the host innate defense against invading microorganisms by using two primary mechanisms-locomotion toward and phagocytosis of the prey. Recent research points to pivotal roles for water channels known as AQPs in cell motility. Here, we focused on the role of AQP9 in chemoattractant-induced polarization and migration of primary mouse neutrophils and neutrophil-like HL60 cells. We found that AQP9 is phosphorylated downstream of fMLFR or PMA stimulation in primary human neutrophils. The dynamics of AQP9 were assessed using GFP-tagged AQP9 constructs and other fluorescent markers through various live-cell imaging techniques. Expression of WT or the phosphomimic S11D AQP9 changed cell volume regulation as a response to hyperosmotic changes and enhanced neutrophil polarization and chemotaxis. WT AQP9 and S11D AQP9 displayed a very dynamic distribution at the cell membrane, whereas the phosphorylation-deficient S11A AQP9 failed to localize to the plasma membrane. Furthermore, we found that Rac1 regulated the translocation of AQP9 to the plasma membrane. Our results show that AQP9 plays an active role in neutrophil volume regulation and migration. The display of AQP9 at the plasma membrane depends on AQP9 phosphorylation, which appeared to be regulated through a Rac1-dependent pathway. PMID:21873454

  3. Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase.

    PubMed

    Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Liu, Ming C; Shetty, Sreerama

    2010-02-01

    The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1-100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth. PMID:19784757

  4. Regulation of the autophagy protein LC3 by phosphorylation

    PubMed Central

    Cherra, Salvatore J.; Kulich, Scott M.; Uechi, Guy; Balasubramani, Manimalha; Mountzouris, John; Day, Billy W.

    2010-01-01

    Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP+) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease–associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl–cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity. PMID:20713600

  5. Probing the stoichiometry of ?2-adrenergic receptor phosphorylation by targeted mass spectrometry

    PubMed Central

    2014-01-01

    Background Protein phosphorylation of G-protein-coupled receptors (GPCR) is central to the myriad of functions that these ubiquitous receptors perform in biology. Although readily addressable with the use of phospho-specific antibodies, analysis phosphorylation at the level of stoichiometry requires receptor isolation and advanced proteomics. We chose two key sites of potential phosphorylation of human beta2-adrenergic receptor (?2AR residues S355 and S356) to ascertain the feasibility of applying targeted mass spectrometry to establishing the stoichiometry of the phosphorylation. Method We stimulated HEK293 cells stably expressing Flag-tagged ?2AR-eGFP with 10 ?M beta-adrenergic agonist (isoproterenol) and made use of proteomics and targeted mass spectrometry (MS) to quantify the molar ration of phosphorylation on S355 and S356 versus non-phosphorylated receptor in agonist-treated cells. Results Phosphorylation of either S355 or S356 residue occurred only for agonist-occupied ?2AR. The results demonstrated that pS356 is the dominant site of protein phosphorylation. The abundance of the p356 was 8.6-fold more than that of pS355. Calculation of the molar ratio of phosphorylated (pS355 plus pS356) versus non-phosphorylated receptor reveals that at high occupancy of the receptor only 12.4% of the ?2AR is phosphorylated at these sites. Conclusions Application of advanced proteomics and use of the most sensitive targeted MS strategy makes possible the detection and quantification of phosphorylation of very low abundance peptide digests of ?2AR. Establishing the stoichiometry of two key sites of agonist-stimulated phosphorylation with ?2AR is an essential first-step to global analysis of the stoichiometry of GPCR phosphorylation. PMID:24690384

  6. Stable Isotope Labeling of Phosphoproteins for Large-scale Phosphorylation Rate Determination*

    PubMed Central

    Molden, Rosalynn C.; Goya, Jonathan; Khan, Zia; Garcia, Benjamin A.

    2014-01-01

    Signals that control responses to stimuli and cellular function are transmitted through the dynamic phosphorylation of thousands of proteins by protein kinases. Many techniques have been developed to study phosphorylation dynamics, including several mass spectrometry (MS)-based methods. Over the past few decades, substantial developments have been made in MS techniques for the large-scale identification of proteins and their post-translational modifications. Nevertheless, all of the current MS-based techniques for quantifying protein phosphorylation dynamics rely on the measurement of changes in peptide abundance levels, and many methods suffer from low confidence in phosphopeptide identification due to poor fragmentation. Here we have optimized an approach for the stable isotope labeling of amino acids by phosphate using [?-18O4]ATP in nucleo to determine global site-specific phosphorylation rates. The advantages of this metabolic labeling technique are increased confidence in phosphorylated peptide identification, direct labeling of phosphorylation sites, measurement phosphorylation rates, and the identification of actively phosphorylated sites in a cell-like environment. In this study we calculated approximate rate constants for over 1,000 phosphorylation sites based on labeling progress curves. We measured a wide range of phosphorylation rate constants from 0.34 min?1 to 0.001 min?1. Finally, we applied stable isotope labeling of amino acids by phosphate to identify sites that have different phosphorylation kinetics during G1/S and M phase. We found that most sites had very similar phosphorylation rates under both conditions; however, a small subset of sites on proteins involved in the mitotic spindle were more actively phosphorylated during M phase, whereas proteins involved in DNA replication and transcription were more actively phosphorylated during G1/S phase. The data have been deposited to the ProteomeXchange with the identifier PXD000680. PMID:24532841

