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Sample records for enzymatic phosphoryl transfer

  1. Arginine Coordination in Enzymatic Phosphoryl Transfer: Evaluation of the Effect of Arg166 Mutations in Escherichia coli Alkaline Phosphatase,

    E-print Network

    Herschlag, Dan

    Arginine Coordination in Enzymatic Phosphoryl Transfer: Evaluation of the Effect of Arg166Vised Manuscript ReceiVed May 19, 2008 ABSTRACT: Arginine residues are commonly found in the active sites the role of arginine residues in the phosphoryl transfer reaction, we have measured the consequences

  2. Theoretical Investigation of the Enzymatic Phosphoryl Transfer of ?-phosphoglucomutase: Revisiting Both Steps of the Catalytic Cycle

    SciTech Connect

    Elsasser, Brigitta M.; Dohmeier-Fischer, Silvia; Fels, Gregor

    2012-07-12

    Enzyme catalyzed phosphate transfer is a part of almost all metabolic processes. Such reactions are of central importance for the energy balance in all organisms and play important roles in cellular control at all levels. Mutases transfer a phosphoryl group while nucleases cleave the phosphodiester linkages between two nucleotides. The subject of our present study is the Lactococcus lactis ?-phosphoglucomutase (?-PGM), which effectively catalyzes the interconversion of ?-D-glucose-1-phosphate (?-G1P) to ?- D-glucose-6-phosphate (?-G6P) and vice versa via stabile intermediate ?-D-glucose-1,6-(bis)phosphate (?-G1,6diP) in the presence of Mg2+. In this paper we revisited the reaction mechanism of the phosphoryl transfer starting from the bisphosphate ?-G1,6diP in both directions (toward ?-G1P and ?-G6P) combining docking techniques and QM/MM theoretical method at the DFT/PBE0 level of theory. In addition we performed NEB (nudged elastic band) and free energy calculations to optimize the path and to identify the transition states and the energies involved in the catalytic cycle. Our calculations reveal that both steps proceed via dissociative pentacoordinated phosphorane, which is not a stabile intermediate but rather a transition state. In addition to the Mg2+ ion, Ser114 and Lys145 also play important roles in stabilizing the large negative charge on the phosphate through strong coordination with the phosphate oxygens and guiding the phosphate group throughout the catalytic process. The calculated energy barrier of the reaction for the ?-G1P to ?-G1,6diP step is only slightly higher than for the ?-G1,6diP to ?-G6P step (16.10 kcal mol-1 versus 15.10 kcal mol-1) and is in excellent agreement with experimental findings (14.65 kcal mol-1).

  3. Arginine Coordination in Enzymatic Phosphoryl Transfer: Evaluation of the Effect of Arg166 Mutations in Escherichia Coli Alkaline Phosphatase

    SciTech Connect

    O'Brien, P.J.; Lassila, J.K.; Fenn, T.D.; Zalatan, J.G.; Herschlag, D.

    2009-05-22

    Arginine residues are commonly found in the active sites of enzymes catalyzing phosphoryl transfer reactions. Numerous site-directed mutagenesis experiments establish the importance of these residues for efficient catalysis, but their role in catalysis is not clear. To examine the role of arginine residues in the phosphoryl transfer reaction, we have measured the consequences of mutations to arginine 166 in Escherichia coli alkaline phosphatase on hydrolysis of ethyl phosphate, on individual reaction steps in the hydrolysis of the covalent enzyme-phosphoryl intermediate, and on thio substitution effects. The results show that the role of the arginine side chain extends beyond its positive charge, as the Arg166Lys mutant is as compromised in activity as Arg166Ser. Through measurement of individual reaction steps, we construct a free energy profile for the hydrolysis of the enzyme-phosphate intermediate. This analysis indicates that the arginine side chain strengthens binding by {approx}3 kcal/mol and provides an additional 1-2 kcal/mol stabilization of the chemical transition state. A 2.1 {angstrom} X-ray diffraction structure of Arg166Ser AP is presented, which shows little difference in enzyme structure compared to the wild-type enzyme but shows a significant reorientation of the bound phosphate. Altogether, these results support a model in which the arginine contributes to catalysis through binding interactions and through additional transition state stabilization that may arise from complementarity of the guanidinum group to the geometry of the trigonal bipyramidal transition state.

  4. Enzymatic Catalysis of Proton Transfer at Carbon

    PubMed Central

    Amyes, Tina L.; Richard, John P.

    2008-01-01

    More than 80% of the rate acceleration for enzymatic catalysis of the aldose-ketose isomerization of (R)-glyceraldehyde 3-phosphate (GAP) by triosephosphate isomerase (TIM) can be attributed to the 3?-phosphodianion group of GAP. We examine here the necessity of the covalent connection between the phosphodianion and triose sugar portions of the substrate by “carving up” GAP into the minimal neutral two-carbon sugar glycolaldehyde and phosphite dianion pieces. This “two-part substrate” preserves both the ?-hydroxycarbonyl and oxydianion portions of GAP. TIM catalyzes proton transfer from glycolaldehyde in D2O, resulting in deuterium incorporation that can be monitored by 1H NMR spectroscopy, with kcat/Km = 0.26 M-1 s-1. Exogenous phosphite dianion results in a very large increase in the observed second-order rate constant (kcat/Km)obsd for turnover of glycolaldehyde, and the dependence of (kcat/Km)obsd on [HPO32-] exhibits saturation. The data give kcat/Km = 185 M-1s-1 for turnover of glycolaldehyde by TIM that is saturated with phosphite dianion, so that the separate binding of phosphite dianion to TIM results in a 700-fold acceleration of proton transfer from carbon. The binding of phosphite dianion to the free enzyme (Kd = 38 mM) is 700-fold weaker than its binding to the fleeting complex of TIM with the altered substrate in the transition state (Kd‡ = 53 ?M); the total intrinsic binding energy of phosphite dianion in the transition state is 5.8 kcal/mol. We propose a physical model for catalysis by TIM in which the intrinsic binding energy of the substrate phosphodianion group is utilized to drive closing of the “mobile loop” and a protein conformational change that leads to formation of an active site environment that is optimally organized for stabilization of the transition state for proton transfer from ?-carbonyl carbon. PMID:17444661

  5. Systematic development of an enzymatic phosphorylation assay compatible with mass spectrometric detection.

    PubMed

    de Boer, A R; Letzel, T; Lingeman, H; Irth, H

    2005-02-01

    The enzymatic peptide phosphorylation by cAMP-dependent protein kinase A (PKA) was optimized and monitored by means of electrospray ionization mass spectrometry (ESI-MS). The direct detection of phosphorylated peptides by MS renders labeling unnecessary, reduces time and labor, due to less initial sample pretreatment. In this study the phosphorylation of the peptide malantide by PKA was performed in batch and reaction compounds were detected by ESI-MS after the incubation time. The subsequent product quantitation was accomplished by using one-point normalization. Applying this set-up, optimum solvent conditions (such as salt and modifier content), concentrations of essential reaction compounds (such as cAMP, Mg2+ and ATP), and the influence of reaction properties (such as pH and reaction time) were determined. The reaction milieu has to be suitable for both, the enzymatic reaction and the mass spectrometric detection. We found that the modifier content and the pH value had to be changed after the enzymatic reaction occurred. Through the addition of methanol and acetic acid, the reaction stopped immediately and a more sensitive mass spectrometric detection could be obtained simultaneously. Furthermore, an inhibitor study was performed, testing the inhibition potency of three protein kinase A inhibitors (PKIs). IC50 values were determined and used to calculate the Ki values, that were 7.4, 19.0 and 340.0 nmol/L for PKI(6-22)amide, PKI(5-24)amide, and PKI(14-24)amide, respectively. These data vary between factor 4.4 (for PKI(6-22)amide) and 8.3 (for PKI(5-24)amide) compared to the Ki values described in literature. However, the Ki values are in good agreement with the data mainly obtained by fluorescence- or radioactivity-based methods. Nevertheless, our results indicate that ESI-MS is a realistic alternative to radioactivity and fluorescence detection in determining enzymatic activity. Furthermore we were able to illustrate its high potential as a quantitative detection method. PMID:15703914

  6. Phosphoryl Transfer from ?-d-Glucose 1-Phosphate Catalyzed by Escherichia coli Sugar-Phosphate Phosphatases of Two Protein Superfamily Types

    PubMed Central

    Wildberger, Patricia; Pfeiffer, Martin; Brecker, Lothar; Rechberger, Gerald N.; Birner-Gruenberger, Ruth

    2014-01-01

    The Cori ester ?-d-glucose 1-phosphate (?Glc 1-P) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes ?Glc 1-P for flexible exploitation in glucosyl and phosphoryl transfer reactions. Two structurally and mechanistically distinct sugar-phosphate phosphatases from Escherichia coli were characterized in this study for utilization of ?Glc 1-P as a phosphoryl donor substrate. The agp gene encodes a periplasmic ?Glc 1-P phosphatase (Agp) belonging to the histidine acid phosphatase family. Had13 is from the haloacid dehydrogenase-like phosphatase family. Cytoplasmic expression of Agp (in E. coli Origami B) gave a functional enzyme preparation (kcat for phosphoryl transfer from ?Glc 1-P to water, 40 s?1) that was shown by mass spectrometry to exhibit no free cysteines and the native intramolecular disulfide bond between Cys189 and Cys195. Enzymatic phosphoryl transfer from ?Glc 1-P to water in H218O solvent proceeded with complete 18O label incorporation into the phosphate released, consistent with catalytic reaction through O-1–P, but not C-1–O, bond cleavage. Hydrolase activity of both enzymes was not restricted to a glycosidic phosphomonoester substrate, and d-glucose 6-phosphate was converted with a kcat similar to that of ?Glc 1-P. By examining phosphoryl transfer from ?Glc 1-P to an acceptor substrate other than water (d-fructose or d-glucose), we discovered that Agp exhibited pronounced synthetic activity, unlike Had13, which utilized ?Glc 1-P mainly for phosphoryl transfer to water. By applying d-fructose in 10-fold molar excess over ?Glc 1-P (20 mM), enzymatic conversion furnished d-fructose 1-phosphate as the main product in a 55% overall yield. Agp is a promising biocatalyst for use in transphosphorylation from ?Glc 1-P. PMID:25527541

  7. Phosphoryl transfer reaction catalyzed by membrane diacylglycerol kinase: a theoretical mechanism study.

    PubMed

    Jiang, Yafei; Tan, Hongwei; Zheng, Jimin; Li, Xichen; Chen, Guangju; Jia, Zongchao

    2015-10-14

    Diacylglycerol kinase is an integral membrane protein which catalyzes phosphoryl transfer from ATP to diacylglycerol. As the smallest kinase known, it shares no sequence homology with conventional kinases and possesses a distinct trimer structure. Thus far, its catalytic mechanism remains elusive. Using molecular dynamics and quantum mechanics calculations, we investigated the co-factor and the substrate binding and phosphoryl transfer mechanism. Based on the analysis of density functional theory calculations, we reveal that the phosphorylation reaction of diacylglycerol kinase features the same phosphoryl transfer mechanism as other kinases, despite its unique structural properties. Our results further show that the active site is relatively open and able to accommodate ligands in multiple orientations, suggesting that the optimization of binding orientations and conformational changes would occur prior to actual phosphoryl transfer. PMID:26352441

  8. Distinct tyrosine phosphorylation sites in JAK3 kinase domain positively and negatively regulate its enzymatic?activity

    PubMed Central

    Zhou, Yong-Jie; Hanson, Eric P.; Chen, Yi-Qing; Magnuson, Kelly; Chen, Min; Swann, Patrick G.; Wange, Ronald L.; Changelian, Paul S.; O’Shea, John J.

    1997-01-01

    Cytokines are critically important for the growth and development of a variety of cells. Janus kinases (JAKs) associate with cytokine receptors and are essential for transmitting downstream cytokine signals. However, the regulation of the enzymatic activity of the JAKs is not well understood. Here, we investigated the role of tyrosine phosphorylation of JAK3 in regulating its kinase activity by analyzing mutations of tyrosine residues within the putative activation loop of the kinase domain. Specifically, tyrosine residues 980 and 981 of JAK3 were mutated to phenylalanine individually or doubly. We found that JAK3 is autophosphorylated on multiple sites including Y980 and Y981. Compared with the activity of wild-type (WT) JAK3, mutant Y980F demonstrated markedly decreased kinase activity, and optimal phosphorylation of JAK3 on other sites was dependent on Y980 phosphorylation. The mutant Y980F also exhibited reduced phosphorylation of its substrates, ?c and STAT5A. In contrast, mutant Y981F had greatly increased kinase activity, whereas the double mutant, YY980/981FF, had intermediate activity. These results indicate that Y980 positively regulates JAK3 kinase activity whereas Y981 negatively regulates JAK3 kinase activity. These observations in JAK3 are similar to the findings in the kinase that is closely related to the JAK family, ZAP-70; mutations of tyrosine residues within the putative activation loop of ZAP-70 also have opposing actions. Thus, it will be important to determine whether this feature of regulation is unique to JAK3 or if it is also a feature of other JAKs. Given the importance of JAKs and particularly JAK3, it will be critical to fully dissect the positive and negative regulatory function of these and other tyrosine residues in the control of kinase activity and hence cytokine signaling. PMID:9391116

  9. Solution Structure of the Phosphoryl Transfer Complex between the Signal-transducing Protein IIAGlucose

    E-print Network

    Clore, G. Marius

    . The proteins downstream from HPr are sugar- specific, comprising four distinct families of IIA permeases (2Solution Structure of the Phosphoryl Transfer Complex between the Signal-transducing Protein are supplemented by pe- ripheral electrostatic interactions involving an alter- nating distribution of positively

  10. Phosphoryl Transfer Reaction Snapshots in Crystals: INSIGHTS INTO THE MECHANISM OF PROTEIN KINASE A CATALYTIC SUBUNIT.

    PubMed

    Gerlits, Oksana; Tian, Jianhui; Das, Amit; Langan, Paul; Heller, William T; Kovalevsky, Andrey

    2015-06-19

    To study the catalytic mechanism of phosphorylation catalyzed by cAMP-dependent protein kinase (PKA) a structure of the enzyme-substrate complex representing the Michaelis complex is of specific interest as it can shed light on the structure of the transition state. However, all previous crystal structures of the Michaelis complex mimics of the PKA catalytic subunit (PKAc) were obtained with either peptide inhibitors or ATP analogs. Here we utilized Ca(2+) ions and sulfur in place of the nucleophilic oxygen in a 20-residue pseudo-substrate peptide (CP20) and ATP to produce a close mimic of the Michaelis complex. In the ternary reactant complex, the thiol group of Cys-21 of the peptide is facing Asp-166 and the sulfur atom is positioned for an in-line phosphoryl transfer. Replacement of Ca(2+) cations with Mg(2+) ions resulted in a complex with trapped products of ATP hydrolysis: phosphate ion and ADP. The present structural results in combination with the previously reported structures of the transition state mimic and phosphorylated product complexes complete the snapshots of the phosphoryl transfer reaction by PKAc, providing us with the most thorough picture of the catalytic mechanism to date. PMID:25925954

  11. 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.

  12. 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.

  13. Enzymatic cellulose oxidation is linked to lignin by long-range electron transfer

    PubMed Central

    Westereng, Bjørge; Cannella, David; Wittrup Agger, Jane; Jørgensen, Henning; Larsen Andersen, Mogens; Eijsink, Vincent G.H.; Felby, Claus

    2015-01-01

    Enzymatic oxidation of cell wall polysaccharides by lytic polysaccharide monooxygenases (LPMOs) plays a pivotal role in the degradation of plant biomass. While experiments have shown that LPMOs are copper dependent enzymes requiring an electron donor, the mechanism and origin of the electron supply in biological systems are only partly understood. We show here that insoluble high molecular weight lignin functions as a reservoir of electrons facilitating LPMO activity. The electrons are donated to the enzyme by long-range electron transfer involving soluble low molecular weight lignins present in plant cell walls. Electron transfer was confirmed by electron paramagnetic resonance spectroscopy showing that LPMO activity on cellulose changes the level of unpaired electrons in the lignin. The discovery of a long-range electron transfer mechanism links the biodegradation of cellulose and lignin and sheds new light on how oxidative enzymes present in plant degraders may act in concert. PMID:26686263

  14. Enzymatic cellulose oxidation is linked to lignin by long-range electron transfer.

    PubMed

    Westereng, Bjørge; Cannella, David; Wittrup Agger, Jane; Jørgensen, Henning; Larsen Andersen, Mogens; Eijsink, Vincent G H; Felby, Claus

    2015-01-01

    Enzymatic oxidation of cell wall polysaccharides by lytic polysaccharide monooxygenases (LPMOs) plays a pivotal role in the degradation of plant biomass. While experiments have shown that LPMOs are copper dependent enzymes requiring an electron donor, the mechanism and origin of the electron supply in biological systems are only partly understood. We show here that insoluble high molecular weight lignin functions as a reservoir of electrons facilitating LPMO activity. The electrons are donated to the enzyme by long-range electron transfer involving soluble low molecular weight lignins present in plant cell walls. Electron transfer was confirmed by electron paramagnetic resonance spectroscopy showing that LPMO activity on cellulose changes the level of unpaired electrons in the lignin. The discovery of a long-range electron transfer mechanism links the biodegradation of cellulose and lignin and sheds new light on how oxidative enzymes present in plant degraders may act in concert. PMID:26686263

  15. Computational study of the phosphoryl transfer catalyzed by a cyclin-dependent kinase.

    PubMed

    De Vivo, Marco; Cavalli, Andrea; Carloni, Paolo; Recanatini, Maurizio

    2007-01-01

    A cyclin-dependent kinase, Cdk2, catalyzes the transfer of the gamma-phosphate from ATP to a threonine or serine residue of its polypeptide substrates. Here, we investigate aspects of the reaction mechanism of Cdk2 by gas-phase density functional calculations, classical molecular dynamics, and Car-Parrinello QM/MM simulations. We focus on the role of the conserved Asp127 and on the nature of the phosphoryl transfer reaction mechanism catalyzed by Cdk2. Our findings suggest that Asp127 is active in its deprotonated form by assisting the formation of the near-attack orientation of the substrate serine or threonine. Therefore, the residue does not act as a general base during the catalysis. The mechanism for the phosphoryl transfer is a single SN2-like concerted step, which shows a phosphorane-like transition state geometry. Although the resulting reaction mechanism is in agreement with a previous density functional study of the same catalytic reaction mechanism (Cavalli et al., Chem. Comm. 2003, 1308-1309), the reaction barrier is considerably lower when QM/MM calculations are performed, as in this study ( approximately 42 kcal mol(-1) QM vs. approximately 24 kcal mol(-1) QM/MM); this indicates that important roles for the catalysis are played by the protein environment and solvent waters. Because of the high amino acid sequence conservation among the whole family of cyclin-dependent kinases (CDKs), these results could be general for the CDK family. PMID:17636466

  16. Non-enzymatic synthesis of the coenzymes, uridine diphosphate glucose and cytidine diphosphate choline, and other phosphorylated metabolic intermediates

    NASA Technical Reports Server (NTRS)

    Mar, A.; Dworkin, J.; Oro, J.

    1987-01-01

    Using urea and cyanamide, the two condensing agents considered to have been present on the primitive earth, uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P), and glucose-6-phosphate (G6P) were synthesized under simulated prebiotic conditions. The reaction products were separated and identified using paper chromatography, thin layer chromatography, enzymatic analyses, and ion-pair reverse-phase high performance liquid chromatography. The possibility of nonenzymatic synthesis of metabolic intermediates on the primitive earth from simple precursors was thus demonstrated.

  17. Rates and Routes of Electron Transfer of [NiFe]-Hydrogenase in an Enzymatic Fuel Cell.

    PubMed

    Petrenko, Alexander; Stein, Matthias

    2015-10-29

    Hydrogenase enzymes are being used in enzymatic fuel cells immobilized on a graphite or carbon electrode surface, for example. The enzyme is used for the anodic oxidation of molecular hydrogen (H2) to produce protons and electrons. The association and orientation of the enzyme at the anode electrode for a direct electron transfer is not completely resolved. The distal FeS-cluster in [NiFe]-hydrogenases contains a histidine residue which is known to play a critical role in the intermolecular electron transfer between the enzyme and the electrode surface. The [NiFe]-hydrogenase graphite electrode association was investigated using Brownian Dynamics simulations. Residues that were shown to be in proximity to the electrode surface were identified (His184, Ser196, Glu461, Glu464), and electron transfer routes connecting the distal FeS-cluster with the surface residues were investigated. Several possible pathways for electron transfer between the distal FeS-cluster and the terminal amino acid residues were probed in terms of their rates of electron transfer using DFT methods. The reorganization energies ? of the distal iron-sulfur cluster and coronene as a molecular model for graphite were calculated. The reorganization energy of the distal (His)(Cys)3 cluster was found to be not very different from that of a standard cubane clusters with a (Cys)4 coordination. Electronic coupling matrix elements and rates of electron transfer for the different pathways were calculated according to the Marcus equation. The rates for glutamate-mediated electrode binding were found to be incompatible with experimental data. A direct electron transfer from the histidine ligand of the distal FeS-cluster to the electrode yielded rates of electron transfer in excellent agreement with experiment. A second pathway, however, from the distal FeS-cluster to the Ser196 residue was found to be equally efficient and feasible. PMID:26218232

  18. A computational study of the phosphoryl transfer reaction between ATP and Dha in aqueous solution.

    PubMed

    Bordes, I; Ruiz-Pernía, J J; Castillo, R; Moliner, V

    2015-10-28

    Phosphoryl transfer reactions are ubiquitous in biology, being involved in processes ranging from energy and signal transduction to the replication genetic material. Dihydroxyacetone phosphate (Dha-P), an intermediate of the synthesis of pyruvate and a very important building block in nature, can be generated by converting free dihydroxyacetone (Dha) through the action of the dihydroxyacetone kinase enzyme. In this paper the reference uncatalyzed reaction in solution has been studied in order to define the foundations of the chemical reaction and to determine the most adequate computational method to describe this electronically complex reaction. In particular, the phosphorylation reaction mechanism between adenosine triphosphate (ATP) and Dha in aqueous solution has been studied by means of quantum mechanics/molecular mechanics (QM/MM) Molecular Dynamics (MD) simulations with the QM subset of atoms described with semi-empirical and DFT methods. The results appear to be strongly dependent on the level of calculation, which will have to be taken into account for future studies of the reaction catalyzed by enzymes. In particular, PM3/MM renders lower free energy barriers and a less endergonic process than AM1d/MM and PM6/MM methods. Nevertheless, the concerted pathway was not located with the former combination of potentials. PMID:26303076

  19. Copper-containing monooxygenases: enzymatic and biomimetic studies of the O-atom transfer catalysis.

    PubMed

    Blain, Ingrid; Slama, Patrick; Giorgi, Michel; Tron, Thierry; Réglier, Marius

    2002-04-01

    This review reports our recent studies or the mechanism of O-atom transfer to a benzylic C-H bond promoted by Dopamine beta-Hydroxylase (DBH) and its biomimetic models. We demonstrate that it is possible to carry out parallel and comparative studies on this enzyme (DBH) and its biomimetic models with the same substrate: 2-aminoindane (3). It was chosen because its two stereogenic centers, both in benzylic positions, make it very powerful for studying the stereochemistry of an O-atom transfer reaction. DBH-catalyzed hydroxylation of 3 produced exclusively 14% of trans-(1S,2S)-2-amino-1-indanol (4) (93% ee). Studies with stereospecifically deuterium-labeled 2-aminoindanes (1R,2S)-3b and (1S,2S)-3a showed that the formation of 4 was the rcsult of an overall process with retention of configuration where an O-atom is stereospecifically inserted in the trans pro-S position of the substrate. With copper(I) and (II) complexes of IndPY2 ligands we studied the reaction with dioxygen and observed an O-atom transfer to a benzylic C-H bond which was performed in the same manner as that of DBH. With the deuterium-labeled cis-2-d-IndPY2 ligand, we demonstrated that the reaction occurs by a stereospecific process with retention of configuration. In both cases (enzymatic vs. biomimetic) the O-atom transfers occur in a two-step process involving radical intermediates. PMID:12069196

  20. Dynamic and steady state 1-D model of mediated electron transfer in a porous enzymatic electrode.

    PubMed

    Do, T Q N; Varni?i?, M; Flassig, R J; Vidakovi?-Koch, T; Sundmacher, K

    2015-12-01

    A 1-D mathematical model of a porous enzymatic electrode exhibiting the mediated electron transfer (MET) mechanism has been developed. As a model system, glucose oxidation catalyzed by immobilized glucose oxidase (GOx) in the presence of a co-immobilized tetrathiafulvalene (TTF) mediator in the porous electrode matrix has been selected. The balance equations for potential fields in the electron- and ion-conducting phases as well as concentration field have been formulated, solved numerically and validated experimentally under steady state conditions. The relevant kinetic parameters of the lumped reaction kinetics have been obtained by global optimization. The confidence intervals (CIs) of each parameter have been extracted from the respective likelihood. The parameter study has shown that the parameters related to mediator consumption/regeneration steps can be responsible for the shift of the reaction onset potential. Additionally, the model has shown that diffusion of the oxidized mediator out of the catalyst layer (CL) plays a significant role only at more positive potentials and low glucose concentrations. Only concentration profiles in different layers influence the electrode performance while other state fields like potential distributions in different phases have no impact on the performance. The concentration profiles reveal that all electrodes work through; the observed limiting currents are diffusion-reaction limiting. The normalized electrode activity decreases with an increase of enzyme loading. According to the model, the reason for this observation is glucose depletion along the CL at higher enzyme loadings. Comparison with experiments advices a decrease of enzyme utilization at higher enzyme loadings. PMID:26257008

  1. 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.

  2. 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.

  3. The guanidinium unit in the catalysis of phosphoryl transfer reactions: from molecular spacers to nanostructured supports.

    PubMed

    Salvio, Riccardo

    2015-07-27

    Examples of guanidinium-based artificial phosphodiesterases are illustrated in this review article. A wide set of collected catalytic systems are presented, from the early examples to the most recent developments of the use of this unit in the design of supramolecular catalysts. Special attention is dedicated to illustrate the operating catalytic mechanism and the role of guanidine/ium units in the catalysis. One or more of these units can act by themselves or in conjunction with other active units. The analogy with the mechanism of enzymatic systems is presented and discussed. In the last part of this overview, recent examples of guanidinophosphodiesterases based on nanostructured supports are reported, namely gold-monolayer-protected clusters and polymer brushes grafted to silica nanoparticles. The issue of the dependence of the catalytic performance on the preorganization of the spacer is tackled and discussed in terms of effective molarity, a parameter that can be taken as a quantitative measurement of this preorganization for both conventional molecular linker and nanosized supports. PMID:25940903

  4. Ground State Destabilization by Anionic Nucleophiles Contributes to the Activity of Phosphoryl Transfer

    E-print Network

    Herschlag, Dan

    and Systems Biology, Stanford University, Stanford, California, United States of America, 2 Department utilizing interactions with only the five atoms attached to the transferred phosphorus. We tested a model substrate. Removal of the Ser102 alkoxide by mutation to glycine or alanine increases the observed Pi

  5. Enzymatic catalysis and transfers in solution. I. Theory and computations, a unified view

    NASA Astrophysics Data System (ADS)

    Marcus, R. A.

    2006-11-01

    The transfer of hydride, proton, or H atom between substrate and cofactor in enzymes has been extensively studied for many systems, both experimentally and computationally. A simple equation for the reaction rate, an analog of an equation obtained earlier for electron transfer rates, is obtained, but now containing an approximate analytic expression for the bond rupture-bond forming feature of these H transfers. A "symmetrization," of the potential energy surfaces is again introduced [R. A. Marcus, J. Chem. Phys. 43, 679 (1965); J. Phys. Chem. 72, 891 (1968)], together with Gaussian fluctuations of the remaining coordinates of the enzyme and solution needed for reaching the transition state. Combining the two expressions for the changes in the difference of the two bond lengths of the substrate-cofactor subsystem and in the fluctuation coordinates of the protein leading to the transition state, an expression is obtained for the free energy barrier. To this end a two-dimensional reaction space (m,n) is used that contains the relative coordinates of the H in the reactants, the heavy atoms to which it is bonded, and the protein/solution reorganization coordinate, all leading to the transition state. The resulting expression may serve to characterize in terms of specific parameters (two "reorganization" terms, thermodynamics, and work terms), experimental and computational data for different enzymes, and different cofactor-substrate systems. A related characterization was used for electron transfers. To isolate these factors from nuclear tunneling, when the H-tunneling effect is large, use of deuterium and tritium transfers is of course helpful, although tunneling has frequently and understandably dominated the discussions. A functional form is suggested for the dependence of the deuterium kinetic isotope effect (KIE) on ?G ° and a different form for the C13 KIE. Pressure effects on deuterium and C13 KIEs are also discussed. Although formulated for a one-step transfer of a light particle in an enzyme, the results would also apply to single-step transfers of other atoms and groups in enzymes and in solution.

  6. Enzymatic-induced upconversion photoinduced electron transfer for sensing tyrosine in human serum.

    PubMed

    Wu, Qiongqiong; Fang, Aijin; Li, Haitao; Zhang, Youyu; Yao, Shouzhuo

    2016-03-15

    This paper reports a novel nanosensor for tyrosine based on photoinduced electron-transfer (PET) between NaYF4:Yb, Tm upconversion nanoparticles (UCNPs) and melanin-like polymers. Melanin-like films were obtained from catalytic oxidation of tyrosine by tyrosinase, and deposited on the surface of UCNPs, and then quenched the fluorescence of UCNPs. Under the optimized conditions, the fluorescence quenching of UCNPs showed a good linear response to tyrosine concentration in the range of 0.8-100?? with a detection limit of 1.1??. Meanwhile, it showed good sensitivity, stability and has been successfully applied to the detection of tyrosine in human serum. PMID:26544870

  7. Photosystem II Core Phosphorylation Heterogeneity, Differential Herbicide Binding, and Regulation of Electron Transfer in Photosystem II Preparations from Spinach 1

    PubMed Central

    Giardi, Maria T.; Rigoni, Fernanda; Barbato, Roberto

    1992-01-01

    The effect of photosystem II core phosphorylation on the secondary quinone acceptor of photosystem II (QB) domain environment was analyzed by comparative herbicide-binding studies with photosystem II preparations from spinach (Spinacia oleracea L.). It was found that phosphorylation reduces the binding affinity for most photosynthetic herbicides. The binding of synthetic quinones and of the electron acceptor 2,6-dichlorophenolindophenol is also reduced by photosystem II phosphorylation. Four photosystem II core populations isolated from membranes showed different extents of phosphorylation as well as different degrees of affinity for photosynthetic herbicides. These findings support the idea that heterogeneity of photosystem II observed in vivo could be, in part, due to phosphorylation. Images Figure 1 PMID:16653222

  8. 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

    SciTech Connect

    Barry, R.J.

    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 and NMR spectral analysis. Incubation of ({sup 32}P)-phosphoenolpyruvate (PEP) with cell-free preparations yielded ({sup 32}P)-labelled material that was chromatographically identical to authentic phosphonopyruvate (p-pry). A reexamination of AEP biosynthesis was initiated. In the second portion of this study the kinetic mechanism of yeast inorganic pyrophosphatase (PPase) was examined by carrying-out initial velocity studies. Ca{sup 2+} and Rh(H{sub 2}O){sub 4} (methylenediphosphonate) (Rh(H{sub 2}O){sub 4}PCP) were used as dead-end inhibitors to study the order of binding of Cr(H{sub 2}O){sub 4}PP to the substrate site and Mg{sup 2+} to the low affinity activator site on the enzyme.

  9. Enzymatic regeneration of adenosine triphosphate cofactor

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1974-01-01

    Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

  10. Vibrational studies of phosphoryl transfer enzymes: ras- p21(*)magnesium-GTP and Myosin S1(*)magnesium-ADP- vanadate

    NASA Astrophysics Data System (ADS)

    Wang, Jianghua

    1999-07-01

    We have measured the Raman spectra of monophosphate compounds in aqueous solution. The measured frequencies were correlated with P••O valence bond order by using a modification of the Hardcastle- Wachs procedure. The P••O bond order and bond length in phosphates can be determined from vibrational spectra by using the derived bond order/stretching frequency correlation and the bond length/bond order correlation of Brown and Wu. The Raman and infrared spectra of guanosine 5'-diphosphate (GDP) and guanosine 5'-triphosphate (GTP) in aqueous solution were also examined. Frequency shifts were observed as Mg2+ complexes with GDP and GTP in aqueous solution. These results suggested that Mg2+ binds to GDP in a bidentate manner to the ?,? P••O bonds and in a tridentate manner to the ?,? and ? P••O bonds of Mg•GTP . We have analyzed the previously obtained isotope edited Raman difference spectra of 1:1 complexes of Mg•GDP and Mg•GTP in ras-p21. Frequency changes of the phosphate groups were observed when Mg•GDP , Mg•GTP bind to the protein. Employing both the previous empirical relationships between bond orders/lengths and frequencies as well as vibrational analysis from ab initio calculations, the spectral changes can be explained by the change of the Mg2+ binding sites and hydrogen-bonding. Implications of these structural results for the reaction mechanism of GTP hydrolysis catalyzed by the GTPase are discussed. We have analyzed previously obtained isotope edited Raman difference spectra of the non-bridging V••O bonds of vanadates, both in solution, and when bound to the myosin S1•MgADP complex. By use of ab initio calculations on a model of the vanadate binding site in myosin, the angles between the non-bridging V••O bonds and between these bonds and the apical bonds in the myosin S1•MgADP -Vi complex were determined. The summed bond order of the two apical bonds between the attacking and leaving group oxygens with the central vanadium ion in the S1•MgADP -Vi complex was found to increase only slightly compared with the bond order of the ester V-O bond of a monoester vanadate model compound in solution, suggesting an SN2 like mechanism for the phosphoryl transfer reaction catalyzed by myosin.

  11. 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; Sakurai, Takeshi

    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.

  12. An enzymatically-sensitized sequential and concentric energy transfer relay self-assembled around semiconductor quantum dots

    NASA Astrophysics Data System (ADS)

    Samanta, Anirban; Walper, Scott A.; Susumu, Kimihiro; Dwyer, Chris L.; Medintz, Igor L.