  7. Stable isotope labeling of phosphoproteins for large-scale phosphorylation rate determination.

    PubMed

    Molden, Rosalynn C; Goya, Jonathan; Khan, Zia; Garcia, Benjamin A

    2014-04-01

    Signals that control responses to stimuli and cellular function are transmitted through the dynamic phosphorylation of thousands of proteins by protein kinases. Many techniques have been developed to study phosphorylation dynamics, including several mass spectrometry (MS)-based methods. Over the past few decades, substantial developments have been made in MS techniques for the large-scale identification of proteins and their post-translational modifications. Nevertheless, all of the current MS-based techniques for quantifying protein phosphorylation dynamics rely on the measurement of changes in peptide abundance levels, and many methods suffer from low confidence in phosphopeptide identification due to poor fragmentation. Here we have optimized an approach for the stable isotope labeling of amino acids by phosphate using [?-¹?O?]ATP in nucleo to determine global site-specific phosphorylation rates. The advantages of this metabolic labeling technique are increased confidence in phosphorylated peptide identification, direct labeling of phosphorylation sites, measurement phosphorylation rates, and the identification of actively phosphorylated sites in a cell-like environment. In this study we calculated approximate rate constants for over 1,000 phosphorylation sites based on labeling progress curves. We measured a wide range of phosphorylation rate constants from 0.34 min?¹ to 0.001 min?¹. Finally, we applied stable isotope labeling of amino acids by phosphate to identify sites that have different phosphorylation kinetics during G1/S and M phase. We found that most sites had very similar phosphorylation rates under both conditions; however, a small subset of sites on proteins involved in the mitotic spindle were more actively phosphorylated during M phase, whereas proteins involved in DNA replication and transcription were more actively phosphorylated during G1/S phase. The data have been deposited to the ProteomeXchange with the identifier PXD000680. PMID:24532841

  8. The Physiological Link between Metabolic Rate Depression and Tau Phosphorylation in Mammalian Hibernation

    PubMed Central

    Stieler, Jens T.; Bullmann, Torsten; Kohl, Franziska; Tøien, Øivind; Brückner, Martina K.; Härtig, Wolfgang; Barnes, Brian M.; Arendt, Thomas

    2011-01-01

    Abnormal phosphorylation and aggregation of tau protein are hallmarks of a variety of neurological disorders, including Alzheimer's disease (AD). Increased tau phosphorylation is assumed to represent an early event in pathogenesis and a pivotal aspect for aggregation and formation of neurofibrillary tangles. However, the regulation of tau phosphorylation in vivo and the causes for its increased stage of phosphorylation in AD are still not well understood, a fact that is primarily based on the lack of adequate animal models. Recently we described the reversible formation of highly phosphorylated tau protein in hibernating European ground squirrels. Hence, mammalian hibernation represents a model system very well suited to study molecular mechanisms of both tau phosphorylation and dephosphorylation under in vivo physiological conditions. Here, we analysed the extent and kinetics of hibernation-state dependent tau phosphorylation in various brain regions of three species of hibernating mammals: arctic ground squirrels, Syrian hamsters and black bears. Overall, tau protein was highly phosphorylated in torpor states and phosphorylation levels decreased after arousal in all species. Differences between brain regions, hibernation-states and phosphosites were observed with respect to degree and kinetics of tau phosphorylation. Furthermore, we tested the phosphate net turnover of tau protein to analyse potential alterations in kinase and/or phosphatase activities during hibernation. Our results demonstrate that the hibernation-state dependent phosphorylation of tau protein is specifically regulated but involves, in addition, passive, temperature driven regulatory mechanisms. By determining the activity-state profile for key enzymes of tau phosphorylation we could identify kinases potentially involved in the differentially regulated, reversible tau phosphorylation that occurs during hibernation. We show that in black bears hibernation is associated with conformational changes of highly phosphorylated tau protein that are typically related to neuropathological alterations. The particular hibernation characteristics of black bears with a continuous torpor period and an only slightly decreased body temperature, therefore, potentially reflects the limitations of this adaptive reaction pattern and, thus, might indicate a transitional state of a physiological process. PMID:21267079

  9. Enzymatic activity of glucose oxidase covalently wired via viologen to electrically conductive polypyrrole films.

    PubMed

    Liu, X; Neoh, K G; Cen, Lian; Kang, E T

    2004-03-15

    The surface functionalization of an electrically conductive polypyrrole film (PPY) with a viologen, (N-(2-carboxyl-ethyl)-N'-(4-vinyl-benzyl)-4,4'-bipyridinium dichloride, or CVV) for the covalent immobilization of glucose oxidase (GOD) has been carried out. The viologen was first synthesized and graft polymerized on PPY film. It then served as an anchor via its carboxyl groups for the covalent immobilization of GOD. The surface composition of the as-functionalized substrates was characterized by X-ray photoelectron spectroscopy (XPS). The effects of the CVV monomer concentration on the CVV-graft polymer concentration and the amount of GOD immobilized on the surface were investigated. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also obtained. The cyclic voltammetric (CV) response of the GOD-functionalized PPY substrates was studied in a phosphate buffer solution under an argon atmosphere. The CV results support the mechanism in which CVV acts as a mediator to transfer electron between the electrode and enzyme, and hence regenerating the enzyme in the enzymatic reaction with glucose. High sensitivity and linear response of the enzyme electrode was observed with glucose concentration ranging from 0 to 20 mM. PMID:15128101

  10. One-step enzymatic modification of the cell surface redirects cellular cytotoxicity and parasite tropism.

    PubMed

    Swee, Lee Kim; Lourido, Sebastian; Bell, George W; Ingram, Jessica R; Ploegh, Hidde L

    2015-02-20

    Surface display of engineered proteins has many useful applications. The expression of a synthetic chimeric antigen receptor composed of an extracellular tumor-specific antibody fragment linked to a cytosolic activating motif in engineered T cells is now considered a viable approach for the treatment of leukemias. The risk of de novo tumor development, inherent in the transfer of genetically engineered cells, calls for alternative approaches for the functionalization of the lymphocyte plasma membrane. We demonstrate the conjugation of LPXTG-tagged probes and LPXTG-bearing proteins to endogenous acceptors at the plasma membrane in a single step using sortase A. We successfully conjugated biotin probes not only to mouse hematopoietic cells but also to yeast cells, 293T cells, and Toxoplasma gondii. Installation of single domain antibodies on activated CD8 T cell redirects cell-specific cytotoxicity to cells that bear the relevant antigen. Likewise, conjugation of Toxoplasma gondii with single domain antibodies targets the pathogen to cells that express the antigen recognized by these single domain antibodies. This simple and robust enzymatic approach enables engineering of the plasma membrane for research or therapy under physiological reaction conditions that ensure the viability of the modified cells. PMID:25360987

  11. Determination of phosphate in soil extracts in the field: A green chemistry enzymatic method

    PubMed Central

    Campbell, Ellen R.; Warsko, Kayla; Davidson, Anna-Marie; (Bill) Campbell, Wilbur H.