    2015-04-01

    The ability to control light energy within de novo nanoscale structures and devices will greatly benefit their continuing development and ultimate application. Ideally, this control should extend from generating the light itself to its spatial propagation within the device along with providing defined emission wavelength(s), all in a stand-alone modality. Here we design and characterize macromolecular nanoassemblies consisting of semiconductor quantum dots (QDs), several differentially dye-labeled peptides and the enzyme luciferase which cumulatively demonstrate many of these capabilities by engaging in multiple-sequential energy transfer steps. To create these structures, recombinantly-expressed luciferase and the dye-labeled peptides were appended with a terminal polyhistidine sequence allowing for controlled ratiometric self-assembly around the QDs via metal-affinity coordination. The QDs serve to provide multiple roles in these structures including as central assembly platforms or nanoscaffolds along with acting as a potent energy harvesting and transfer relay. The devices are activated by addition of coelenterazine H substrate which is oxidized by luciferase producing light energy which sensitizes the central 625 nm emitting QD acceptor by bioluminescence resonance energy transfer (BRET). The sensitized QD, in turn, acts as a relay and transfers the energy to a first peptide-labeled Alexa Fluor 647 acceptor dye displayed on its surface. This dye then transfers energy to a second red-shifted peptide-labeled dye acceptor on the QD surface through a second concentric Förster resonance energy transfer (FRET) process. Alexa Fluor 700 and Cy5.5 are both tested in the role of this terminal FRET acceptor. Photophysical analysis of spectral profiles from the resulting sequential BRET-FRET-FRET processes allow us to estimate the efficiency of each of the transfer steps. Importantly, the efficiency of each step within this energy transfer cascade can be controlled to some extent by the number of enzymes/peptides displayed on the QD. Further optimization of the energy transfer process(es) along with potential applications of such devices are finally discussed.The ability to control light energy within de novo nanoscale structures and devices will greatly benefit their continuing development and ultimate application. Ideally, this control should extend from generating the light itself to its spatial propagation within the device along with providing defined emission wavelength(s), all in a stand-alone modality. Here we design and characterize macromolecular nanoassemblies consisting of semiconductor quantum dots (QDs), several differentially dye-labeled peptides and the enzyme luciferase which cumulatively demonstrate many of these capabilities by engaging in multiple-sequential energy transfer steps. To create these structures, recombinantly-expressed luciferase and the dye-labeled peptides were appended with a terminal polyhistidine sequence allowing for controlled ratiometric self-assembly around the QDs via metal-affinity coordination. The QDs serve to provide multiple roles in these structures including as central assembly platforms or nanoscaffolds along with acting as a potent energy harvesting and transfer relay. The devices are activated by addition of coelenterazine H substrate which is oxidized by luciferase producing light energy which sensitizes the central 625 nm emitting QD acceptor by bioluminescence resonance energy transfer (BRET). The sensitized QD, in turn, acts as a relay and transfers the energy to a first peptide-labeled Alexa Fluor 647 acceptor dye displayed on its surface. This dye then transfers energy to a second red-shifted peptide-labeled dye acceptor on the QD surface through a second concentric Förster resonance energy transfer (FRET) process. Alexa Fluor 700 and Cy5.5 are both tested in the role of this terminal FRET acceptor. Photophysical analysis of spectral profiles from the resulting sequential BRET-FRET-FRET processes allow us to estimate the efficiency of each of the transfer steps. Impor

  13. Optimal alignment for enzymatic proton transfer: Structure of the Michaelis complex of triosephosphate isomerase at 1.2-? resolution

    PubMed Central

    Jogl, Gerwald; Rozovsky, Sharon; McDermott, Ann E.; Tong, Liang

    2003-01-01

    In enzyme catalysis, where exquisitely positioned functionality is the sine qua non, atomic coordinates for a Michaelis complex can provide powerful insights into activation of the substrate. We focus here on the initial proton transfer of the isomerization reaction catalyzed by triosephosphate isomerase and present the crystal structure of its Michaelis complex with the substrate dihydroxyacetone phosphate at near-atomic resolution. The active site is highly compact, with unusually short and bifurcated hydrogen bonds for both catalytic Glu-165 and His-95 residues. The carboxylate oxygen of the catalytic base Glu-165 is positioned in an unprecedented close interaction with the ketone and the ?-hydroxy carbons of the substrate (C… O ? 3.0 ?), which is optimal for the proton transfer involving these centers. The electrophile that polarizes the substrate, His-95, has close contacts to the substrate's O1 and O2 (N… O ? 3.0 and 2.6 ?, respectively). The substrate is conformationally relaxed in the Michaelis complex: the phosphate group is out of the plane of the ketone group, and the hydroxy and ketone oxygen atoms are not in the cisoid configuration. The ? ammonium group of the electrophilic Lys-12 is within hydrogen-bonding distance of the substrate's ketone oxygen, the bridging oxygen, and a terminal phosphate's oxygen, suggesting a role for this residue in both catalysis and in controlling the flexibility of active-site loop. PMID:12509510

  14. Structural Characterizations of Glycerol Kinase: Unraveling Phosphorylation-Induced Long-Range Activation

    SciTech Connect

    Yeh, Joanne I.; Kettering, Regina; Saxl, Ruth; Bourand, Alexa; Darbon, Emmanuelle; Joly, Nathalie; Briozzo, Pierre; Deutscher, Josef

    2009-09-11

    Glycerol metabolism provides a central link between sugar and fatty acid catabolism. In most bacteria, glycerol kinase plays a crucial role in regulating channel/facilitator-dependent uptake of glycerol into the cell. In the firmicute Enterococcus casseliflavus, this enzyme's activity is enhanced by phosphorylation of the histidine residue (His232) located in its activation loop, approximately 25 A from its catalytic cleft. We reported earlier that some mutations of His232 altered enzyme activities; we present here the crystal structures of these mutant GlpK enzymes. The structure of a mutant enzyme with enhanced enzymatic activity, His232Arg, reveals that residues at the catalytic cleft are more optimally aligned to bind ATP and mediate phosphoryl transfer. Specifically, the position of Arg18 in His232Arg shifts by approximately 1 A when compared to its position in wild-type (WT), His232Ala, and His232Glu enzymes. This new conformation of Arg18 is more optimally positioned at the presumed gamma-phosphate location of ATP, close to the glycerol substrate. In addition to structural changes exhibited at the active site, the conformational stability of the activation loop is decreased, as reflected by an approximately 35% increase in B factors ('thermal factors') in a mutant enzyme displaying diminished activity, His232Glu. Correlating conformational changes to alteration of enzymatic activities in the mutant enzymes identifies distinct localized regions that can have profound effects on intramolecular signal transduction. Alterations in pairwise interactions across the dimer interface can communicate phosphorylation states over 25 A from the activation loop to the catalytic cleft, positioning Arg18 to form favorable interactions at the beta,gamma-bridging position with ATP. This would offset loss of the hydrogen bonds at the gamma-phosphate of ATP during phosphoryl transfer to glycerol, suggesting that appropriate alignment of the second substrate of glycerol kinase, the ATP molecule, may largely determine the rate of glycerol 3-phosphate production.

  15. Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy.

    PubMed

    Ordureau, Alban; Heo, Jin-Mi; Duda, David M; Paulo, Joao A; Olszewski, Jennifer L; Yanishevski, David; Rinehart, Jesse; Schulman, Brenda A; Harper, J Wade

    2015-05-26

    The PTEN-induced putative kinase protein 1 (PINK1) and ubiquitin (UB) ligase PARKIN direct damaged mitochondria for mitophagy. PINK1 promotes PARKIN recruitment to the mitochondrial outer membrane (MOM) for ubiquitylation of MOM proteins with canonical and noncanonical UB chains. PINK1 phosphorylates both Ser65 (S65) in the UB-like domain of PARKIN and the conserved Ser in UB itself, but the temporal sequence and relative importance of these events during PARKIN activation and mitochondria quality control remain poorly understood. Using "UB(S65A)-replacement," we find that PARKIN phosphorylation and activation, and ubiquitylation of Lys residues on a cohort of MOM proteins, occur similarly irrespective of the ability of the UB-replacement to be phosphorylated on S65. In contrast, polyubiquitin (poly-UB) chain synthesis, PARKIN retention on the MOM, and mitophagy are reduced in UB(S65A)-replacement cells. Analogous experiments examining roles of individual UB chain linkage types revealed the importance of K6 and K63 chain linkages in mitophagy, but phosphorylation of K63 chains by PINK1 did not enhance binding to candidate mitophagy receptors optineurin (OPTN), sequestosome-1 (p62), and nuclear dot protein 52 (NDP52) in vitro. Parallel reaction monitoring proteomics of total mitochondria revealed the absence of p-S65-UB when PARKIN cannot build UB chains, and <0.16% of the monomeric UB pool underwent S65 phosphorylation upon mitochondrial damage. Combining p-S65-UB and p-S65-PARKIN in vitro showed accelerated transfer of nonphosphorylated UB to PARKIN itself, its substrate mitochondrial Rho GTPase (MIRO), and UB. Our data further define a feed-forward mitochondrial ubiquitylation pathway involving PARKIN activation upon phosphorylation, UB chain synthesis on the MOM, UB chain phosphorylation, and further PARKIN recruitment and enzymatic amplification via binding to phosphorylated UB chains. PMID:25969509

  16. What’s New in Enzymatic Halogenations

    PubMed Central

    Fujimori, Danica Galoniæ; Walsh, Christopher T.

    2007-01-01

    Summary The halogenation of thousands of natural products occurs during biosynthesis and often confers important functional properties. While haloperoxidases had been the default paradigm for enzymatic incorporation of halogens, via X+ equivalents into organic scaffolds, a combination of microbial genome sequencing, enzymatic studies and structural biology have provided deep new insights into enzymatic transfer of halide equivalents in three oxidation states. These are: (1) the halide ions (X?) abundant in nature, (2) halogen atoms (X•), and (3) the X+ equivalents. The mechanism of halogen incorporation is tailored to the electronic demands of specific substrates and involves enzymes with distinct redox coenzyme requirements. PMID:17881282

  17. 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; Yi, Bin; Institute of Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037 ; Bai, Li; Xia, Yongzhi; Wang, Guansong; Qian, Guisheng; Feng, Hua

    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.

  18. Structural and Biochemical Insights into the Mechanism of Fosfomycin Phosphorylation by Fosfomycin Resistance Kinase FomA

    PubMed Central

    Pakhomova, Svetlana; Bartlett, Sue G.; Doerner, Pamela A.; Newcomer, Marcia E.

    2011-01-01

    We present here the crystal structures of fosfomycin resistance protein (FomA) complexed with MgATP, with ATP and fosfomycin, with MgADP and fosfomycin vanadate, with MgADP and the product of the enzymatic reaction, fosfomycin monophosphate, and with ADP at 1.87, 1.58, 1.85, 1.57, and 1.85 Å resolution, respectively. Structures of these complexes that approximate different reaction steps allowed us to distinguish the catalytically active conformation of ATP and to reconstruct the model of the MgATP·fosfomycin complex. According to the model, the triphosphate tail of the nucleotide is aligned toward the phosphonate moiety of fosfomycin, in contast to the previously published MgAMPPNP complex, with the attacking fosfomycin oxygen positioned 4 Å from the ?-phosphorus of ATP. Site-directed mutagenesis studies and comparison of these structures with that of homologous N-acetyl-l-glutamate and isopentenyl phosphate kinases allowed us to propose a model of phosphorylation of fosfomycin by FomA enzyme. A Mg cation ligates all three phosphate groups of ATP and together with positively charged K216, K9, K18, and H58 participates in the dissipation of negative charge during phosphoryl transfer, indicating that the transferred phosphate group is highly negatively charged, which would be expected for an associative mechanism. K216 polarizes the ?-phosphoryl group of ATP. K9, K18, and H58 participate in stabilization of the transition state. D150 and D208 play organizational roles in catalysis. S148, S149, and T210 participate in fosfomycin binding, with T210 being crucial for catalysis. Hence, it appears that as in the homologous enzymes, FomA-catalyzed phosphoryl transfer takes place by an in-line predominantly associative mechanism. PMID:21728358

  19. Regulation of protein phosphorylation in oat mitochondria

    SciTech Connect

    Pike, C.; Kopeck, K.; Sceppa, E. )

    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.

  20. Enzymatic desulfurization of coal

    SciTech Connect

    Marquis, J.K. . School of Medicine); Kitchell, J.P. )

    1988-12-15

    Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

  1. Synthesis of Red-Shifted 8-Hydroxyquinoline Derivatives Using Click Chemistry and Their Incorporation into Phosphorylation Chemosensors

    E-print Network

    Gonzalez-Vera, Juan A.

    Protein phosphorylation is a ubiquitous post-translational modification, and protein kinases, the enzymes that catalyze the phosphoryl transfer, are involved in nearly every aspect of normal, as well as aberrant, cell ...

  2. Enzymatic Defects in Three Genetic Classes of Serine-Requiring Mutants of Bacillus pumilus

    PubMed Central

    Lovett, Paul S.; Pizer, Lewis I.; Isom, Harriet C.

    1973-01-01

    Serine-requiring mutants of Bacillus pumilus NRRL B-3275 have been divided into three groups based on the position of the mutant loci on the linkage map of this organism. Representatives of each group were found deficient in enzymatic activities that constitute the phosphorylated pathway for serine biosynthesis. The evidence suggests that the genes coding for the enzymes of the phosphorylated pathway of serine biosynthesis are not clustered in B. pumilus. PMID:4745428

  3. A graphene oxide based fluorescence resonance energy transfer (FRET) biosensor for ultrasensitive detection of botulinum neurotoxin A (BoNT/A) enzymatic activity.

    PubMed

    Shi, Jingyu; Guo, Jiubiao; Bai, Gongxun; Chan, Chunyu; Liu, Xuan; Ye, Weiwei; Hao, Jianhua; Chen, Sheng; Yang, Mo

    2014-10-23

    Botulinum neurotoxins (BoNTs) are among the most potent toxic bacterial proteins for humans, which make them potential agents for bioterrorism. Therefore, an ultrasensitive detection of BoNTs and their active states is in great need as field-deployable systems for anti-terrorism applications. We report the construction of a novel graphene oxide (GO)-peptide based fluorescence resonance energy transfer (FRET) biosensor for ultrasensitive detection of the BoNT serotype A light chain (BoNT-LcA) protease activity. A green fluorescence protein (GFP) modified SNAP-25 peptide substrate (SNAP-25-GFP) was optimally designed and synthesized with the centralized recognition/cleavage sites. This FRET platform was constructed by covalent immobilization of peptide substrate on GO with BSA passivation which have advantages of low non-specific adsorption and high stability in protein abundant solution. BoNT-LcA can specifically cleave SNAP-25-GFP substrate covalently immobilized on GO to release the fragment with GFP. Based on fluorescence signal recovery measurement, the target BoNT-LcA was detected sensitively and selectively with the linear detection range from 1fg/mL to 1pg/mL. The limit of detection (LOD) for BoNT-LcA is around 1fg/mL. PMID:25461164

  4. 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

  5. Enzymatic synthesis of magnetic nanoparticles.

    PubMed

    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

  6. Regulation of VASP by phosphorylation

    PubMed Central

    Döppler, Heike; Storz, Peter

    2013-01-01

    Phosphorylations control all aspects of vasodilator-stimulated phospho-protein (VASP) function. Mapped phosphorylation sites include Y39, S157, S239, T278, and S322, and multiple kinases have been shown to mediate their phosphorylation. Recently, Protein Kinase D1 (PKD1) as a direct kinase for S157 and S322 joined this group. While S157 phosphorylation generally seems to serve as a signal for membrane localization, phosphorylations at S322 or at S239 and T278 have opposite effects on F-actin accumulation. In migrating cells, S322 phosphorylation increases filopodia numbers and length, while S239/T278 phosphorylations decrease these and also disrupt formation of focal adhesions. Therefore, the kinases mediating these phosphorylations can be seen as switches needed to facilitate cell motility. PMID:24401601

  7. 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.

  8. Enzymatic and mitochondrial responses to 5 months of aerial exposure in the slender lungfish

    E-print Network

    McClelland, Grant B.

    Enzymatic and mitochondrial responses to 5 months of aerial exposure in the slender lungfish control aquatic slender lungfish Protopterus dolloi, and those exposed to air for 5 months. Activities; ornithine-urea cycle; oxidative phosphorylation. INTRODUCTION The habitat of the African slender lungfish

  9. Protein phosphorylation in Streptomyces albus.

    PubMed

    Dobrová, Z; Jiresová, M; Petrík, T; Rysavý, P; Náprstek, J; Janecek, J

    1990-09-01

    The phosphorylated proteins of Streptomyces albus, radioactively labeled with [32P]orthophosphate have been analyzed by gel electrophoresis and autoradiography. More than 10 protein species were found to be phosphorylated. With [32P]ATP as substrate cell free extracts phosphorylated endogenous proteins in vitro which were predominantly phosphorylated in vivo. From cell extract which exhibited active phosphorylated in vitro, a protein kinase has been partially purified. The kinase activity was identified in fractions corresponding to a 90 kDa protein. PMID:2276604

  10. Enzymatic desulfurization of coal

    SciTech Connect

    Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V.

    1991-05-16

    The overall objective of this program was to investigate the feasibility of an enzymatic desulfurization process specifically intended for organic sulfur removal from coal. Toward that end, a series of specific objectives were defined: (1) establish the feasibility of (bio)oxidative pretreatment followed by biochemical sulfate cleavage for representative sulfur-containing model compounds and coals using commercially-available enzymes; (2) investigate the potential for the isolation and selective use of enzyme preparations from coal-utilizing microbial systems for desulfurization of sulfur-containing model compounds and coals; and (3) develop a conceptual design and economic analysis of a process for enzymatic removal of organic sulfur from coal. Within the scope of this program, it was proposed to carry out a portion of each of these efforts concurrently. (VC)

  11. 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.

  12. Oxidative phosphorylation at the fin de siècle.

    PubMed

    Saraste, M

    1999-03-01

    Mitochondria produce most of the energy in animal cells by a process called oxidative phosphorylation. Electrons are passed along a series of respiratory enzyme complexes located in the inner mitochondrial membrane, and the energy released by this electron transfer is used to pump protons across the membrane. The resultant electrochemical gradient enables another complex, adenosine 5'-triphosphate (ATP) synthase, to synthesize the energy carrier ATP. Important new mechanistic insights into oxidative phosphorylation have emerged from recent three-dimensional structural analyses of ATP synthase and two of the respiratory enzyme complexes, cytochrome bc1 and cytochrome c oxidase. This work, and new enzymological studies of ATP synthase's unusual catalytic mechanism, are reviewed here. PMID:10066163

  13. 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

  14. The Synthesis of Methylated, Phosphorylated, and Phosphonated 3?-Aminoacyl-tRNASec Mimics**

    PubMed Central

    Rigger, Lukas; Schmidt, Rachel L.; Holman, Kaitlyn M.