    2015-01-01

    Measurement of ortho-phosphate in soil extracts usually involves sending dried samples of soil to a laboratory for analysis and waiting several weeks for the results. Phosphate determination methods often involve use of strong acids, heavy metals, and organic dyes. To overcome limitations of this approach, we have developed a phosphate determination method which can be carried out in the field to obtain results on the spot. This new method uses: • Small volumes. • An enzymatic reaction. • Green chemistry. First, the soil sample is extracted with deionized water and filtered. Next, an aliquot of the soil extract (0.5 mL) is transferred to a disposable cuvette, containing 0.5 mL of reaction mixture [200 mM HEPES, pH 7.6, 20 mM MgCl2, with 80 nmol 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) and 1 unit of recombinant purine nucleoside phosphorylase (PNP; EC 2.4.2.1)], mixed, and incubated for 10 min at field temperature. Absorbance of the completed reaction is measured at 360 nm in open-source, portable photometer linked by bluetooth to a smartphone. The phosphate and phosphorus content of the soil is determined by comparison of its absorbance at 360 nm to a previously prepared standard phosphate curve, which is stored in the smartphone app.

  12. A computational study on enzymatically driven oxidative coupling of chlorophenols: an indirect dehalogenation reaction.

    PubMed

    Szatkowski, Lukasz; Dybala-Defratyka, Agnieszka

    2013-04-01

    Density functional theory calculations have been used to describe the mechanism of the dimerization reaction catalyzed by peroxidases and predict whether it could be accompanied by chlorine isotopic fractionation when various chlorophenols are used as their substrates. Since free radicals formed during the catalytic cycle of peroxidases can undergo coupling reactions (either radical-radical or radical-molecule) that can lead to dimers and also polymers formation four different pathways have been considered: radical-anion, radical-cation, radical-radical (singlet state) and radical-radical (triplet state). The following substrates have been investigated: 2-chlorophenol, 4-chlorophenol, 2,4,6-trichlorophenol and 4-chloro-2,6-dimethylphenol. Based on the obtained energetic profiles radical-cation and radical-radical (singlet) coupling seem to be the most probable. Radical-anion coupling although energetically more expensive should not be disregarded taking into account the excess of the anionic form of the substrate being provided in the course of enzymatic reaction. Upon dimer formation halogen is released during radical-anion and radical-radical triplet coupling. However only the latter pathway exhibits chlorine isotopic fractionation. Radical-cation and radical-radical singlet couplings are two-step reactions where the second step comprises intramolecular chlorine transfer accompanied by large chlorine isotope effect. PMID:23273327

  13. Langmuir and Langmuir-Blodgett films of lipids and penicillinase: Studies on adsorption and enzymatic activity.

    PubMed

    Scholl, Fabio Antonio; Caseli, Luciano

    2015-02-01

    Bioelectronic devices, such as biosensors, can be constructed with enzymes immobilized in ultrathin solid films, for which preserving the enzymatic catalytic activity is fundamental for optimal performance. In this sense, nanostructured films in which molecular architectures can be controlled are of interest. In this present work, the adsorption of the enzyme penicillinase onto Langmuir monolayers of the phospholipid dimyristoylphosphatidic acid was investigated and characterized with surface pressure-area isotherms and polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS). The incorporation of the enzyme in the lipid monolayer not only caused the film to expand, but also could be identified through amide bands in the PM-IRRAS spectra, with the CN and CO dipole moments being identified, lying parallel to monolayer plane. Structuring of the enzyme into ?-helices was identified in the mixed enzyme-phospholipid monolayer and preserved when transferred to solid as a Langmuir-Blodgett (LB) film. The enzyme-lipid LB films were then characterized with PM-IRRAS, atomic force microscopy and fluorescence spectroscopy. Measurements of the catalytic activity showed that the enzyme accommodated in the LB films preserved 76% of the enzyme activity in relation to the homogeneous medium. The method presented here not only allows for enhanced catalytic activity toward penicillin, but also can be useful to explain why certain film architectures exhibit better enzyme activity. PMID:25576807

  14. Ultrasound-assisted enzymatic extraction and antioxidant activity of polysaccharides from pumpkin (Cucurbita moschata).

    PubMed

    Wu, Hao; Zhu, Junxiang; Diao, Wenchao; Wang, Chengrong

    2014-11-26

    An efficient ultrasound-assisted enzymatic extraction (UAEE) of Cucurbita moschata polysaccharides (CMCP) was established and the CMCP antioxidant activities were studied. The UAEE operating parameters (extraction temperature, ultrasonic power, pH, and liquid-to-material ratio) were optimized using the central composite design (CCD) and the mass transfer kinetic study in UAEE procedure was used to select the optimal extraction time. Enzymolysis and ultrasonication that were simultaneously conducted was selected as the UAEE synergistic model and the optimum extraction conditions with a maximum polysaccharide yield of 4.33 ± 0.15% were as follows: extraction temperature, 51.5 °C; ultrasonic power, 440 W; pH, 5.0; liquid-to-material ratio, 5.70:1 mL/g; and extraction time, 20 min. Evaluation of the antioxidant activity in vitro suggested that CMCP has good potential as a natural antioxidant used in the food or medicine industry because of their high reducing power and positive radical scavenging activity for DPPH radical. PMID:25256490

  15. Miniaturised enzymatic conductometric biosensor with Nafion membrane for the direct determination of formaldehyde in water samples.