    2014-01-01

    The twenty first amino acid, selenocysteine (Sec), is the only amino acid that is synthesized on its cognate transfer RNA (tRNASec) in all domains of life. The multistep pathway involves O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS), an enzyme that catalyzes the terminal chemical reaction during which the phosphoseryl–tRNASec intermediate is converted into selenocysteinyl-tRNASec. The SepSecS architecture and the mode of tRNASec recognition have been recently determined at atomic resolution. The crystal structure provided valuable insights that gave rise to mechanistic proposals that could not be validated because of the lack of appropriate molecular probes. To further improve our understanding of the mechanism of the biosynthesis of selenocysteine in general and the mechanism of SepSecS in particular, stable tRNASec substrates carrying aminoacyl moieties that mimic particular reaction intermediates are needed. Here, we report on the accurate synthesis of methylated, phosphorylated, and phosphonated serinyl-derived tRNASec mimics that contain a hydrolysis-resistant ribose 3?-amide linkage instead of the natural ester bond. The procedures introduced allow for efficient site-specific methylation and/or phosphorylation directly on the solid support utilized in the automated RNA synthesis. For the preparation of (S)-2-amino-4-phosphonobutyric acid–oligoribonucleotide conjugates, a separate solid support was generated. Furthermore, we developed a three-strand enzymatic ligation protocol to obtain the corresponding full-length tRNASec derivatives. Finally, we developed an electrophoretic mobility shift assay (EMSA) for rapid, qualitative characterization of the SepSecS-tRNA interactions. The novel tRNASec mimics are promising candidates for further elucidation of the biosynthesis of selenocysteine by X-ray crystallography and other biochemical approaches, and could be attractive for similar studies on other tRNA-dependent enzymes. PMID:24127424

  15. The synthesis of methylated, phosphorylated, and phosphonated 3'-aminoacyl-tRNA(Sec) mimics.

    PubMed

    Rigger, Lukas; Schmidt, Rachel L; Holman, Kaitlyn M; Simonovi?, Miljan; Micura, Ronald

    2013-11-18

    The twenty first amino acid, selenocysteine (Sec), is the only amino acid that is synthesized on its cognate transfer RNA (tRNA(Sec)) in all domains of life. The multistep pathway involves O-phosphoseryl-tRNA:selenocysteinyl-tRNA synthase (SepSecS), an enzyme that catalyzes the terminal chemical reaction during which the phosphoseryl-tRNA(Sec) intermediate is converted into selenocysteinyl-tRNA(Sec). The SepSecS architecture and the mode of tRNA(Sec) recognition have been recently determined at atomic resolution. The crystal structure provided valuable insights that gave rise to mechanistic proposals that could not be validated because of the lack of appropriate molecular probes. To further improve our understanding of the mechanism of the biosynthesis of selenocysteine in general and the mechanism of SepSecS in particular, stable tRNA(Sec) substrates carrying aminoacyl moieties that mimic particular reaction intermediates are needed. Here, we report on the accurate synthesis of methylated, phosphorylated, and phosphonated serinyl-derived tRNA(Sec) mimics that contain a hydrolysis-resistant ribose 3'-amide linkage instead of the natural ester bond. The procedures introduced allow for efficient site-specific methylation and/or phosphorylation directly on the solid support utilized in the automated RNA synthesis. For the preparation of (S)-2-amino-4-phosphonobutyric acid-oligoribonucleotide conjugates, a separate solid support was generated. Furthermore, we developed a three-strand enzymatic ligation protocol to obtain the corresponding full-length tRNA(Sec) derivatives. Finally, we developed an electrophoretic mobility shift assay (EMSA) for rapid, qualitative characterization of the SepSecS-tRNA interactions. The novel tRNA(Sec) mimics are promising candidates for further elucidation of the biosynthesis of selenocysteine by X-ray crystallography and other biochemical approaches, and could be attractive for similar studies on other tRNA-dependent enzymes. PMID:24127424

  16. 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.

  17. Enzymatic temperature change indicator

    DOEpatents

    Klibanov, Alexander M. (Newton, MA); Dordick, Jonathan S. (Iowa City, IA)

    1989-01-21

    A temperature change indicator is described which is composed of an enzyme and a substrate for that enzyme suspended in a solid organic solvent or mixture of solvents as a support medium. The organic solvent or solvents are chosen so as to melt at a specific temperature or in a specific temperature range. When the temperature of the indicator is elevated above the chosen, or critical temperature, the solid organic solvent support will melt, and the enzymatic reaction will occur, producing a visually detectable product which is stable to further temperature variation.

  18. Covalent Protein Labeling by Enzymatic Phosphocholination.

    PubMed

    Heller, Katharina; Ochtrop, Philipp; Albers, Michael F; Zauner, Florian B; Itzen, Aymelt; Hedberg, Christian

    2015-08-24

    We present a new protein labeling method based on the covalent enzymatic phosphocholination of a specific octapeptide amino acid sequence in intact proteins. The bacterial enzyme AnkX from Legionella pneumophila has been established to transfer functional phosphocholine moieties from synthetically produced CDP-choline derivatives to N-termini, C-termini, and internal loop regions in proteins of interest. Furthermore, the covalent modification can be hydrolytically removed by the action of the Legionella enzyme Lem3. Only a short peptide sequence (eight amino acids) is required for efficient protein labeling and a small linker group (PEG-phosphocholine) is introduced to attach the conjugated cargo. PMID:26147231

  19. Kinetic basis for the stimulatory effect of phosphorylation on the methylesterase activity of CheB.

    PubMed

    Anand, Ganesh S; Stock, Ann M

    2002-05-28

    Response regulators are activated to elicit a specific cellular response to an extracellular stimulus via phosphotransfer from a cognate sensor histidine kinase to a specific aspartate residue. Phosphorylation at the conserved aspartate residue modulates the activity of the response regulator. Methylesterase CheB is a two-domain response regulator composed of a regulatory domain and an effector domain with enzymatic activity. CheB functions within the bacterial chemotaxis pathway to control the level of chemoreceptor methylation. In its unphosphorylated state, the regulatory domain inhibits methylesterase activity of the effector domain. Phosphorylation of the regulatory domain leads to an enhancement of methylesterase activity through a relief of inhibition and a stimulatory effect on catalysis. CheB is a useful model protein for understanding the effects of phosphorylation of the regulatory domain on interdomain interactions and stimulation of enzymatic activity of the effector domain. Kinetic analyses of CheB activation indicate that the basis for the nearly 100-fold methylesterase activation upon phosphorylation is due to a change in the catalytic rate constant for the methylesterase reaction. It is also shown that the P2 domain of histidine kinase CheA inhibits the methylesterase activity of CheB and that this inhibition is decreased upon phosphorylation of CheB. Finally, studies of methylesterase catalysis by the free catalytic domain in the presence and absence of the regulatory domain have enabled detection of an association between the two domains in the absence of the linker. PMID:12022879

  20. The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin

    PubMed Central

    Brauksiepe, Bastienne; Mujica, Alejandro O; Herrmann, Harald; Schmidt, Erwin R

    2008-01-01

    Background Colocalization of Stk33 with vimentin by double immunofluorescence in certain cells indicated that vimentin might be a target for phosphorylation by the novel kinase Stk33. We therefore tested in vitro the ability of Stk33 to phosphorylate recombinant full length vimentin and amino-terminal truncated versions thereof. In order to prove that Stk33 and vimentin are also in vivo associated proteins co-immunoprecipitation experiments were carried out. For testing the enzymatic activity of immunoprecipitated Stk33 we incubated precipitated Stk33 with recombinant vimentin proteins. To investigate whether Stk33 binds directly to vimentin, an in vitro co-sedimentation assay was performed. Results The results of the kinase assays demonstrate that Stk33 is able to specifically phosphorylate the non-?-helical amino-terminal domain of vimentin in vitro. Furthermore, co-immunoprecipitation experiments employing cultured cell extracts indicate that Stk33 and vimentin are associated in vivo. Immunoprecipitated Stk33 has enzymatic activity as shown by successful phosphorylation of recombinant vimentin proteins. The results of the co-sedimentation assay suggest that vimentin binds directly to Stk33 and that no additional protein mediates the association. Conclusion We hypothesize that Stk33 is involved in the in vivo dynamics of the intermediate filament cytoskeleton by phosphorylating vimentin. PMID:18811945

  1. Semisynthesis and Enzymatic Preparation of Post-translationally Modified ?-Synuclein.

    PubMed

    Fauvet, Bruno; Lashuel, Hilal A

    2016-01-01

    Posttranslational modifications (PTMs) serve as molecular switches for regulating protein folding, function, and interactome and have been implicated in the misfolding and amyloid formation by several proteins linked to neurodegenerative diseases, including Alzheimer's and Parkinson's disease. Understanding the role of individual PTMs in protein misfolding and aggregation requires the preparation of site-specifically modified proteins, as well as the identification of the enzymes involved in regulating these PTMs. Recently, our group has pioneered the development of enzymatic, synthetic, and semisynthetic strategies that allow site-specific introduction of PTMs at single or multiple sites and generation of modified proteins in milligram quantities. In this chapter, we provide detailed description of enzymatic and semisynthetic strategies for the generation of the phosphorylated ?-Synuclein (?-Syn) at S129, (pS129), which has been identified as a pathological hallmark of Parkinson's disease. The semisynthetic method described for generation of ?-Syn-pS129 requires expertise with protein chemical ligation, but can be used to incorporate other PTMs (single or multiple) within the ?-Syn C-terminus if desired. On the other hand, the in vitro kinase-mediated phosphorylation strategy does not require any special setup and is rather easy to apply, but its application is restricted to the generation of ?-Syn_pS129. These methods have the potential to increase the availability of pure and homogenous modified ?-Syn reagents, which may be used as standards in numerous applications, including the search for potential biomarkers of synucleinopathies. PMID:26453202

  2. 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)

  3. 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)

  4. Properties of phosphorylated thymidylate synthase.

    PubMed

    Fr?czyk, Tomasz; Ruman, Tomasz; Wilk, Piotr; Palmowski, Pawe?; Rogowska-Wrzesinska, Adelina; Cie?la, Joanna; Zieli?ski, Zbigniew; Nizio?, Joanna; Jarmu?a, Adam; Maj, Piotr; Go?os, Barbara; Wi?ska, Patrycja; Ostafil, Sylwia; Wa?ajtys-Rode, El?bieta; Shugar, David; Rode, Wojciech

    2015-12-01

    Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent. PMID:26315778

  5. Electron transfer and protein engineering studies of the soluble methane monooxygenase from Methylococcus capsulatus (Bath)

    E-print Network

    Blazyk, Jessica L. (Jessica Lee), 1974-

    2003-01-01

    Chapter 1. Introduction: Electron Transfer in Biological Systems In many biological processes, including oxidative phosphorylation and photosynthesis, electron transfer reactions play vital roles. Electrons must be transported ...

  6. Phosphorylation Regulates myo-Inositol-3-phosphate Synthase

    PubMed Central

    Deranieh, Rania M.; He, Quan; Caruso, Joseph A.; Greenberg, Miriam L.

    2013-01-01

    myo-Inositol-3-phosphate synthase (MIPS) plays a crucial role in inositol homeostasis. Transcription of the coding gene INO1 is highly regulated. However, regulation of the enzyme is not well defined. We previously showed that MIPS is indirectly inhibited by valproate, suggesting that the enzyme is post-translationally regulated. Using 32Pi labeling and phosphoamino acid analysis, we show that yeast MIPS is a phosphoprotein. Mass spectrometry analysis identified five phosphosites, three of which are conserved in the human MIPS. Analysis of phosphorylation-deficient and phosphomimetic site mutants indicated that the three conserved sites in yeast (Ser-184, Ser-296, and Ser-374) and humans (Ser-177, Ser-279, and Ser-357) affect MIPS activity. Both S296A and S296D yeast mutants and S177A and S177D human mutants exhibited decreased enzymatic activity, suggesting that a serine residue is critical at that location. The phosphomimetic mutations S184D (human S279D) and S374D (human S357D) but not the phosphodeficient mutations decreased activity, suggesting that phosphorylation of these two sites is inhibitory. The double mutation S184A/S374A caused an increase in MIPS activity, conferred a growth advantage, and partially rescued sensitivity to valproate. Our findings identify a novel mechanism of regulation of inositol synthesis by phosphorylation of MIPS. PMID:23902760

  7. Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization

    SciTech Connect

    Geiss, Brian J.; Cano, Gina L.; Tavis, John E.; Morrison, Lynda A. . E-mail: morrisla@slu.edu

    2004-12-05

    Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22. Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.

  8. Spatiotemporal Investigation of Phosphorylation Events During Cell Cycle Progression.

    PubMed

    Gheghiani, Lilia; Gavet, Olivier

    2016-01-01

    Polo-like kinase 1 (Plk1) is an essential kinase for mitotic commitment and progression through mitosis. In contrast to its well characterized roles during mitosis, the precise molecular events controlled by Plk1 during G2/M progression and their spatiotemporal regulation are still poorly elucidated. We recently investigated Plk1-dependent regulation of Cdc25C phosphatase, an activator of the master mitotic driver Cyclin B1-Cdk1. To this end, we generated a genetically encoded FRET (Förster Resonance Energy Transfer)-based Cdc25C phosphorylation biosensor to observe Cdc25 spatiotemporal phosphorylation during cell cycle progression in live single cell assays. Because this approach proved to be powerful, we provide here guidelines for the development of biosensors for any phosphorylation site of interest. PMID:26254922

  9. Click conjugation of a binuclear terbium(III) complex for real-time detection of tyrosine phosphorylation.

    PubMed

    Akiba, Hiroki; Sumaoka, Jun; Tsumoto, Kouhei; Komiyama, Makoto

    2015-04-01

    Phosphorylation of proteins is closely associated with various diseases, and, therefore, its detection is vitally important in molecular biology and drug discovery. Previously, we developed a binuclear Tb(III) complex, which emits notable luminescence only in the presence of phosphotyrosine. In this study, we conjugated a newly synthesized binuclear Tb(III) complex to substrate peptides by using click chemistry. Using these conjugates, we were able to detect tyrosine phosphorylation in real time. These conjugates were superior to nonconjugated Tb(III) complexes for the detection of tyrosine phosphorylation, especially when the substrate peptides used were positively charged. Luminescence intensity upon phosphorylation was enhanced 10-fold, making the luminescence intensity of this system one of the largest among lanthanide luminescence-based systems. We also determined Michaelis-Menten parameters for the phosphorylation of various kinase/peptide combinations and quantitatively analyzed the effects of mutations in the peptide substrates. Furthermore, we successfully monitored the inhibition of enzymatic phosphorylation by inhibitors in real time. Advantageously, this system detects only the phosphorylation of tyrosine (phosphorylated serine and threonine are virtually silent) and is applicable to versatile peptide substrates. Our study thus demonstrates the applicability of this system for the analysis of kinase activity, which could lead to drug discovery. PMID:25757507

  10. Phosphorylation-dependent regulation of Notch1 signaling: the fulcrum of Notch1 signaling

    PubMed Central

    Lee, Hye-Jin; Kim, Mi-Yeon; Park, Hee-Sae

    2015-01-01

    Notch signaling plays a pivotal role in cell fate determination, cellular development, cellular self-renewal, tumor progression, and has been linked to developmental disorders and carcinogenesis. Notch1 is activated through interactions with the ligands of neighboring cells, and acts as a transcriptional activator in the nucleus. The Notch1 intracellular domain (Notch1-IC) regulates the expression of target genes related to tumor development and progression. The Notch1 protein undergoes modification after translation by posttranslational modification enzymes. Phosphorylation modification is critical for enzymatic activation, complex formation, degradation, and subcellular localization. According to the nuclear cycle, Notch1-IC is degraded by E3 ligase, FBW7 in the nucleus via phosphorylation-dependent degradation. Here, we summarize the Notch signaling pathway, and resolve to understand the role of phosphorylation in the regulation of Notch signaling as well as to understand its relation to cancer. [BMB Reports 2015; 48(8): 431-437] PMID:26058398

  11. Glycogen phosphorylation and Lafora disease.

    PubMed

    Roach, Peter J

    2015-12-01

    Covalent phosphorylation of glycogen, first described 35 years ago, was put on firm ground through the work of the Whelan laboratory in the 1990s. But glycogen phosphorylation lay fallow until interest was rekindled in the mid 2000s by the finding that it could be removed by a glycogen-binding phosphatase, laforin, and that mutations in laforin cause a fatal teenage-onset epilepsy, called Lafora disease. Glycogen phosphorylation is due to phosphomonoesters at C2, C3 and C6 of glucose residues. Phosphate is rare, ranging from 1:500 to 1:5000 phosphates/glucose depending on the glycogen source. The mechanisms of glycogen phosphorylation remain under investigation but one hypothesis to explain C2 and perhaps C3 phosphate is that it results from a rare side reaction of the normal synthetic enzyme glycogen synthase. Lafora disease is likely caused by over-accumulation of abnormal glycogen in insoluble deposits termed Lafora bodies in neurons. The abnormality in the glycogen correlates with elevated phosphorylation (at C2, C3 and C6), reduced branching, insolubility and an enhanced tendency to aggregate and become insoluble. Hyperphosphorylation of glycogen is emerging as an important feature of this deadly childhood disease. PMID:26278984

  12. 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

  13. Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

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

    2011-08-22

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

  14. 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.

  15. Autophagy proteins regulate ERK phosphorylation.

    PubMed

    Martinez-Lopez, Nuria; Athonvarangkul, Diana; Mishall, Priti; Sahu, Srabani; Singh, Rajat

    2013-01-01

    Autophagy is a conserved pathway that maintains cellular quality control. Extracellular signal-regulated kinase (ERK) controls various aspects of cell physiology including proliferation. Multiple signalling cascades, including ERK, have been shown to regulate autophagy, however whether autophagy proteins (ATG) regulate cell signalling is unknown. Here we show that growth factor exposure increases the interaction of ERK cascade components with ATG proteins in the cytosol and nucleus. ERK and its upstream kinase MEK localize to the extra-luminal face of autophagosomes. ERK2 interacts with ATG proteins via its substrate-binding domains. Deleting Atg7 or Atg5 or blocking LC3 lipidation or ATG5-ATG12 conjugation decreases ERK phosphorylation. Conversely, increasing LC3-II availability by silencing the cysteine protease ATG4B or acute trehalose exposure increases ERK phosphorylation. Decreased ERK phosphorylation in Atg5?/? cells does not occur from overactive phosphatases. Our findings thus reveal an unconventional function of ATG proteins as cellular scaffolds in the regulation of ERK phosphorylation. PMID:24240988

  16. Stress-induced phosphorylation of SNAP-25.

    PubMed

    Yamamori, Saori; Sugaya, Daichi; Iida, Yuuki; Kokubo, Hirotoshi; Itakura, Makoto; Suzuki, Eiji; Kataoka, Masakazu; Miyaoka, Hitoshi; Takahashi, Masami

    2014-02-21

    Synaptosomal-associated protein of 25 kDa (SNAP-25), a t-SNARE protein, plays a crucial role in neurotransmitter release by exocytosis. Protein kinase C phosphorylates SNAP-25 at Ser(187), however the physiological significance of this phosphorylation event in brain function remains unclear. In the present study, we found that SNAP-25 phosphorylation increased rapidly in the mouse brain following cold-water restraint stress. Both basal and stress-induced phosphorylation of SNAP-25 were high in stress-related brain regions, including the cerebral cortex, hippocampus, and amygdala, and the extent of phosphorylation increased with increasing amounts of stress. Intravenous administration of adrenaline increased SNAP-25 phosphorylation, although stress-induced phosphorylation was still observed in adrenalectomized mice. These results indicate that SNAP-25 phosphorylation is regulated in a stress-dependent manner through both central and peripheral mechanisms. PMID:24374286

  17. Bioluminescence methods for enzymatic determinations

    DOEpatents

    Bostick, William D. (Oak Ridge, TN); Denton, Mark S. (Clinton, TN); Dinsmore, Stanley R. (Norris, TN)

    1982-01-01

    An enzymatic method for continuous, on-line and rapid detection of diagnostically useful biomarkers, which are symptomatic of disease or trauma-related tissue damage, is disclosed. The method is characterized by operability on authentic samples of complex biological fluids which contain the biomarkers.

  18. Bioluminescence methods for enzymatic determinations

    SciTech Connect

    Bostick, W.D.; Denton, M.S.; Dinsmore, S.R.

    1982-11-02

    An enzymatic method for continuous, on-line and rapid detection of diagnostically useful biomarkers, which are symptomatic of disease or trauma-related tissue damage, is disclosed. The method is characterized by operability on authentic samples of complex biological fluids which contain the biomarkers.

  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. PMID:26029234

  20. Phosphorylation of Smads by Intracellular Kinases.

    PubMed

    Liu, Fang; Matsuura, Isao

    2016-01-01

    Smad proteins transduce the TGF-ß family signal at the cell surface into gene regulation in the nucleus. In addition to being phosphorylated by the TGF-ß family receptors, Smads are phosphorylated by a variety of intracellular kinases. The most studied are by cyclin-dependent kinases, the MAP kinase family members, and GSK-3. Phosphorylation by these kinases regulates Smad activities, leading to various biological effects. This chapter describes the methods for analyzing Smad phosphorylation by these kinases. PMID:26520119

  1. Phosphorylation-Elicited Quaternary Changes of GA Binding Protein in Transcriptional Activation

    PubMed Central

    Sunesen, Morten; Huchet-Dymanus, Monique; Christensen, Morten O.; Changeux, Jean-Pierre

    2003-01-01

    Enrichment of nicotinic acetylcholine receptors (nAChR) on the tip of the subjunctional folds of the postsynaptic membrane is a central event in the development of the vertebrate neuromuscular junction. This is attained, in part, through a selective transcription in the subsynaptic nuclei, and it has recently been shown that the GA binding protein (GABP) plays an important role in this compartmentalized expression. The neural factor heregulin (HRG) activates nAChR transcription in cultured cells by stimulating a signaling cascade of protein kinases. Hence, it is speculated that GABP becomes activated by phosphorylation, but the mechanism has remained elusive. To fully understand the consequences of GABP phosphorylation, we examined the effect of heregulin-elicited GABP phosphorylation on cellular localization, DNA binding, transcription, and mobility. We demonstrate that HRG-elicited phosphorylation dramatically changes the transcriptional activity and mobility of GABP. While phosphorylation of GABP? seems to be dispensable for these changes, phosphorylation of GABP? is crucial. Using fluorescence resonance energy transfer, we furthermore showed that phosphorylation of threonine 280 in GABP? triggers reorganizations of the quaternary structure of GABP. Taken together, these results support a model in which phosphorylation-elicited structural changes of GABP enable engagement in certain interactions leading to transcriptional activation. PMID:14585962

  2. Enzymatic conversion of carbon dioxide.

    PubMed

    Shi, Jiafu; Jiang, Yanjun; Jiang, Zhongyi; Wang, Xueyan; Wang, Xiaoli; Zhang, Shaohua; Han, Pingping; Yang, Chen

    2015-10-01

    With the continuous increase in fossil fuels consumption and the rapid growth of atmospheric CO2 concentration, the harmonious state between human and nature faces severe challenges. Exploring green and sustainable energy resources and devising efficient methods for CO2 capture, sequestration and utilization are urgently required. Converting CO2 into fuels/chemicals/materials as an indispensable element for CO2 capture, sequestration and utilization may offer a win-win strategy to both decrease the CO2 concentration and achieve the efficient exploitation of carbon resources. Among the current major methods (including chemical, photochemical, electrochemical and enzymatic methods), the enzymatic method, which is inspired by the CO2 metabolic process in cells, offers a green and potent alternative for efficient CO2 conversion due to its superior stereo-specificity and region/chemo-selectivity. Thus, in this tutorial review, we firstly provide a brief background about enzymatic conversion for CO2 capture, sequestration and utilization. Next, we depict six major routes of the CO2 metabolic process in cells, which are taken as the inspiration source for the construction of enzymatic systems in vitro. Next, we focus on the state-of-the-art routes for the catalytic conversion of CO2 by a single enzyme system and by a multienzyme system. Some emerging approaches and materials utilized for constructing single-enzyme/multienzyme systems to enhance the catalytic activity/stability will be highlighted. Finally, a summary about the current advances and the future perspectives of the enzymatic conversion of CO2 will be presented. PMID:26055659

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

    PubMed Central

    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

  4. Molecular Cell Phosphorylation of LSD1

    E-print Network

    Just, Armin

    Molecular Cell Article Phosphorylation of LSD1 by PKCa Is Crucial for Circadian Rhythmicity (LSD1) is phosphory- lated by protein kinase Ca (PKCa) in a circadian manner and the phosphorylated LSD mice bearing phosphorylation-defective Lsd1SA/SA alleles exhibited altered circadian rhythms

  5. Phosphorylation of Single Stranded RNA Virus Proteins and Potential for Novel Therapeutic Strategies

    PubMed Central

    Keck, Forrest; Ataey, Pouya; Amaya, Moushimi; Bailey, Charles; Narayanan, Aarthi

    2015-01-01

    Post translational modification of proteins is a critical requirement that regulates function. Among the diverse kinds of protein post translational modifications, phosphorylation plays essential roles in protein folding, protein:protein interactions, signal transduction, intracellular localization, transcription regulation, cell cycle progression, survival and apoptosis. Protein phosphorylation is also essential for many intracellular pathogens to establish a productive infection cycle. Preservation of protein phosphorylation moieties in pathogens in a manner that mirrors the host components underscores the co-evolutionary trajectory of pathogens and hosts, and sheds light on how successful pathogens have usurped, either in part or as a whole, the host enzymatic machinery. Phosphorylation of viral proteins for many acute RNA viruses including Flaviviruses and Alphaviruses has been demonstrated to be critical for protein functionality. This review focuses on phosphorylation modifications that have been documented to occur on viral proteins with emphasis on acutely infectious, single stranded RNA viruses. The review additionally explores the possibility of repurposing Food and Drug Administration (FDA) approved inhibitors as antivirals for the treatment of acute RNA viral infections. PMID:26473910

  6. Phosphorylation of Single Stranded RNA Virus Proteins and Potential for Novel Therapeutic Strategies.

    PubMed

    Keck, Forrest; Ataey, Pouya; Amaya, Moushimi; Bailey, Charles; Narayanan, Aarthi

    2015-10-01

    Post translational modification of proteins is a critical requirement that regulates function. Among the diverse kinds of protein post translational modifications, phosphorylation plays essential roles in protein folding, protein:protein interactions, signal transduction, intracellular localization, transcription regulation, cell cycle progression, survival and apoptosis. Protein phosphorylation is also essential for many intracellular pathogens to establish a productive infection cycle. Preservation of protein phosphorylation moieties in pathogens in a manner that mirrors the host components underscores the co-evolutionary trajectory of pathogens and hosts, and sheds light on how successful pathogens have usurped, either in part or as a whole, the host enzymatic machinery. Phosphorylation of viral proteins for many acute RNA viruses including Flaviviruses and Alphaviruses has been demonstrated to be critical for protein functionality. This review focuses on phosphorylation modifications that have been documented to occur on viral proteins with emphasis on acutely infectious, single stranded RNA viruses. The review additionally explores the possibility of repurposing Food and Drug Administration (FDA) approved inhibitors as antivirals for the treatment of acute RNA viral infections. PMID:26473910

  7. Electrochemical enzymatic biosensors using carbon nanofiber nanoelectrode arrays

    NASA Astrophysics Data System (ADS)

    Li, Jun; Li, Yi-fen; Swisher, Luxi Z.; Syed, Lateef U.; Prior, Allan M.; Nguyen, Thu A.; Hua, Duy H.

    2012-10-01

    The reduction of electrode size down to nanometers could dramatically enhance detection sensitivity and temporal resolution. Nanoelectrode arrays (NEAs) are of particular interest for ultrasensitive biosensors. Here we report the study of two types of biosensors for measuring enzyme activities using NEAs fabricated with vertically aligned carbon nanofibers (VACNFs). VACNFs of ~100 nm in average diameter and 3-5 ?m in length were grown on conductive substrates as uniform vertical arrays which were then encapsulated in SiO2 matrix leaving only the tips exposed. We demonstrate that such VACNF NEAs can be used in profiling enzyme activities through monitoring the change in electrochemical signals induced by enzymatic reactions to the peptides attached to the VACNF tip. The cleavage of the tetrapeptide with a ferrocene tag by a cancerrelated protease (legumain) was monitored with AC voltammetry. Real-time electrochemical impedance spectroscopy (REIS) was used for fast label-free detection of two reversible processes, i.e. phosphorylation by c-Src tyrosine kinase and dephosphorylation by protein tyrosine phosphatase 1B (PTP1B). The REIS data of phosphorylation were slow and unreliable, but those of dephosphorylation showed large and fast exponential decay due to much higher activity of phosphatase PTP1B. The kinetic data were analyzed with a heterogeneous Michaelis-Menten model to derive the "specificity constant" kcat/Km, which is 8.2x103 M-1s-1 for legumain and (2.1 ± 0.1) x 107 M-1s-1 for phosphatase (PTP1B), well consistent with literature. It is promising to develop VACNF NEA based electrochemical enzymatic biosensors as portable multiplex electronic techniques for rapid cancer diagnosis and treatment monitoring.

  8. Agonist-Induced Endocytosis and Receptor Phosphorylation Mediate Resensitization of Dopamine D2 Receptors

    PubMed Central

    Cho, Dongim; Zheng, Mei; Min, Chengchun; Ma, Lan; Kurose, Hitoshi; Park, Jae H.; Kim, Kyeong-Man

    2010-01-01

    The regulatory mechanisms and functional roles of agonist-induced internalization of G protein-coupled receptors (GPCRs) were analyzed using mutant dopamine D2 receptors (D2Rs) in which all possible GPCR kinase (GRK) phosphorylation sites were mutated or the affinity for ?-arrestins was altered. Agonist-induced internalization of D2Rs involved a phosphorylation-dependent component, which was mediated by serine/threonine (S/T) residues in the second loop and T225 in the third loop, and a phosphorylation-independent component. GRK2-mediated enhancement of the internalization and inhibition of D2R signaling did not involve receptor phosphorylation, and only the former required the enzymatic activity of GRK2. The phosphorylation-deficient mutant (D2R-intracellular loop 2/3) recycled more slowly and showed more agonist-induced desensitization than did the wild-type D2R, suggesting that receptor phosphorylation mediates the recycling of the internalized receptors and enhances receptor resensitization. Blockade of the agonist-induced internalization of D2R-intracellular loop 2/3 provoked desensitization as in wild-type D2R, suggesting that certain cellular processes other than receptor dephosphorylation occurring within the endocytic vesicle are responsible for the resensitization of D2R. When dissociation between D2R and ?-arrestin was inhibited or when the expression of cellular ?-arrestins was decreased, agonist-induced desensitization of D2R did not occur, suggesting that dissociation from ?-arrestin is the main cellular process required for resensitization of D2R and is achieved through agonist-induced internalization. These results indicate that, in the regulation of some GPCRs, phosphorylation-independent association with ?-arrestin plays a major role in agonist-induced desensitization. PMID:20160122

  9. Mycobacterium tuberculosis supports protein tyrosine phosphorylation

    PubMed Central

    Kusebauch, Ulrike; Ortega, Corrie; Ollodart, Anja; Rogers, Richard S.; Sherman, David R.; Moritz, Robert L.; Grundner, Christoph

    2014-01-01

    Reversible protein phosphorylation determines growth and adaptive decisions in Mycobacterium tuberculosis (Mtb). At least 11 two-component systems and 11 Ser/Thr protein kinases (STPKs) mediate phosphorylation on Asp, His, Ser, and Thr. In contrast, protein phosphorylation on Tyr has not been described previously in Mtb. Here, using a combination of phospho-enrichment and highly sensitive mass spectrometry, we show extensive protein Tyr phosphorylation of diverse Mtb proteins, including STPKs. Several STPKs function as dual-specificity kinases that phosphorylate Tyr in cis and in trans, suggesting that dual-specificity kinases have a major role in bacterial phospho-signaling. Mutation of a phosphotyrosine site of the essential STPK PknB reduces its activity in vitro and in live Mtb, indicating that Tyr phosphorylation has a functional role in bacterial growth. These data identify a previously unrecognized phosphorylation system in a human pathogen that claims ?1.4 million lives every year. PMID:24927537

  10. Phosphorylated nano-diamond/ Polyimide Nanocomposites

    NASA Astrophysics Data System (ADS)

    Beyler-Çi?il, Asli; Çakmakçi, Emrah; Vezir Kahraman, Memet

    2014-08-01

    In this study, a novel route to synthesize polyimide (PI)/phosphorylated nanodiamond films with improved thermal and mechanical properties was developed. Surface phosphorylation of nano-diamond was performed in dichloromethane. Phosphorylation dramatically enhanced the thermal stability of nano-diamond. Poly(amic acid) (PAA), which is the precursor of PI, was successfully synthesized with 3,3',4,4'-Benzophenonetetracarboxylic dianhydride (BTDA) and 4,4'-oxydianiline (4,4'-ODA) in the solution of N,N- dimethylformamide (DMF). Pure BTDA-ODA polyimide films and phosphorylated nanodiamond containing BTDA-ODA PI films were prepared. The PAA displayed good compatibility with phosphorylated nano-diamond. The morphology of the polyimide (PI)/phosphorylated nano-diamond was characterized by scanning electron microscopy (SEM). Chemical structure of polyimide and polyimide (PI)/phosphorylated nano-diamond was characterized by FTIR. SEM and FTIR results showed that the phosphorylated nano-diamond was successfully prepared. Thermal properties of the polyimide (PI)/phosphorylated nanodiamond was characterized by thermogravimetric analysis (TGA). TGA results showed that the thermal stability of (PI)/phosphorylated nano-diamond film was increased.

  11. Phosphorylation of brain proteins in generalized convulsions

    SciTech Connect

    Horan, M.P.

    1986-01-01

    Phosphorylation of neuronal proteins is being proposed as a modulating influence on several aspects of neuronal function. By labeling proteins with radioactive phosphorus (/sup 32/P) and then separating these proteins by polyacrylamide gel electrophoresis, the author can determine what factors change the phosphorylation of these proteins. They have used such a system to analyze the effects of generalized convulsions on protein phosphorylation. Electroshock (ES) and pentylenetetrazol (PTZ) were utilized to produce generalized convulsions. Brain membranes, taken from rats immediately after a convulsion, exhibited an increase in protein phosphorylation in vitro. The most noticeable change took place in proteins in the 18,000-20,000 MW range. They have designated these proteins as the low molecular weight (LMW) proteins. The change in phosphorylation was basically the same after one convulsions as after six daily convulsions. Twenty-four hours after a single convulsion no change in phosphorylation was observed. When rat membranes are exposed to PTZ in vitro, phosphorylation is increased at 20 sec but has returned to control level at 90 sec of incubation. This effect is produced without a convulsion. In general, as the concentration of magnesium is increased from 5 mM to 10 mM phosphorylation is increased. Increasing the incubation time from 20 sec to 90 sec and increasing the calcium concentration to 10 mM both decrease phosphorylation of the LMW proteins. Human temporal cortex samples present with phosphorylated proteins having patterns very similar to those in rat membranes.

  12. Photoinitiated proton-coupled electron transfer and radical transport kinetics in class la ribonucleotide reductase

    E-print Network

    Pizano, Arturo A. (Arturo Alejandro)

    2013-01-01

    Proton-coupled electron transfer (PCET) is a critical mechanism in biology, underpinning key processes such as radical transport, energy transduction, and enzymatic substrate activation. Ribonucleotide reductases (RNRs) ...