    PubMed

    Nguyen-Boisse, Thanh-Thuy; Saulnier, Joëlle; Jaffrezic-Renault, Nicole; Lagarde, Florence

    2014-02-01

    A new conductometric enzyme-based biosensor was developed for the determination of formaldehyde (FA) in aqueous solutions. The biosensor was prepared by cross-linking formaldehyde dehydrogenase from Pseudomonas putida with bovine serum albumin in saturated glutaraldehyde vapours (GA) at the surface of interdigitated gold microelectrodes. Nicotinamide adenine dinucleotide cofactor (NAD(+)) was added in solution at each measurement to maintain enzyme activity. Addition of a Nafion layer over the enzyme modified electrode resulted in a significant increase of biosensor signal due to enhanced accumulation of protons generated by enzymatic reaction at the electrode surface. Different parameters affecting enzyme activity or playing a role in ionic transfer through the Nafion membrane were optimised. In optimal conditions (0.045 mg enzyme, 30 min exposure to GA, 0.3 ?L of a 1% (v/v) Nafion solution deposit, measurement in 5 mM phosphate buffer pH 7 containing 20 ?M NAD(+)), the biosensor signal was linear up to 10 mM FA, and the detection limit was 18 ?M. Relative standard deviations calculated from five consecutive replicates of FA solutions were lower than 5% in the 1-10 mM range. The biosensor was successfully applied to the determination of FA in spiked water samples (tap water and Rhone river water), with recoveries in the 95-110% range. PMID:23907681

  16. Development of a hybrid fermentation-enzymatic bioprocess for the production of ethyl lactate from dairy waste.

    PubMed

    Koutinas, Michalis; Menelaou, Maria; Nicolaou, Evrydiki N

    2014-08-01

    This work explores the potential for the development of a hybrid fermentation-enzymatic process for the production of ethyl lactate from dairy waste. Cheese whey was used in Kluyveromyces marxianus and Lactobacillus bulgaricus batch cultures to produce ethanol and lactic acid respectively. Subsequently, the fermentation products were transferred into an organic phase through liquid-liquid extraction and ethyl lactate was formed in an esterification reaction catalyzed by lipases. The production of ethanol and lactic acid achieved under different conditions was 23gL(-1) and 29gL(-1), respectively. Furthermore, the efficiency of various organic solvents for the esterification reaction was evaluated and toluene was chosen for application in the process. The effect of water content was determined aiming to maximize the product yield and 40mgml(-1) was the optimal enzyme concentration. The bioprocess achieved maximum conversion of 33% constituting a valuable alternative to the application of energy demanding chemically derived methods. PMID:24785788

  17. An integrated photocatalytic/enzymatic system for the reduction of CO2 to methanol in bioglycerol–water

    PubMed Central

    Baran, Tomasz; Angelini, Antonella; ?abuz, Przemys?aw; Macyk, Wojciech

    2014-01-01

    Summary A hybrid enzymatic/photocatalytic approach for the conversion of CO2 into methanol is described. For the approach discussed here, the production of one mol of CH3OH from CO2 requires three enzymes and the consumption of three mol of NADH. Regeneration of the cofactor NADH from NAD+ was achieved by using visible-light-active, heterogeneous, TiO2-based photocatalysts. The efficiency of the regeneration process is enhanced by using a Rh(III)-complex for facilitating the electron and hydride transfer from the H-donor (water or a water–glycerol solution) to NAD+. This resulted in the production of 100 to 1000 mol of CH3OH from one mol of NADH, providing the possibility for practical application. PMID:25383127

  18. Enzymatic and molecular strategies to diagnose Pompe disease.

    PubMed

    Reuser, Ajj; Verheijen, Fw; Kroos, Ma; Okumiya, T; Van Diggelen, Op; Van der Ploeg, At; Halley, Djj

    2010-01-01

    Enzyme replacement therapy for Pompe disease, a neuromuscular disorder characterized by lysosomal glycogen storage due to acid ?-glucosidase deficiency, has entered the clinic. There is more than ever a need for early and reliable diagnosis. The objective of this review is to present a critical review of the recent literature on laboratory procedures to diagnose Pompe disease by enzymatic assay and DNA analysis. The methods we used were Compilation and expert interpretation of recent and relevant publications. The introduction of new and the updating of existing laboratory procedures have facilitated the diagnosis of Pompe disease (glycogen storage disease type II; acid maltase deficiency; OMIM 232300). With regard to enzymatic analysis, the application of acarbose as inhibitor of maltase-glucoamylase has enabled the use of mixed leukocyte preparations as diagnostic material. The use of glycogen as a natural substrate in the reaction mixture adds to the selectivity of this procedure. Newborn screening is envisaged and facilitated by the introduction of high-throughput procedures. DNA analysis has become an integral part of the diagnostic procedure for confirmation and completion, for carrier detection, and for genetic counseling. PMID:23496111

  19. Enzymatic hydrolysis: a method in alleviating legume allergenicity.

    PubMed

    Kasera, Ramkrashan; Singh, A B; Lavasa, S; Prasad, Komarla Nagendra; Arora, Naveen

    2015-02-01

    Legumes are involved in IgE mediated food allergy in many countries. Avoidance of allergenic food is the only way to avoid symptomatic reaction. The present study investigated the effect of enzymatic hydrolysis on the allergenicity of three legumes - kidney bean (Phaseolus vulgaris), black gram (Vigna mungo) and peanut (Arachis hypogaea). Soluble protein extracts of the study legumes were sequentially treated by Alcalase(®) and Flavourzyme(®). Allergenicity of hydrolysates was then determined by ELISA, immunoblot, stripped basophil histamine release and skin prick test (SPT). Hydrolysis resulted in the loss of all IgE binding fractions determined by immunoblot in the three legumes. Specific IgE binding in ELISA was reduced by 62.2?±?7.7%, 87.1?±?9.6% and 91.8?±?7.2% in the hydrolysates of kidney bean, black gram and peanut, respectively (p?enzymatic hydrolysis is effective in attenuating allergenicity of legume proteins and may be employed for preparing hypoallergenic food extracts. PMID:25481434

  20. A Minimalist Substrate for Enzymatic Peptide and Protein Conjugation

    PubMed Central

    Wollack, James W.; Silverman, Julie M.; Petzold, Christopher J.; Mougous, Joseph D.; Distefano, Mark D.