  13. Enzymatic reaction paths as determined by transition path sampling

    NASA Astrophysics Data System (ADS)

    Masterson, Jean Emily

    Enzymes are biological catalysts capable of enhancing the rates of chemical reactions by many orders of magnitude as compared to solution chemistry. Since the catalytic power of enzymes routinely exceeds that of the best artificial catalysts available, there is much interest in understanding the complete nature of chemical barrier crossing in enzymatic reactions. Two specific questions pertaining to the source of enzymatic rate enhancements are investigated in this work. The first is the issue of how fast protein motions of an enzyme contribute to chemical barrier crossing. Our group has previously identified sub-picosecond protein motions, termed promoting vibrations (PVs), that dynamically modulate chemical transformation in several enzymes. In the case of human heart lactate dehydrogenase (hhLDH), prior studies have shown that a specific axis of residues undergoes a compressional fluctuation towards the active site, decreasing a hydride and a proton donor--acceptor distance on a sub-picosecond timescale to promote particle transfer. To more thoroughly understand the contribution of this dynamic motion to the enzymatic reaction coordinate of hhLDH, we conducted transition path sampling (TPS) using four versions of the enzymatic system: a wild type enzyme with natural isotopic abundance; a heavy enzyme where all the carbons, nitrogens, and non-exchangeable hydrogens were replaced with heavy isotopes; and two versions of the enzyme with mutations in the axis of PV residues. We generated four separate ensembles of reaction paths and analyzed each in terms of the reaction mechanism, time of barrier crossing, dynamics of the PV, and residues involved in the enzymatic reaction coordinate. We found that heavy isotopic substitution of hhLDH altered the sub-picosecond dynamics of the PV, changed the favored reaction mechanism, dramatically increased the time of barrier crossing, but did not have an effect on the specific residues involved in the PV. In the mutant systems, we observed changes in the reaction mechanism and altered contributions of the mutated residues to the enzymatic reaction coordinate, but we did not detect a substantial change in the time of barrier crossing. These results confirm the importance of maintaining the dynamics and structural scaffolding of the hhLDH PV in order to facilitate facile barrier passage. We also utilized TPS to investigate the possible role of fast protein dynamics in the enzymatic reaction coordinate of human dihydrofolate reductase (hsDHFR). We found that sub-picosecond dynamics of hsDHFR do contribute to the reaction coordinate, whereas this is not the case in the E. coli version of the enzyme. This result indicates a shift in the DHFR family to a more dynamic version of catalysis. The second inquiry we addressed in this thesis regarding enzymatic barrier passage concerns the variability of paths through reactive phase space for a given enzymatic reaction. We further investigated the hhLDH-catalyzed reaction using a high-perturbation TPS algorithm. Though we saw that alternate reaction paths were possible, the dominant reaction path we observed corresponded to that previously elucidated in prior hhLDH TPS studies. Since the additional reaction paths we observed were likely high-energy, these results indicate that only the dominant reaction path contributes significantly to the overall reaction rate. In conclusion, we show that the enzymes hhLDH and hsDHFR exhibit paths through reactive phase space where fast protein motions are involved in the enzymatic reaction coordinate and exhibit a non-negligible contribution to chemical barrier crossing.

  14. 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

  15. Protein phosphorylations in poliovirus infected cells.

    PubMed

    James, L A; Tershak, D R

    1981-01-01

    In vivo phosphorylation of proteins that are associated with polysomes of poliovirus-infected VERO (African green monkey kidney) and HeLa (Henrietta Lacks) cells differed from phosphorylations observed with uninfected cells that were fed fresh medium. With both types of cells infection stimulated phosphorylation of proteins with molecular weights of 40 000-41 000, 39 000, 34 000, 32 000, and 24 000. Similarities of phosphorylations in VERO and HeLa cells suggest that they are a specific consequence of infection and might serve a regulatory function during protein synthesis. PMID:6260321

  16. Quantification of Protein Kinase Enzymatic Activity in Unfractionated Cell Lysates Using CSox-Based Sensors

    PubMed Central

    Beck, Jon R.; Peterson, Laura B.; Imperiali, Barbara; Stains, Cliff I.

    2014-01-01

    Defining perturbations in protein kinase activity within biological samples can provide insight into disease mechanisms as well as potential targets for drug development. In this protocol, we present a method that utilizes a phosphorylation-sensitive amino acid, termed CSox, to afford kinase-selective biosensors capable of reporting on enzymatic activity directly in biological samples. These sensors produce an increase in fluorescence in response to phosphorylation of an amino acid residue adjacent to CSox. Probes can be designed for either serine/threonine or tyrosine kinases and analysis can be performed using standard fluorescence equipment. The procedures provided herein represent our optimized protocols for the design, validation and application of CSox-based protein kinase activity sensors. PMID:25205563

  17. Method for the enzymatic production of hydrogen

    DOEpatents

    Woodward, J.; Mattingly, S.M.

    1999-08-24

    The present invention is an enzymatic method for producing hydrogen comprising the steps of: (a) forming a reaction mixture within a reaction vessel comprising a substrate capable of undergoing oxidation within a catabolic reaction, such as glucose, galactose, xylose, mannose, sucrose, lactose, cellulose, xylan and starch; the reaction mixture also comprising an amount of glucose dehydrogenase in an amount sufficient to catalyze the oxidation of the substrate, an amount of hydrogenase sufficient to catalyze an electron-requiring reaction wherein a stoichiometric yield of hydrogen is produced, an amount of pH buffer in an amount sufficient to provide an environment that allows the hydrogenase and the glucose dehydrogenase to retain sufficient activity for the production of hydrogen to occur and also comprising an amount of nicotinamide adenine dinucleotide phosphate sufficient to transfer electrons from the catabolic reaction to the electron-requiring reaction; (b) heating the reaction mixture at a temperature sufficient for glucose dehydrogenase and the hydrogenase to retain sufficient activity and sufficient for the production of hydrogen to occur, and heating for a period of time that continues until the hydrogen is no longer produced by the reaction mixture, wherein the catabolic reaction and the electron-requiring reactions have rates of reaction dependent upon the temperature; and (c) detecting the hydrogen produced from the reaction mixture. 8 figs.

  18. Method for the enzymatic production of hydrogen

    DOEpatents

    Woodward, Jonathan (Kingston, TN); Mattingly, Susan M. (State College, PA)

    1999-01-01

    The present invention is an enzymatic method for producing hydrogen comprising the steps of: a) forming a reaction mixture within a reaction vessel comprising a substrate capable of undergoing oxidation within a catabolic reaction, such as glucose, galactose, xylose, mannose, sucrose, lactose, cellulose, xylan and starch. The reaction mixture further comprises an amount of glucose dehydrogenase in an amount sufficient to catalyze the oxidation of the substrate, an amount of hydrogenase sufficient to catalyze an electron-requiring reaction wherein a stoichiometric yield of hydrogen is produced, an amount of pH buffer in an amount sufficient to provide an environment that allows the hydrogenase and the glucose dehydrogenase to retain sufficient activity for the production of hydrogen to occur and also comprising an amount of nicotinamide adenine dinucleotide phosphate sufficient to transfer electrons from the catabolic reaction to the electron-requiring reaction; b) heating the reaction mixture at a temperature sufficient for glucose dehydrogenase and the hydrogenase to retain sufficient activity and sufficient for the production of hydrogen to occur, and heating for a period of time that continues until the hydrogen is no longer produced by the reaction mixture, wherein the catabolic reaction and the electron-requiring reactions have rates of reaction dependent upon the temperature; and c) detecting the hydrogen produced from the reaction mixture.

  19. Accelerated Articles Enzymatically Amplified Surface Plasmon

    E-print Network

    enhance nucleic acid detection using RNA micro- arrays.12 This new enzymatic amplification method 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

  20. Kinetics of Enzymatic Depolymerization of Guar Galactomannan

    E-print Network

    Khan, Saad A.

    Kinetics of Enzymatic Depolymerization of Guar Galactomannan Shamsheer Mahammad, Robert K. Prud, 2006 A new mathematical model based on Michaelis Menten (MM) kinetics is developed to predict types of bonds with different MM kinetic parameters. The overall kinetics of the enzymatic reactions

  1. Dispersing biofilms with engineered enzymatic bacteriophage

    E-print Network

    Collins, James J.

    Dispersing biofilms with engineered enzymatic bacteriophage Timothy K. Lu* and James J. Collins engineered bacteriophage to express a biofilm-degrading en- zyme during infection to simultaneously attack substances. We show that the efficacy of biofilm removal by this two-pronged enzymatic bacteriophage strategy

  2. 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

  3. Modulation of P1798 lymphosarcoma proliferation by protein phosphorylation

    SciTech Connect

    Michnoff, C.A.H.

    1983-01-01

    The role of protein kinases in modulating cell proliferation was examined. Studies characterized the regulation of cell proliferation by adenosine 3':5'-monophosphate-dependent protein kinase (cA-Pk). Calcium/calmodulin-dependent myosin light chain kinase (MLCK) was isolated and examined as a potential substrate regulated by cA-PK in the rapidly proliferating P1798 lymphosarcoma. Modulation of cell proliferation by cA-PK was characterized by quantitating cell division by (methyl-/sup 3/H) thymidine ((/sup 3/H)-dT) incorporation into DNA, cAMP accumulations, and activation of cA-PK using P1798 lymphosarcoma cells. Epinephrine and prostaglandin E/sub 1/ (PGE/sub 1/) were demonstrated to suppress (/sup 3/H)-dT incorporation into DNA, to stimulate cAMP accumulation, and to activate cA-PK with dose-dependency. Calcium/calmodulin-dependent MLCK was partially purified from P1798 lymphosarcoma. P1798 MLCK phosphorylated myosin regulatory light chains (P-LC) from thymus, cardiac and skeletal muscles. One mol (/sup 32/Pi) was transferred into one mol cardiac or skeletal P-LC by P1798 MLCK. Apparent Km values of 65 ..mu..M and 51 ..mu..M were determined for ATP and cardiac P-LC, respectively. The apparent molecular weight of P1798 MLCK was 135,000. P1798 MLCK was phosphorylated by cA-PK. Phosphorylated MLCK showed a 41% decrease in calcium-dependent activity. Two additional protein kinases from P1798 lymphosarcoma phosphorylated cardiac and skeletal light chains (MLC).

  4. Dendritic cells require STAT-1 phosphorylated at its transactivating domain for the induction of peptide-specific CTL.

    PubMed

    Pilz, Andreas; Kratky, Wolfgang; Stockinger, Silvia; Simma, Olivia; Kalinke, Ulrich; Lingnau, Karen; von Gabain, Alexander; Stoiber, Dagmar; Sexl, Veronika; Kolbe, Thomas; Rülicke, Thomas; Müller, Mathias; Decker, Thomas

    2009-08-15

    Phosphorylation of transcription factor STAT-1 on Y701 regulates subcellular localization whereas phosphorylation of the transactivating domain at S727 enhances transcriptional activity. In this study, we investigate the impact of STAT-1 and the importance of transactivating domain phosphorylation on the induction of peptide-specific CTL in presence of the TLR9-dependent immune adjuvant IC31. STAT-1 deficiency completely abolished CTL induction upon immunization, which was strongly reduced in animals carrying the mutation of the S727 phospho-acceptor site. A comparable reduction of CTL was found in mice lacking the type I IFN (IFN-I) receptor, whereas IFN-gamma-deficient mice behaved like wild-type controls. This finding suggests that S727-phosphorylated STAT-1 supports IFN-I-dependent induction of CTL. In adoptive transfer experiments, IFN-I- and S727-phosphorylated STAT-1 were critical for the activation and function of dendritic cells. Mice with a T cell-specific IFN-I receptor ablation did not show impaired CTL responses. Unlike the situation observed for CTL development S727-phosphorylated STAT-1 restrained proliferation of naive CD8(+) T cells both in vitro and following transfer into Rag-deficient mice. In summary, our data reveal a dual role of S727-phosphorylated STAT-1 for dendritic cell maturation as a prerequisite for the induction of CTL activity and for T cell autonomous control of activation-induced or homeostatic proliferation. PMID:19620292

  5. The impact of N-terminal phosphorylation on LHCII conformation in state transition

    NASA Astrophysics Data System (ADS)

    Ding, Jin-Hong; Li, Ning; Wang, Man-Liu; Zhang, Yan; Lü, Shou-Qin; Long, Mian

    2014-06-01

    State transition is an important protection mechanism of plants for maintaining optimal efficiency through redistributing unbalanced excitation energy between photo-system II (PSII) and photosystem I (PSI). This process depends on the reversible phosphorylation/dephosphorylation of the major light-harvesting complex II (LHCII) and its bi-directional migration between PSII and PSI. But it remains unclear how phosphorylation/dephosphorylation modulates the LHCII conformation and further regulates its reversible migration. Here molecular dynamics simulations (MDS) were employed to elucidate the impact of phosphorylation on LHCII conformation. The results indicated that N-terminal phosphorylation loosened LHCII trimer with decreased hydrogen bond (H-bond) interactions and extended the distances between neighboring monomers, which stemmed from the conformational adjustment of each monomer itself. Global conformational change of LHCII monomer started from its stromal Nterminal (including the phosphorylation sites) by enhancing its interaction to lipid membrane and by adjusting the interaction network with surrounded inter-monomer and intra-monomer transmembrane helixes of B, C, and A, and finally triggered the reorientation of transmembrane helixes and transferred the conformational change to luminal side helixes and loops. These results further our understanding in molecular mechanism of LHCII migration during state transition from the phosphorylation-induced microstructural feature of LHCII.

  6. 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

  7. Propylthiouracil-induced congenital hypothyroidism upregulates vimentin phosphorylation and depletes antioxidant defenses in immature rat testis.

    PubMed

    Zamoner, Ariane; Barreto, Kátia Padilha; Filho, Danilo Wilhelm; Sell, Fabíola; Woehl, Viviane Mara; Guma, Fátima Costa Rodrigues; Pessoa-Pureur, Regina; Silva, Fátima Regina Mena Barreto

    2008-03-01

    Congenital hypothyroidism was induced in rats by adding 0.05% 6-propyl-2-thiouracil in the drinking water from day 9 of gestation, and continually up to postnatal day 15. Structural alterations observed by light microscopy of seminiferous tubules and by transmission electron microscopy of Sertoli cells of treated animals were consistent with hypothyroid condition. Hypothyroidism was also associated with high phospho-p38 mitogen-activated protein kinase and decreased phospho-extracellular signal-regulated kinase 1/2 levels. Furthermore, the phosphorylation and the immunoreactivity of cytoskeletal-associated vimentin were increased without altering vimentin expression, suggesting an accumulation of insoluble and phosphorylated vimentin. These alterations in intermediate filament dynamics could result in loss of Sertoli cell cytoskeletal integrity and be somewhat related to the deleterious effects of hypothyroidism in testis. In addition, the mitochondrial alterations observed could also be related to defective cytoskeletal dynamics implying in cell damage. Moreover, we observed decreased oxygen consumption and unaltered lipid peroxidation in hypothyroid testis. However, we demonstrated decreased enzymatic and non-enzymatic antioxidant defenses, supporting an increased mitochondrial reactive oxygen species (ROS) generation, contributing to biochemical changes in hypothyroid testis. In addition, the changes in the testis histoarchitecture could be ascribed to cytoskeletal alterations, decreased antioxidant defenses, and increased ROS generation, leading to oxidative stress in the organ. PMID:18316471

  8. 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

  9. Tyrosine phosphorylation of the asialoglycoprotein receptor

    SciTech Connect

    Fallon, R.J. )

    1990-02-25

    The asialoglycoprotein (ASGP) receptor undergoes constitutive endocytosis through the coated pit/coated vesicle pathway in hepatocytes. Studies on HepG2 cells have shown that the receptor is phosphorylated at serine under control conditions and following protein kinase C stimulation. This study examined whether the ASGP receptor could also serve as a substrate for a tyrosine kinase in HepG2 cells. 32P labeling was performed in membrane preparations, in permeabilized cells at 4 degrees C, and in intact cells at 37 degrees C. The phosphorylated ASGP receptor was isolated by immunoprecipitation, hydrolyzed in 6 N HCl at 110 degrees C, and analyzed by two-dimensional high voltage electrophoresis. The receptor isolated from a membrane preparation incubated in vitro with (gamma-32P)ATP incorporated radiolabel predominantly (greater than 90%) into phosphotyrosine. ASGP receptor phosphorylation at both tyrosine and serine was detected in intact cells incubated with phosphatase inhibitors for 60 min at 37 degrees C. The presence of both phenylarsine oxide (20 microM) and sodium orthovanadate (200 microM) was required for tyrosine phosphorylation. Use of these inhibitors together resulted in a 16.4-fold increase in phosphorylation of the immunoprecipitated ASGP receptor, whereas phosphorylation of total HepG2 membrane proteins was not significantly augmented by this procedure. Selective proteolytic digestion of ASGP receptors in isolated vesicles demonstrated that the phosphorylation site identified in these studies is located at tyrosine 5 in the cytoplasmic tail.

  10. Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation.