    2010-01-01

    Recently a number of non-natural prenyl groups containing alkynes and azides have been developed as handles to perform click chemistry on proteins and peptides ending in the sequence “CAAX”, where C is a cysteine that becomes alkylated, A is an aliphatic amino acid and X is any amino acid. When such molecules are modified, a tag containing a prenyl analog and the “CAAX box” sequence remains. Here we report the synthesis of an alkyne-containing substrate comprised of only nine non-hydrogen atoms. This substrate was synthesized in six steps from 3-methyl-2-buten-1-ol and has been enzymatically incorporated into both proteins and peptides using protein farnesyltransferase. After prenylation the final three amino acids required for enzymatic recognition can be removed using carboxypeptidase Y, leaving a single residue (the cysteine from the “CAAX box”) and the prenyl analog as the only modifications. We also demonstrate that this small tag minimizes the impact of the modification on the solubility of the targeted protein. Hence, this new approach should be useful for applications in which the presence of a large tag hinders the modified protein's solubility, reactivity or utility. PMID:19856367

  1. Queueing up for enzymatic processing: correlated signaling through coupled degradation

    PubMed Central

    Cookson, Natalie A; Mather, William H; Danino, Tal; Mondragón-Palomino, Octavio; Williams, Ruth J; Tsimring, Lev S; Hasty, Jeff

    2011-01-01

    High-throughput technologies have led to the generation of complex wiring diagrams as a post-sequencing paradigm for depicting the interactions between vast and diverse cellular species. While these diagrams are useful for analyzing biological systems on a large scale, a detailed understanding of the molecular mechanisms that underlie the observed network connections is critical for the further development of systems and synthetic biology. Here, we use queueing theory to investigate how ‘waiting lines' can lead to correlations between protein ‘customers' that are coupled solely through a downstream set of enzymatic ‘servers'. Using the E. coli ClpXP degradation machine as a model processing system, we observe significant cross-talk between two networks that are indirectly coupled through a common set of processors. We further illustrate the implications of enzymatic queueing using a synthetic biology application, in which two independent synthetic networks demonstrate synchronized behavior when common ClpXP machinery is overburdened. Our results demonstrate that such post-translational processes can lead to dynamic connections in cellular networks and may provide a mechanistic understanding of existing but currently inexplicable links. PMID:22186735

  2. Immobilized Aspartase-Containing Microbial Cells: Preparation and Enzymatic Properties

    PubMed Central

    Chibata, Ichiro; Tosa, Tetsuya; Sato, Tadashi

    1974-01-01

    The immobilization of asparatase-containing Escherichia coli was investigated by various methods, and the most active immobilized cells were obtained by entrapment in a polyacrylamide gel lattice. Other asparatase-containing bacteria were also entrapped by the same method, and the enzymatically active immobilized cells were obtained. The aspartase activity of the immobilized E. coli cells was increased nine- to tenfold by autolysis of the cells entrapped in the gel lattice. Enzymatic properties of the immobilized E. coli cells were investigated and compared with those of the intact cells. The optimal pH was 8.5 for the immobilized cells and 10.5 for the intact cells. The aspartase activities of immobilized and intact cells were not activated by Mn2+, which can activate the immobilized and native aspartases. The heat stability of the immobilized cells was somewhat higher than that of the intact cells. Bivalent metal ions such as Mn2+, Mg2+, Ca2+ protected against thermal inactivation of the aspartase activity of the immobilized and intact cells. Images PMID:4208512

  3. Synergism and Mutualism in Non-Enzymatic RNA Polymerization

    PubMed Central

    Kaddour, Hussein; Sahai, Nita

    2014-01-01

    The link between non-enzymatic RNA polymerization and RNA self-replication is a key step towards the “RNA world” and still far from being solved, despite extensive research. Clay minerals, lipids and, more recently, peptides were found to catalyze the non-enzymatic synthesis of RNA oligomers. Herein, a review of the main models for the formation of the first RNA polymers is presented in such a way as to emphasize the cooperation between life’s building blocks in their emergence and evolution. A logical outcome of the previous results is a combination of these models, in which RNA polymerization might have been catalyzed cooperatively by clays, lipids and peptides in one multi-component prebiotic soup. The resulting RNAs and oligopeptides might have mutualistically evolved towards functional RNAs and catalytic peptides, preceding the first RNA replication, thus supporting an RNA-peptide world. The investigation of such a system is a formidable challenge, given its complexity deriving from a tremendously large number of reactants and innumerable products. A rudimentary experimental design is outlined, which could be used in an initial attempt to study a quaternary component system. PMID:25370531

  4. Glycoprotein analysis using enzymatic digestion and MALDI-TOF MS

    NASA Astrophysics Data System (ADS)

    Kornfeld, Rich; Kenny, James W.; Weinberger, Scot R.; Yang, Yi; Orlando, Ron

    1996-04-01

    A sensitive and facile method is described to identify the glycosylation sites and site-specific heterogeneity in the carbohydrate portion of glycoproteins. In this procedure, the peptide backbone of the glycoprotein is cleaved enzymatically. The peptide/glycopeptide mixture is divided into three fractions. The first fraction is analyzed directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), while the other two aliquots are analyzed by MALDI-TOF MS after enzymatic release of the N-linked and N- and O-linked chains. Comparison of these mass spectra provides the molecular weight of each carbohydrate side chain and of the peptide to which it is attached. This information combined with the protein's amino acid sequence identifies the glycosylation sites and provides information concerning site-specific oligosaccharide heterogeneity. This approach is faster and simpler than procedures currently used for glycosylation site mapping and can be performed on as little as 10 picomoles of glycoprotein.

  5. Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated IIIGlc, a signal-transducing protein from Escherichia coli, using two-dimensional heteronuclear NMR techniques.

    PubMed Central

    Pelton, J. G.; Torchia, D. A.; Meadow, N. D.; Roseman, S.

    1993-01-01

    IIIGlc is an 18.1-kDa signal-transducing phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system from Escherichia coli. The 1H, 15N, and 13C histidine ring NMR signals of both the phosphorylated and unphosphorylated forms of IIIGlc have been assigned using two-dimensional 1H-15N and 1H-13C heteronuclear multiple-quantum coherence (HMQC) experiments and a two-dimensional 13C-13C-1H correlation spectroscopy via JCC coupling experiment. The data were acquired on uniformly 15N-labeled and uniformly 15N/13C-labeled protein samples. The experiments rely on one-bond and two-bond J couplings that allowed for assignment of the signals without the need for the analysis of through-space (nuclear Overhauser effect spectroscopy) correlations. The 15N and 13C chemical shifts were used to determine that His-75 exists predominantly in the N epsilon 2-H tautomeric state in both the phosphorylated and unphosphorylated forms of IIIGlc, and that His-90 exists primarily in the N delta 1-H state in the unphosphorylated protein. Upon phosphorylation of the N epsilon 2 nitrogen of His-90, the N delta 1 nitrogen remains protonated, resulting in the formation of a charged phospho-His-90 moiety. The 1H, 15N, and 13C signals of the phosphorylated and unphosphorylated proteins showed only minor shifts in the pH range from 6.0 to 9.0. These data indicate that the pK alpha values for both His-75 and His-90 in IIIGlc and His-75 in phospho-IIIGlc are less than 5.0, and that the pK alpha value for phospho-His-90 is greater than 10. The results are presented in relation to previously obtained structural data on IIIGlc, and implications for proposed mechanisms of phosphoryl transfer are discussed. PMID:8518729

  6. Soil solid materials affect the kinetics of extracellular enzymatic reactions

    NASA Astrophysics Data System (ADS)

    Lammirato, C.; Miltner, A.; Kästner, M.

    2009-04-01

    INTRODUCTION Soil solid materials affect the degradation processes of many organic compounds by decreasing the bioavailability of substrates and by interacting with degraders. The magnitude of this effect in the environment is shown by the fact that xenobiotics which are readily metabolized in aquatic environments can have long residence times in soil. Extracellular enzymatic hydrolysis of cellobiose (enzyme: beta-glucosidase from Aspergillus niger) was chosen as model degradation process since it is easier to control and more reproducible than a whole cell processes. Furthermore extracellular enzymes play an important role in the environment since they are responsible for the first steps in the degradation of organic macromolecules; beta-glucosidase is key enzyme in the degradation of cellulose and therefore it is fundamental in the carbon cycle and for soil in general. The aims of the project are: 1) quantification of solid material effect on degradation, 2) separation of the effects of minerals on enzyme (adsorption ?change in activity) and substrate (adsorption ?change in bioavailability). Our hypothesis is that a rate reduction in the enzymatic reaction in the presence of a solid phase results from the sum of decreased bioavailability of the substrate and decreased activity of enzyme molecules. The relative contribution of the two terms to the overall effect can vary widely depending on the chemical nature of the substrate, the properties of the enzyme and on the surface properties of the solid materials. Furthermore we hypothesize that by immobilizing the enzyme in an appropriate carrier the adsorption of enzymes to soil materials can be eliminated and that therefore immobilization can increase the overall reaction rate (activity loss caused by immobilization < activity loss caused by adsorption to soil minerals). MATERIALS AND METHODS Enzymatic kinetic experiments are carried out in homogeneous liquid systems and in heterogeneous systems where solid materials (bentonite, kaolinite, goethite, activated charcoal) are suspended in a mixed liquid (standard experimental conditions: 66 mM phosphate buffer, pH 5, 25°C, 20 mg solid/ml buffer). The enzyme in an immobilized form (covalent bonding to oxirane groups on the surfaces of macroporous Eupergit® C particles) is used to exclude a direct effect of soil solid materials on the enzyme without excluding their effect on the availability of the substrate.The progress of the reactions is determined by measuring the accumulation of the product (i.e. glucose) in the systems at different times (after destroying enzymatic activity by boiling the samples) with a coupled enzymatic assay and an automatic microplate spectrophotometer. A regression analysis on the data points is performed to calculate the initial reaction rates, which is the parameter that allows to compare the different systems. RESULTS AND DISCUSSION The results show that, under the standard experimental conditions, cellobiose is not adsorbed by the clay minerals bentonite and kaolinite and by the iron oxyhydroxide goethite. In the case of activated charcoal a rapid adsorption phase in the first 20' is followed by a much slower process; after 4h 30' approximately 98% of cellobiose was adsorbed. The results from the adsorption experiments of beta-glucosidase to bentonite, kaolinite, goethite and activated charcoal show that, under the standard experimental conditions, the adsorption process is rapid in all cases (more than 80% of the adsorption takes place in the first 20 minutes). After 1h 20min the following fractions of enzyme were adsorbed: 30 % to bentonite, 60% to kaolinite, 67% to goethite, 100% to activated charcoal. The effect of kaolinite on the reaction rate was quantified: under the standard experimental conditions the initial reaction rate in presence of the mineral was 22% less then in the control. The fraction of enzyme molecules which are adsorbed to kaolinite (60%) loses 37% of its activity. CONCLUSIONS The results from the adsorption experiments lead to the conclusion that, among the sol

  7. Animal Transfer Agreement -1 ANIMAL TRANSFER AGREEMENT

    E-print Network

    Bandettini, Peter A.

    Animal Transfer Agreement - 1 ANIMAL TRANSFER AGREEMENT This Animal Transfer Agreement has been adopted for use by the National Institutes of Health (NIH) for use in transferring animals for research transferring the animal) Recipient: (name of laboratory/institution receiving the animal) The Provider agrees

  8. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates

    NASA Astrophysics Data System (ADS)

    Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

    2014-03-01

    Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by using our developed Phosphorylation Set Enrichment Analysis (PSEA) method. We evaluated the efficiency of our method with independent test and concluded that our approach is reliable for identifying kinases responsible for phosphorylated substrates. In addition, we found that Mitogen-activated protein kinase (MAPK) and Glycogen synthase kinase (GSK) families are more associated with abnormal phosphorylation. It can be anticipated that our method might be helpful to identify the mechanism of phosphorylation and the relationship between kinase and phosphorylation related diseases. A user-friendly web interface is now freely available at http://bioinfo.ncu.edu.cn/PKPred_Home.aspx.