    PubMed

    Swetha, Medchalmi; Ramaiah, Kolluru V A

    2015-11-01

    Tyrosine phosphorylation of insulin receptor beta (IR?) in insulin treated HepG2 cells is inversely correlated to ser(51) phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2?) that regulates protein synthesis. Insulin stimulates interaction between IR? and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IR? and anti-phosphotyrosine antibodies, recombinant IR? and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IR? and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IR?, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2? phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IR?, reduces PKR association with the receptor, IR? in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells. PMID:26321373

  11. Phosphorylation of the hepatitis delta virus antigens.

    PubMed

    Bichko, V; Barik, S; Taylor, J

    1997-01-01

    We used two-dimensional electrophoresis (nonequilibrium pH gradient electrophoresis followed by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis) coupled with 32P labeling and immunoblotting detection with 125I-protein A to detect and quantitate phosphorylation of the large and small forms of the delta antigen (deltaAg-L and deltaAg-S, respectively). Analysis of deltaAg species from the serum and liver of an infected woodchuck as well as deltaAg species expressed in and secreted from transfected Huh7 cells revealed the following. (i) No detectable phosphorylation of deltaAg-S occurred. (ii) In virions from the serum of an infected animal and in the particles secreted from cotransfected cells, none of the deltaAg-L was phosphorylated. (iii) Only in the infected liver and in transfected cells was any phosphorylation detected; it corresponded to a monophosphorylated form of deltaAg-L. Given these results, we carried out serine-to-alanine mutagenesis of the deltaAg-L to determine whether the monophosphorylation was predominantly at a specific site on the unique 19-amino-acid (aa) extension. We mutated each of the two serines, aa 207 and 210, on this extension and also the serine at aa 177. These three mutations had no significant effect on phosphorylation. In contrast, mutagenesis to alanine of the cysteine at aa 211, which normally acts as the acceptor for farnesylation, completely inhibited phosphorylation. Our interpretation is that the site(s) of phosphorylation is probably not in the 19-aa extension unique to deltaAg-L and that phosphorylation of deltaAg-L may depend upon prior farnesylation. The possible significance of the intracellular phosphorylated forms of deltaAg-L is discussed. PMID:8985379

  12. Insulin stimulates the tyrosine phosphorylation of caveolin

    PubMed Central

    1995-01-01

    The specialized plasma membrane structures termed caveolae and the caveolar-coat protein caveolin are highly expressed in insulin- sensitive cells such as adipocytes and muscle. Stimulation of 3T3-L1 adipocytes with insulin significantly increased the tyrosine phosphorylation of caveolin and a 29-kD caveolin-associated protein in caveolin-enriched Triton-insoluble complexes. Maximal phosphorylation occurred within 5 min, and the levels of phosphorylation remained elevated for at least 30 min. The insulin-dose responses for the tyrosine phosphorylation of caveolin and the 29-kD caveolin-associated protein paralleled those for the phosphorylation of the insulin receptor. The stimulation of caveolin tyrosine phosphorylation was specific for insulin and was not observed with PDGF or EGF, although PDGF stimulated the tyrosine phosphorylation of the 29-kD caveolin- associated protein. Increased tyrosine phosphorylation of caveolin, its associated 29-kD protein, and a 60-kD protein was observed in an in vitro kinase assay after incubation of the caveolin-enriched Triton- insoluble complexes with Mg-ATP, suggesting the presence of an intrinsic tyrosine kinase in these complexes. These fractions contain only trace amounts of the activated insulin receptor. In addition, these complexes contain a 60-kD kinase detected in an in situ gel kinase assay and an approximately 60 kD protein that cross-reacts with an antibody against the Src-family kinase p59Fyn. Thus, the insulin- dependent tyrosine phosphorylation of caveolin represents a novel, insulin-specific signal transduction pathway that may involve activation of a tyrosine kinase downstream of the insulin receptor. PMID:7540611

  13. Increased phospholipase A2 activity with phosphorylation of peroxiredoxin 6 requires a conformational change in the protein.

    PubMed

    Rahaman, Hamidur; Zhou, Suiping; Dodia, Chandra; Feinstein, Sheldon I; Huang, Shaohui; Speicher, David; Fisher, Aron B

    2012-07-10

    We have shown previously and confirmed in this study that the phospholipase A(2) (PLA(2)) activity of peroxiredoxin 6 (Prdx6) is markedly increased by phosphorylation. This report evaluates the conformation and thermodynamic stability of Prdx6 protein after phosphorylation to understand the physical basis for increased activity. Phosphorylation resulted in decreased negative far-UV CD, strengthened ANS binding, and a lack of rigid tertiary structure, compatible with a change in conformation to that of a molten globule. The ?G°(D) was 3.3 ± 0.3 kcal mol(-1) for Prdx6 and 1.7 ± 0.7 kcal mol(-1) for pPrdx6, suggesting that phosphorylation destabilizes the protein. Phosphorylation of Prdx6 changed the conformation of the N-terminal domain exposing Trp 33, as determined by tryptophan fluorescence and NaI fluorescence quenching. The kinetics of interaction of proteins with unilamellar liposomes (50:25:15:10 DPPC:egg PC:cholesterol:PG molar ratio) were evaluated with tryptophan fluorescence. pPrdx6 bound to liposomes with a higher affinity (K(d) = 5.6 ± 1.2 ?M) than Prdx6 (K(d) = 24.9 ± 4.5 ?M). By isothermal titration calorimetry, pPrdx6 bound to liposomes with a large exothermic heat loss (?H = -31.49 ± 0.22 kcal mol(-1)). Correlating our conformational studies with the published crystal structure of oxidized Prdx6 suggests that phosphorylation results in exposure of hydrophobic residues, thereby providing accessibility to the sites for liposome binding. Because binding of the enzyme to the phospholipid substrate interface is a requirement for PLA(2) activity, these results indicate that a change in the conformation of Prdx6 upon its phosphorylation is the basis for enhancement of PLA(2) enzymatic activity. PMID:22663767

  14. 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...

  15. Phosphorylation based insulation devices design and implementation

    E-print Network

    Rivera Ortiz, Phillip M. (Phillip Michael)

    2013-01-01

    This thesis presents the analysis of a phosphorylation based insulation device implemented in Saccharomyces cerevisae and the minimization of the retroactivity to the input and retroactivity to the output of a single cycle ...

  16. Enzymatic Vitrectomy and Pharmacologic Vitreodynamics.

    PubMed

    Shah, Ankoor R; Trese, Michael T

    2016-01-01

    The field of vitreoretinal surgery has evolved substantially over the last several decades. Scientific advances have improved our understanding of disease pathophysiology, and new surgical adjuncts and techniques have decreased surgical time and improved patient outcomes. Pharmacologic agents have recently been developed for intraocular use in order to enhance vitreous removal and even as a nonsurgical treatment for pathology due to an abnormal vitreoretinal interface. Plasmin can successfully cause vitreous liquefaction and induce a posterior vitreous detachment. Additionally, ocriplasmin has been approved for symptomatic vitreomacular adhesion and others appear to be promising for pharmacologic manipulation of the vitreous. The ability to induce vitreous liquefaction and a complete posterior vitreous detachment (PVD) with a single intravitreal injection has potential implications for the management of multiple vitreoretinopathies. Enzymatic vitrectomy may help to reduce vitreous viscosity, thereby facilitating removal during vitrectomy and reducing surgical time, especially when using smaller-gauge vitrectomy instruments. The induction of a PVD also has the potential to reduce intraoperative complications. As we improve our understanding of the molecular flux in the vitreous cavity, pharmacologic vitreodynamics will likely become more important as it may allow for improved manipulation of intravitreal molecules. PMID:26501959

  17. Detection of Phosphorylation Status of Cytokinetic Components.

    PubMed

    Meitinger, Franz; Palani, Saravanan; Pereira, Gislene

    2016-01-01

    Yeast cells can be easily cultured, synchronized, and genetically modified making them a convenient model system to study molecular mechanisms underlying cytokinesis. Here, we describe simple methods that allow the analysis of the phosphorylation profile of cytokinetic proteins, both in vivo and in vitro, using standard laboratory equipment. In addition, we compare the ability of three different, standard, and optimized acrylamide gel conditions to separate phosphorylated forms, using the protein Inn1 as an example. PMID:26519316

  18. 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.

  19. Protein phosphorylation: Localization in regenerating optic axons

    SciTech Connect

    Larrivee, D. )

    1990-09-01

    A number of axonal proteins display changes in phosphorylation during goldfish optic nerve regeneration. (1) To determine whether the phosphorylation of these proteins was closely linked to their synthesis in the retinal ganglion cell body, cycloheximide was injected intraocularly into goldfish whose optic nerves had been regenerating for 3 weeks. Cycloheximide reduced the incorporation of (3H)proline and 32P orthophosphate into total nerve protein by 84% and 46%, respectively. Of the 20 individual proteins examined, 17 contained less than 15% of the (3H)proline label measured in corresponding controls, whereas 18 proteins contained 50% or more of the 32P label, suggesting that phosphorylation was largely independent of synthesis. (2) To determine whether the proteins were phosphorylated in the ganglion cell axons, axonal transport of proteins was blocked by intraocular injection of vincristine. Vincristine reduced (3H)proline labeling of total protein by 88% and 32P labeling by 49%. Among the individual proteins (3H)proline labeling was reduced by 90% or more in 18 cases but 32P labeling was reduced only by 50% or less. (3) When 32P was injected into the cranial cavity near the ends of the optic axons, all of the phosphoproteins were labeled more intensely in the optic tract than in the optic nerve. These results suggest that most of the major phosphoproteins that undergo changes in phosphorylation in the course of regeneration are phosphorylated in the optic axons.

  20. Protein phosphorylation in neurodegeneration: friend or foe?

    PubMed Central

    Tenreiro, Sandra; Eckermann, Katrin; Outeiro, Tiago F.

    2014-01-01

    Protein misfolding and aggregation is a common hallmark in neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), and fronto-temporal dementia (FTD). In these disorders, the misfolding and aggregation of specific proteins occurs alongside neuronal degeneration in somewhat specific brain areas, depending on the disorder and the stage of the disease. However, we still do not fully understand the mechanisms governing protein aggregation, and whether this constitutes a protective or detrimental process. In PD, alpha-synuclein (aSyn) forms protein aggregates, known as Lewy bodies, and is phosphorylated at serine 129. Other residues have also been shown to be phosphorylated, but the significance of phosphorylation in the biology and pathophysiology of the protein is still controversial. In AD and in FTD, hyperphosphorylation of tau protein causes its misfolding and aggregation. Again, our understanding of the precise consequences of tau phosphorylation in the biology and pathophysiology of the protein is still limited. Through the use of a variety of model organisms and technical approaches, we are now gaining stronger insight into the effects of phosphorylation in the behavior of these proteins. In this review, we cover recent findings in the field and discuss how targeting phosphorylation events might be used for therapeutic intervention in these devastating diseases of the nervous system. PMID:24860424

  1. Citron Rho-interacting kinase mediates arsenite-induced decrease in endothelial nitric oxide synthase activity by increasing phosphorylation at threonine 497: Mechanism underlying arsenite-induced vascular dysfunction.

    PubMed

    Seo, Jungwon; Cho, Du-Hyong; Lee, Hyeon-Ju; Sung, Min-Sun; Lee, Jee Young; Won, Kyung-Jong; Park, Jung-Hyun; Jo, Inho

    2016-01-01

    We reported that arsenite causes an acute decrease in nitric oxide (NO) production by increasing phosphorylation of endothelial NO synthase at threonine 497 (eNOS-Thr(497)); however, the detailed mechanism has not yet been clarified. Here, we investigated the kinase involving in arsenite-stimulated eNOS-Thr(497) phosphorylation. Although treatment with H-89, a known protein kinase A (PKA) inhibitor, inhibited arsenite-stimulated eNOS-Thr(497) phosphorylation, no inhibition was found in cells treated with other PKA inhibitors, including Rp-8-Br-cAMPS or PKI. Based on previous reports, we also tested whether RhoA mediates arsenite-stimulated eNOS-Thr(497) phosphorylation and found that arsenite causes an acute increase in RhoA activity. Ectopic expression of dominant negative (DN)-RhoA significantly reversed arsenite-stimulated eNOS-Thr(497) phosphorylation. An in vitro phosphorylation assay also revealed that the well-known Rho effectors, Rho-associated protein kinase 1/2 (ROCK1/2), directly phosphorylate eNOS-Thr(497). Y27632, a selective ROCK inhibitor, reversed arsenite-stimulated eNOS-Thr(497) phosphorylation. However, overexpression of a small interfering RNA (siRNA) against ROCK1/2 or DN-ROCK did not reverse arsenite-stimulated eNOS-Thr(497) phosphorylation, thereby providing no conclusive evidence of a role for ROCK1/2. Knockdown of PKC-related protein kinase 1/2, another Rho effector, also did not reverse arsenite-stimulated eNOS-Thr(497) phosphorylation. In contrast, we found that transfection with an siRNA against citron Rho-interacting kinase (CRIK), the other downstream effector of Rho, significantly reversed the arsenite-induced eNOS-Thr(497) phosphorylation that was accompanied by restoration of eNOS enzymatic activity repressed by arsenite. Moreover, CRIK directly phosphorylated eNOS-Thr(497)in vitro. Finally, we also found that arsenite increased eNOS-Thr(497) phosphorylation and decreased acetylcholine-induced vessel relaxation in rat aortas. In conclusion, we demonstrate that arsenite acutely inhibits eNOS enzymatic activity and vessel relaxation in part by increasing the RhoA/CRIK/eNOS-Thr(497) phosphorylation signaling axis, which provides a molecular mechanism underlying arsenite-induced impaired vascular diseases. PMID:26593676

  2. Structural dynamics of muscle protein phosphorylation

    PubMed Central

    Colson, Brett A.; Gruber, Simon J.; Thomas, David D.

    2013-01-01

    We have used site-directed spectroscopic probes to detect structural changes, motions, and interactions due to phosphorylation of proteins involved in the regulation of muscle contraction and relaxation. Protein crystal structures provide static snapshots that provide clues to the conformations that are sampled dynamically by proteins in the cellular environment. Our site-directed spectroscopic experiments, combined with computational simulations, extend these studies into functional assemblies in solution, and reveal details of protein regions that are too dynamic or disordered for crystallographic approaches. Here, we discuss phosphorylation-mediated structural transitions in the smooth muscle myosin regulatory light chain (RLC), the striated muscle accessory protein myosin binding protein-C (MyBP-C), and the cardiac membrane Ca2+ pump modulator phospholamban (PLB). In each of these systems, phosphorylation near the N terminus of the regulatory protein relieves an inhibitory interaction between the phosphoprotein and its regulatory target. Several additional unifying themes emerge from our studies: (a) The effect of phosphorylation is not to change the affinity of the phosphoprotein for its regulated binding partner, but to change the structure of the bound complex without dissociation. (b) Phosphorylation induces transitions between order and dynamic disorder. (c) Structural states are only loosely coupled to phosphorylation; i.e., complete phosphorylation induces dramatic functional effects with only a partial shift in the equilibrium between ordered and disordered structural states. These studies, which offer atomic-resolution insight into the structural and functional dynamics of these phosphoproteins, were inspired in part by the ground-breaking work in this field by Michael and Kate Barany. PMID:22930331

  3. Phosphorylated lymphocyte plasma-membrane proteins.

    PubMed Central

    Johnstone, A P; DuBois, J H; Crumpton, M J

    1981-01-01

    Lymphocytes were labelled by incubation with [32P]Pi and their plasma membranes isolated. Analysis by one-dimensional and two-dimensional gel electrophoresis revealed a small number of strongly phosphorylated polypeptides. Two of these were especially prominent; they had molecular weights of about 52000 and 90000, were acidic and were apparently not glycosylated. Similar patterns were obtained for quiescent T- and B-lymphocytes from different species and for cultured lymphoblastoid cells, although the relative amounts of the labelled polypeptides varied. Immunoprecipitation analyses of the detergent-solubilized 32P-labelled plasma membranes indicated that the glycosylated polypeptide of the human major transplantation (HLA-A and HLA-B) antigens and its mouse and pig counterparts are phosphorylated. In contrast, no phosphorylation of the membrane-associated immunoglobulin, the mouse Thy-1 antigen or the human HLA-DRw(Ia) antigen was detected. The phosphorylation patterns of human peripheral blood and nude-mouse spleen lymphocytes did not change during the period 5-30min after mitogen stimulation. Therefore a change in the phosphorylation of plasma-membrane protein(s) is probably not an early biochemical event in the initiation of T-lymphocyte and B-lymphocyte growth, although a rapid transient change cannot be ruled out. Similar plasma-membrane phosphorylation patterns were also obtained by incubating the purified plasma membrane with [gamma-32P]ATP. The phosphorylation of the 90000-mol.wt. polypeptide was particularly rapid and was stimulated by the addition of cyclic AMP. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:7305985

  4. 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

  5. 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.

  6. Regulation of Elg1 activity by phosphorylation.

    PubMed

    Shkedy, Dganit; Singh, Nishant; Shemesh, Keren; Amir, Ayelet; Geiger, Tamar; Liefshitz, Batia; Harari, Yaniv; Kupiec, Martin

    2015-12-01

    ELG1 is a conserved gene with important roles in the maintenance of genome stability. Elg1's activity prevents gross chromosomal rearrangements, maintains proper telomere length regulation, helps repairing DNA damage created by a number of genotoxins and participates in sister chromatid cohesion. Elg1 is evolutionarily conserved, and its Fanconi Anemia-related mammalian ortholog (also known as ATAD5) is embryonic lethal when lost in mice and acts as a tumor suppressor in mice and humans. Elg1 encodes a protein that forms an RFC-like complex that unloads the replicative clamp, PCNA, from DNA, mainly in its SUMOylated form. We have identified 2 different regions in yeast Elg1 that undergo phosphorylation. Phosphorylation of one of them, S112, is dependent on the ATR yeast ortholog, Mec1, and probably is a direct target of this kinase. We show that phosphorylation of Elg1 is important for its role at telomeres. Mutants unable to undergo phosphorylation suppress the DNA damage sensitivity of ?rad5 mutants, defective for an error-free post-replicational bypass pathway. This indicates a role of phosphorylation in the regulation of DNA repair. Our results open the way to investigate the mechanisms by which the activity of Elg1 is regulated during DNA replication and in response to DNA damage. PMID:26177013

  7. Sensitive western blotting for detection of endogenous Ser129-phosphorylated ?-synuclein in intracellular and extracellular spaces

    PubMed Central

    Sasaki, Asuka; Arawaka, Shigeki; Sato, Hiroyasu; Kato, Takeo

    2015-01-01

    ?-Synuclein deposited in Lewy bodies, a pathological hallmark of Parkinson’s disease (PD), is highly phosphorylated at serine 129 (Ser129). In contrast, there is very little Ser129-phosphorylated ?-synuclein in the normal brains. This difference suggests that Ser129-phosphorylation is involved in neurodegenerative processes of PD. However, the role of this modification remains unclear. One limiting factor for relevant biochemical analyses is that it is difficult to detect endogenous Ser129-phosphoryated ?-synuclein by western blotting, because ?-synuclein monomers detached from the transferred membrane during incubation. Here, we reported that combination fixation of the transferred membrane with 4% paraformaldehyde and 0.01?~?0.1% glutaraldehyde produced an approximately 10-fold increase in the sensitivity for Ser129-phosphorylated ?-synuclein monomers, allowing detection of endogenous proteins even in conditioned medium, human cerebrospinal fluid, and extracts from cell lines and human brain. This method may enable more detailed biochemical analyses for ?-synuclein transmission between intra and extracellular spaces under physiological and pathological conditions. PMID:26381815

  8. Review: Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

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

    2011-07-16

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

  9. Gas-Phase Acidities of Phosphorylated Amino Acids.

    PubMed

    Stover, Michele L; Plummer, Chelsea E; Miller, Sean R; Cassady, Carolyn J; Dixon, David A

    2015-11-19

    Gas-phase acidities and heats of formation have been predicted at the G3(MP2)/SCRF-COSMO level of theory for 10 phosphorylated amino acids and their corresponding amides, including phospho-serine (pSer), -threonine (pThr), and -tyrosine (pTyr), providing the first reliable set of these values. The gas-phase acidities (GAs) of the three named phosphorylated amino acids and their amides have been determined using proton transfer reactions in a Fourier transform ion cyclotron mass spectrometer. Excellent agreement was found between the experimental and predicted GAs. The phosphate group is the deprotonation site for pSer and pThr and deprotonation from the carboxylic acid generated the lowest energy anion for pTyr. The infrared spectra were calculated for six low energy anions of pSer, pThr, and pTyr. For deprotonated pSer and pThr, good agreement is found between the experimental IRMPD spectra and the calculated spectra for our lowest energy anion structure. For pTyr, the IR spectra for a higher energy phosphate deprotonated structure is in good agreement with experiment. Additional experiments tested electrospray ionization (ESI) conditions for pTyr and determined that variations in solvent, temperature, and voltage can result in a different experimental GA value, indicating that ESI conditions affect the conformation of the pTyr anion. PMID:26492552

  10. Phosphorylation of Mycobacterium tuberculosis protein tyrosine kinase A PtkA by Ser/Thr protein kinases.

    PubMed

    Zhou, Peifu; Wong, Dennis; Li, Wu; Xie, Jianping; Av-Gay, Yossef

    2015-11-13

    Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has inflicted about one third of mankind and claims millions of deaths worldwide annually. Signalling plays an important role in Mtb pathogenesis and persistence, and thus represents attractive resource for drug target candidates. Here, we show that protein tyrosine kinase A (PtkA) can be phosphorylated by Mtb endogenous eukaryotic-like Ser/Thr protein kinases (eSTPKs). Kinase assays showed that PknA, PknD, PknF, and PknK can phosphorylate PtkA in dose- and time-dependent manner. Enzyme kinetics suggests that PknA has the highest affinity and enzymatic efficiency towards PtkA. Furthermore, protein-protein interaction assay in surrogate host showed that PtkA interacts with multi-eSTPKs in vivo, including PknA. Lastly, we show that PtkA phosphorylation by eSTPKs occurs on threonine residues and may effect tyrosine phosphorylation levels and thus PtkA activity in vitro. These results demonstrate that PtkA can serve as a substrate to many eSTPKs and suggests that's its activity can be regulated. PMID:26417687

  11. Src kinase regulation by phosphorylation and dephosphorylation

    SciTech Connect

    Roskoski, Robert . 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.