  9. Phosphorylation of thylakoid proteins of Oryza sativa: in vitro characterization and effects of chilling temperatures

    SciTech Connect

    Moll, B.A.; Eilmann, M.; Steinback, K.E.

    1987-02-01

    The phosphorylation of thylakoid proteins of rice (Oryza sativa L.) was studied in vitro using (..gamma..-/sup 32/P)ATP. Several thylakoid proteins are labeled, including the light-harvesting complex of photosystem II. Protein phosphorylation is sensitive to temperature, pH, and ADP, ATP, and divalent cation concentrations. In the range pH 7 to 8.2, phosphorylation of the light-harvesting polypeptides declines above pH 7.5, whereas labeling of several other thylakoid polypeptides increases. Increasing divalent cation concentration from 3 to 20 millimolar results in a decrease in phosphorylation of the 26 kilodalton light-harvesting complex polypeptide and increased phosphorylation of several other polypeptides. ADP has an inhibitory effect on the phosphorylation of the light-harvesting complex polypeptides. Phosphorylation of the 26 kilodalton light-harvesting polypeptide requires 0.45 millimolar ATP for half-maximal phosphorylation, compared to 0.3 millimolar for the 32 kilodalton phosphoprotein. Low temperature inhibits the phosphorylation of thylakoid proteins in chilling-sensitive rice. However, phosphorylation of histones by thylakoid-bound kinase(s) is independent of temperature in the range of 25 to 5/sup 0/C.

  10. PINK1 kinase catalytic activity is regulated by phosphorylation on serines 228 and 402.

    PubMed

    Aerts, Liesbeth; Craessaerts, Katleen; De Strooper, Bart; Morais, Vanessa A

    2015-01-30

    Mutations in the PINK1 gene cause early-onset recessive Parkinson disease. PINK1 is a mitochondrially targeted kinase that regulates multiple aspects of mitochondrial biology, from oxidative phosphorylation to mitochondrial clearance. PINK1 itself is also phosphorylated, and this might be linked to the regulation of its multiple activities. Here we systematically analyze four previously identified phosphorylation sites in PINK1 for their role in autophosphorylation, substrate phosphorylation, and mitophagy. Our data indicate that two of these sites, Ser-228 and Ser-402, are autophosphorylated on truncated PINK1 but not on full-length PINK1, suggesting that the N terminus has an inhibitory effect on phosphorylation. We furthermore establish that phosphorylation of these PINK1 residues regulates the phosphorylation of the substrates Parkin and Ubiquitin. Especially Ser-402 phosphorylation appears to be important for PINK1 function because it is involved in Parkin recruitment and the induction of mitophagy. Finally, we identify Thr-313 as a residue that is critical for PINK1 catalytic activity, but, in contrast to previous reports, we find no evidence that this activity is regulated by phosphorylation. These data clarify the regulation of PINK1 through multisite phosphorylation. PMID:25527497

  11. Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules

    PubMed Central

    Guo, Huifang; Gao, Meng; Lu, Yiling; Liang, Jiyong; Lorenzi, Philip L.; Bai, Shanshan; Hawke, David H.; Li, Jie; Dogruluk, Turgut; Scott, Kenneth L.; Jonasch, Eric; Mills, Gordon B.; Ding, Zhiyong

    2013-01-01

    Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty-two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated due to the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. While basal AKT2 phosphorylation was sensitive to inhibition of PI3K, basal AKT1 phosphorylation was essentially resistant. PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1. Thus NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation. PMID:23912456

  12. Conformation-Dependent Human p52Shc Phosphorylation by Human c-Src.

    PubMed

    Tsutsui, Yuko; Johnson, Jennifer M; Demeler, Borries; Kinter, Michael T; Hays, Franklin A

    2015-06-01

    Phosphorylation of the human p52Shc adaptor protein is a key determinant in modulating signaling complex assembly in response to tyrosine kinase signaling cascade activation. The underlying mechanisms that govern p52Shc phosphorylation status are unknown. In this study, p52Shc phosphorylation by human c-Src was investigated using purified proteins to define mechanisms that affect the p52Shc phosphorylation state. We conducted biophysical characterizations of both human p52Shc and human c-Src in solution as well as membrane-mimetic environments using the acidic lipid phosphatidylinositol 4-phosphate or a novel amphipathic detergent (2,2-dihexylpropane-1,3-bis-?-d-glucopyranoside). We then identified p52Shc phosphorylation sites under various solution conditions, and the amount of phosphorylation at each identified site was quantified using mass spectrometry. These data demonstrate that the p52Shc phosphorylation level is altered by the solution environment without affecting the fraction of active c-Src. Mass spectrometry analysis of phosphorylated p52Shc implies functional linkage among phosphorylation sites. This linkage may drive preferential coupling to protein binding partners during signaling complex formation, such as during initial binding interactions with the Grb2 adaptor protein leading to activation of the Ras/MAPK signaling cascade. Remarkably, tyrosine residues involved in Grb2 binding were heavily phosphorylated in a membrane-mimetic environment. The increased phosphorylation level in Grb2 binding residues was also correlated with a decrease in the thermal stability of purified human p52Shc. A schematic for the phosphorylation-dependent interaction between p52Shc and Grb2 is proposed. The results of this study suggest another possible therapeutic strategy for altering protein phosphorylation to regulate signaling cascade activation. PMID:25961473

  13. Functional analysis of discoidin domain receptor 1: effect of adhesion on DDR1 phosphorylation.

    PubMed

    L'hôte, Corine G M; Thomas, Peter H; Ganesan, Trivadi S

    2002-02-01

    Discoidin domain receptor 1 (DDR1), a receptor tyrosine kinase (RTK), has been shown to be activated mainly by soluble fibrillar collagen. Unusually, the kinetics of phosphorylation of the receptor is slow, with maximal phosphorylation observed after 90 min. To understand the reasons for slow phosphorylation of the receptor, we examined several cell lines under different conditions. We confirm that endogenous DDR1 is phosphorylated slowly by collagen in adherent T47D and HCT116 cells. In detached and resuspended cells, collagen induced rapid phosphorylation of DDR1. This was further confirmed with a semiadherent cell line (COLO201) and one that grows as a suspension (K562), both of which express endogenous DDR1. Replating K562 on fibronectin to mimic adherent conditions altered the kinetics of phosphorylation from rapid to slow, similar to those of adherent cells. The slow kinetics of phosphorylation in the adherent state was probably not due to cell-cell contacts because EDTA had no major effect. However, pervanadate in the absence of collagen was able to induce strong DDR1 phosphorylation, indicating that a phosphatase may inhibit or delay the phosphorylation of DDR1. Further, downstream signals after phosphorylation of DDR1 by collagen were not transmitted through the classical mitogen-activated protein kinase pathway. In addition, a chimeric TrkA-DDR1 receptor failed to become phosphorylated on stimulation with nerve growth factor (NGF), although it dimerized normally. This is the first RTK whose kinetics of phosphorylation is dependent on cellular context. The interaction of the cells with the matrix, rather than cell-cell contact, is probably responsible for the inhibition of phosphorylation. PMID:11772944

  14. Identification of Novel in Vivo Phosphorylation Sites of the Human Proapoptotic Protein BAD

    PubMed Central

    Polzien, Lisa; Baljuls, Angela; Rennefahrt, Ulrike E. E.; Fischer, Andreas; Schmitz, Werner; Zahedi, Rene P.; Sickmann, Albert; Metz, Renate; Albert, Stefan; Benz, Roland; Hekman, Mirko; Rapp, Ulf R.

    2009-01-01

    BAD is a proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD, little data are available with respect to phosphorylation of human BAD protein. Using mass spectrometry, we identified here besides the established phosphorylation sites at serines 75, 99, and 118 several novel in vivo phosphorylation sites within human BAD (serines 25, 32/34, 97, and 124). Furthermore, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating serine residues 75, 99, and 118. Our results indicate that RAF kinases represent, besides protein kinase A, PAK, and Akt/protein kinase B, in vivo BAD-phosphorylating kinases. RAF-induced phosphorylation of BAD was reduced to control levels using the RAF inhibitor BAY 43-9006. This phosphorylation was not prevented by MEK inhibitors. Consistently, expression of constitutively active RAF suppressed apoptosis induced by BAD and the inhibition of colony formation caused by BAD could be prevented by RAF. In addition, using the surface plasmon resonance technique, we analyzed the direct consequences of BAD phosphorylation by RAF with respect to association with 14-3-3 and Bcl-2/Bcl-XL proteins. Phosphorylation of BAD by active RAF promotes 14-3-3 protein association, in which the phosphoserine 99 represented the major binding site. Finally, we show here that BAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation. PMID:19667065

  15. Fronts and pulses in an enzymatic reaction catalyzed by glucose oxidase

    E-print Network

    Epstein, Irving R.

    Fronts and pulses in an enzymatic reaction catalyzed by glucose oxidase David G. Míguez* , Vladimir as catalysts and regulators. We present a reaction­diffusion system catalyzed by the enzyme glucose oxidase previously studied the temporal dynamics of the enzymatic autocatalytic reaction between glucose and ferricya

  16. Comparative study of the enzymatic hydrolysis of acid-pretreated white pine and mixed hardwood

    Microsoft Academic Search

    H. E. Grethlein; D. C. Allen; A. O. Converse

    1984-01-01

    Removal of hemicellulose by acid pretreatment in a flow reactor followed by enzymatic hydrolysis of the neutralized slurry has resulted in glucose yields as high as 95% for mixed hardwood. For white pine, however, the maximum glucose yield is 65%. Although pine has a higher extractives content, removal of the extractives prior to enzymatic hydrolysis does not increase the glucose

  17. Analytica Chimica Acta 505 (2004) 183187 Microchip enzymatic assay of organophosphate nerve agents

    E-print Network

    Chen, Wilfred

    2004-01-01

    Analytica Chimica Acta 505 (2004) 183­187 Microchip enzymatic assay of organophosphate nerve agents Abstract An on-chip enzymatic assay for screening organophosphate (OP) nerve agents, based on a pre and nerve agents. The study demonstrates also for the first time the suitability of the contactless

  18. Enzymatic variability in the cattail ( Typha angustifolia and T. latifolia, Typhaceae )

    Microsoft Academic Search

    Marie-Andrée Esnault; André Huon

    1985-01-01

    The enzymatic spectra ofTypha angustifolia, T. latifolia and their hybrids were examined by disc electrophoresis for three enzymatic systems: esterases, alcohol deshydrogenase, and glutamate deshydrogenase. The intermediate position of the hybrids is confirmed. The study of alcohol deshydrogenase reveales the existence of an intraspecific variability inT. angustifolia between individuals from the Massif Armoricain in France and Dellys in Algeria.

  19. Disk-Shaped Amperometric Enzymatic Biosensor for in Vivo Detection of Dserine

    E-print Network

    Ruthazer, Edward

    Disk-Shaped Amperometric Enzymatic Biosensor for in Vivo Detection of Dserine David Polcari,, Annie. We report herein the develop- ment of a disk-shaped amperometric enzymatic biosensor for detection-serine biosensor is 1-5 orders of magnitude smaller than previously reported probes and exhibits a sensitivity

  20. Enzymatic hydrolysis and glucose fermentation of wet oxidized sugarcane bagasse and rice straw for bioethanol production

    Microsoft Academic Search

    Alkaline wet oxidation was used as pretreatment method of sugarcane bagasse (SB) and rice straw (RS) prior to enzymatic hydrolysis and glucose fermentations with Saccharomyces cerevisiae. At high enzyme loadings, the enzymatic hydrolysis of wet oxidized sugarcane bagasse (SBWO) resulted in the highest degree of saccharification compared to wet oxidized rice straw (RSWO). However, at enzyme concentrations below 10 FPU\\/g-cellulose,