  12. Measuring Gli2 Phosphorylation by Selected Reaction Monitoring Mass Spectrometry

    PubMed Central

    Ahrends, Robert; Niewiadomski, Pawel; Teruel, Mary N.; Rohatgi, Rajat

    2015-01-01

    Phosphorylation is an important mechanism by which Gli proteins are regulated. When the Hedgehog (Hh) pathway is activated, multiple serine and threonine residues of Gli2 are dephosphorylated, while at least one residue undergoes phosphorylation. These changes in phosphorylation have functional relevance for the transcriptional activity of Gli proteins, as shown by in vitro and in vivo assays on Gli mutants lacking the phosphorylated residues. Here, we describe a method of quantitatively monitoring the phosphorylation of Gli proteins by triple quadrupole mass spectrometry of Gli2 immunoprecipitated from cell lysates. This method is broadly applicable to the monitoring of phosphorylation changes of immunoprecipitated Gli proteins when the putative phosphosites are known. PMID:26179043

  13. Rapid alteration of protein phosphorylation during postmortem: implication in the study of protein phosphorylation

    PubMed Central

    Wang, Yifan; Zhang, Yanchong; Hu, Wen; Xie, Shutao; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2015-01-01

    Protein phosphorylation is an important post-translational modification of proteins. Postmortem tissues are widely being utilized in the biomedical studies, but the effects of postmortem on protein phosphorylation have not been received enough attention. In the present study, we found here that most proteins in mouse brain, heart, liver, and kidney were rapidly dephosphorylated to various degrees during 20?sec to 10?min postmortem. Phosphorylation of tau at Thr212 and glycogen synthase kinase 3? (GSK-3?) at Ser9 was reduced by 50% in the brain with 40?sec postmortem, a regular time for tissue processing. During postmortem, phosphorylation of cAMP-dependent protein kinase (PKA) and AMP activated kinase (AMPK) was increased in the brain, but not in other organs. Perfusion of the brain with cold or room temperature phosphate-buffered saline (PBS) also caused significant alteration of protein phosphorylation. Cooling down and maintaining mouse brains in the ice-cold buffer prevented the alteration effectively. This study suggests that phosphorylation of proteins is rapidly changed during postmortem. Thus, immediate processing of tissues followed by cooling down in ice-cold buffer is vitally important and perfusion has to be avoided when protein phosphorylation is to be studied. PMID:26511732

  14. Rapid alteration of protein phosphorylation during postmortem: implication in the study of protein phosphorylation.

    PubMed

    Wang, Yifan; Zhang, Yanchong; Hu, Wen; Xie, Shutao; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2015-01-01

    Protein phosphorylation is an important post-translational modification of proteins. Postmortem tissues are widely being utilized in the biomedical studies, but the effects of postmortem on protein phosphorylation have not been received enough attention. In the present study, we found here that most proteins in mouse brain, heart, liver, and kidney were rapidly dephosphorylated to various degrees during 20?sec to 10?min postmortem. Phosphorylation of tau at Thr212 and glycogen synthase kinase 3? (GSK-3?) at Ser9 was reduced by 50% in the brain with 40?sec postmortem, a regular time for tissue processing. During postmortem, phosphorylation of cAMP-dependent protein kinase (PKA) and AMP activated kinase (AMPK) was increased in the brain, but not in other organs. Perfusion of the brain with cold or room temperature phosphate-buffered saline (PBS) also caused significant alteration of protein phosphorylation. Cooling down and maintaining mouse brains in the ice-cold buffer prevented the alteration effectively. This study suggests that phosphorylation of proteins is rapidly changed during postmortem. Thus, immediate processing of tissues followed by cooling down in ice-cold buffer is vitally important and perfusion has to be avoided when protein phosphorylation is to be studied. PMID:26511732

  15. Dynamics of human keratin 18 phosphorylation: polarized distribution of phosphorylated keratins in simple epithelial tissues

    PubMed Central

    1995-01-01

    Phosphorylation of keratin polypeptides 8 and 18 (K8/18) and other intermediate filament proteins results in their reorganization in vitro and in vivo. In order to study functional aspects of human K18 phosphorylation, we generated and purified a polyclonal antibody (termed 3055) that specifically recognizes a major phosphorylation site (ser52) of human K18 but not dephosphorylated K18 or a ser52-->ala K18 mutant. Pulse-chase experiments followed by immunoprecipitation and peptide mapping of in vivo 32PO4-labeled K8/18 indicated that the overall phosphorylation turnover rate is faster for K18 versus K8, and that ser52 of K18 is a highly dynamic phosphorylation site. Isoelectric focusing of 32PO4 labeled K18 followed by immunoblotting with 3055 showed that the major phosphorylated K18 species contain ser52 phosphorylation but that some K18 molecules exist that are preferentially phosphorylated on K18 sites other than ser52. Immunoblotting of total cell lysates obtained from cells at different stages of the cell cycle showed that ser52 phosphorylation increases three to fourfold during the S and G2/M phases of the cell cycle. Immunofluorescence staining of cells at different stages of mitosis, using 3055 or other antibodies that recognize the total keratin pool, resulted in preferential binding of the 3055 antibody to the reorganized keratin fraction. Staining of human tissues or tissues from transgenic mice that express human K18 showed that the phospho-ser52 K18 species are located preferentially in the basolateral and apical domains in the liver and pancreas, respectively, but no preferential localization was noted in other simple epithelial organs examined. Our results support a model whereby phosphorylated intermediate filaments are localized in specific cellular domains depending on the tissue type and site(s) of phosphorylation. In addition, ser52 of human K18 is a highly dynamic phosphorylation site that undergoes modulation during the S and G2/M phases of the cell cycle in association with filament reorganization. PMID:8522590

  16. Nucleoside phosphorylation by the mineral schreibersite

    PubMed Central

    Gull, Maheen; Mojica, Mike A.; Fernández, Facundo M.; Gaul, David A.; Orlando, Thomas M.; Liotta, Charles L.; Pasek, Matthew A.

    2015-01-01

    Phosphorylation of the nucleosides adenosine and uridine by the simple mixing and mild heating of aqueous solutions of the organic compounds with synthetic analogs of the meteoritic mineral schreibersite, (Fe,Ni)3P under slightly basic conditions (pH ~9) is reported. These results suggest a potential role for meteoritic phosphorus in the origin and development of early life. PMID:26606901

  17. Nucleoside phosphorylation by the mineral schreibersite.

    PubMed

    Gull, Maheen; Mojica, Mike A; Fernández, Facundo M; Gaul, David A; Orlando, Thomas M; Liotta, Charles L; Pasek, Matthew A

    2015-01-01

    Phosphorylation of the nucleosides adenosine and uridine by the simple mixing and mild heating of aqueous solutions of the organic compounds with synthetic analogs of the meteoritic mineral schreibersite, (Fe,Ni)3P under slightly basic conditions (pH ~9) is reported. These results suggest a potential role for meteoritic phosphorus in the origin and development of early life. PMID:26606901

  18. Systematic Discovery of In Vivo Phosphorylation Networks

    E-print Network

    .05.052 SUMMARY Protein kinases control cellular decision pro- cesses by phosphorylating specific substrates, and expression, on cellular substrate specific- ity. We have developed an approach (Networ- KIN) that augments and recent experiments employing mass spectrometry (MS) tech- niques have identified thousands of in vivo

  19. The universality of enzymatic rate temperature dependency

    E-print Network

    Tawfik, Dan S.

    temperatures, become >10-fold less powerful catalysts per 25-C temperature increase. Among other factors and reaction rates, the strength of molecular interactions, and other physic-chemical properties. The relationsThe universality of enzymatic rate­ temperature dependency Mikael Elias1 , Grzegorz Wieczorek2

  20. 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...

  1. Frank Westheimer's Early Demonstration of Enzymatic Specificity

    ERIC Educational Resources Information Center

    Ault, Addison

    2008-01-01

    In this article I review one of the most significant accomplishments of Frank H. Westheimer, one of the most respected chemists of the 20th century. This accomplishment was a series of stereospecific enzymatic oxidation and reduction experiments that led chemists to recognize what we now call the enantiotopic and diastereotopic relationships of…

  2. A singular enzymatic megacomplex from Bacillus subtilis

    E-print Network

    Rudner, David

    A singular enzymatic megacomplex from Bacillus subtilis Paul D. Straight*, Michael A. Fischbach% of the Bacillus subtilis genome, encodes the subunits of 2.5 megadalton active hybrid NRPS/PKS. Many copies enzymes in the context of their native producer organisms. The genome of Bacillus subtilis contains

  3. 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…

  4. A Method to Determine the Ionization Efficiency Change of Peptides Caused by Phosphorylation

    PubMed Central

    Gao, Yuan; Wang, Yinsheng

    2007-01-01

    Quantitative assessment of post-translational modifications in proteins by mass spectrometry often requires the consideration of the alteration in ionization efficiency of peptides induced by the modification. Herein, we introduced a method to measure the relative ionization efficiencies of peptides using specifically designed unlabeled peptides. In our design, the peptide under study, in either the unmodified or modified form, is linked with an internal standard peptide via an enzyme cleavage site; thus, after enzymatic digestion, we could obtain readily a 1:1 ratio between the peptide under investigation and the internal standard peptide. The relative ionization efficiencies of the modified and unmodified peptides can then be calculated from the modification-induced change in the ratio of relative abundances of the ion of the peptide of interest over that of the internal standard peptide. We demonstrated the usefulness of the method by assessing the change in ionization efficiencies of four peptides introduced by phosphorylation. PMID:17870613

  5. BSA Treatment to Enhance Enzymatic Hydrolysis of Cellulose in Lignin

    E-print Network

    California at Riverside, University of

    BSA Treatment to Enhance Enzymatic Hydrolysis of Cellulose in Lignin Containing Substrates Bin Yang stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92. Similar improve- ments were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX

  6. Post-Translational Phosphorylation of Serine 74 of Human Deoxycytidine Kinase Favors the Enzyme Adopting the Open Conformation Making It Competent for Nucleoside Binding and Release

    SciTech Connect

    Hazra, Saugata; Szewczak, Andrzej; Ort, Stephan; Konrad, Manfred; Lavie, Arnon

    2012-03-26

    Deoxycytidine kinase (dCK) uses either ATP or UTP as a phosphoryl donor to catalyze the phosphorylation of nucleoside acceptors. The kinetic properties of human dCK are modulated in vivo by phosphorylation of serine 74. This residue is a part of the insert region and is distant from the active site. Replacing the serine with a glutamic acid (S74E variant) can mimic phosphorylation of Ser74. To understand how phosphorylation affects the catalytic properties of dCK, we examined the S74E variant of dCK both structurally and kinetically. We observe that the presence of a glutamic acid at position 74 favors the adoption by the enzyme of the open conformation. Glu74 stabilizes the open conformation by directly interacting with the indole side chain of Trp58, a residue that is in the proximity of the base of the nucleoside substrate. The open dCK conformation is competent for the binding of nucleoside but not for phosphoryl transfer. In contrast, the closed conformation is competent for phosphoryl transfer but not for product release. Thus, dCK must make the transition between the open and closed states during the catalytic cycle. We propose a reaction scheme for dCK that incorporates the transition between the open and closed states, and this serves to rationalize the observed kinetic differences between wild-type dCK and the S74E variant.

  7. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    SciTech Connect

    Karen S. Browning

    2009-06-15

    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

  8. Site of phosphorylation of SpoIIAA, the anti-anti-sigma factor for sporulation-specific sigma F of Bacillus subtilis.

    PubMed Central

    Najafi, S M; Willis, A C; Yudkin, M D

    1995-01-01

    Sigma F is regulated by an anti-sigma factor, SpoIIAB, and an anti-anti-sigma factor, SpoIIAA. SpoIIAB also functions as a phosphokinase which transfers phosphate from ATP to SpoIIAA; this phosphorylation is thought to be involved in the regulatory mechanism. By using [gamma-32P]ATP to phosphorylate SpoIIAA, cleaving the protein proteolytically, and analyzing the one resulting radiolabelled peptide by the Edman degradation procedure, we show that the site of phosphorylation in SpoIIAA is Ser-58. PMID:7751305

  9. [The effect of butylcaptax on oxidative phosphorylation and the activity of multienzyme systems of the mitochondrial membranes in the rat liver].

    PubMed

    Mirakhmedov, A K; Rustamov, R D; Almatov, K T; Agzamov, Kh

    1991-01-01

    The oxidative phosphorylation and the activity of polyenzymatic systems in the rat liver mitochondrial membranes were studied upon a 5 day administration to rats of butylcaptax in the dose of 1/10 LD50. It is established that butylcaptax led to significant disturbances in the system of oxidative phosphorylation and in the transfer of electrons in the rat liver mitochondrial respiratory chain. PMID:1803706

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

    SciTech Connect

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

    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.

  11. Phosphorylation of human DNA polymerase ? by the cyclin-dependent kinase Cdk2/cyclin A complex is modulated by its association with proliferating cell nuclear antigen

    PubMed Central

    Frouin, Isabelle; Toueille, Magali; Ferrari, Elena; Shevelev, Igor; Hübscher, Ulrich

    2005-01-01

    DNA polymerase (Pol) ? is a member of the Pol X family and possesses four different enzymatic activities, being DNA polymerase, terminal transferase, deoxyribose phosphate lyase and polynucleotide synthetase, all localized in its C-terminal region. On the basis of its biochemical properties, Pol ? has been implicated in various DNA repair pathways, such as abasic site translesion DNA synthesis, base excision repair and non-homologous end joining of double strand breaks. However, its role in vivo has not yet been elucidated. In addition, Pol ? has been shown to interact with the replication clamp proliferating cell nuclear antigen (PCNA) in vitro and in vivo. In this work, we searched by affinity chromatography for novel partners and we identified the cyclin-dependent kinase Cdk2 as novel partner of Pol ?. Pol ? is phosphorylated in vitro by several Cdk/cyclin complexes, including Cdk2/cyclin A, in its proline-serine-rich domain. While the polymerase activity of Pol ? was not affected by Cdk2/cyclin A phosphorylation, phosphorylation of Pol ? was decreased by its interaction with PCNA. Finally, Pol ? is also phosphorylated in vivo in human cells and this phosphorylation is modulated during the cell cycle. PMID:16174846

  12. Hamartin-Hsp70 interaction is necessary for Akt-dependent tuberin phosphorylation during heat shock.

    PubMed

    Inoue, Hirohumi; Ndong, Moussa; Suzuki, Tsukasa; Kazami, Machiko; Uyama, Takumi; Kobayashi, Ken-Ichi; Tadokoro, Tadahiro; Yamamoto, Yuji

    2009-11-01

    Hamartin and tuberin interact directly to regulate cell growth negatively. In this study, far-western blotting revealed that hamartin binds directly Heat shock protein 70 (Hsp70), even in the absence of tuberin. While the hamartin-tuberin complex acts as a sensor for a variety of types of stress, it is unclear how the complex is regulated under stress conditions. We found that the hamartin-Hsp70 interaction is stabilized during heat shock. On the other hand, tuberin underwent degradation through phosphorylation in an Akt-dependent manner. Furthermore, we found that when Hsp70 expression was inhibited by N-formyl-3,4-methylenedioxy-benzylidene-gamma-butyrolactam (KNK437), Akt phosphorylation on site Ser308 diminished and tuberin was not phosphorylated at Thr1462 during heat shock. We conclude that both hamartin and Hsp70 increase in response to heat shock, whereas tuberin is phosphorylated and thereafter degraded via the PI3K/Akt pathway. Through this pathway, hamartin-Hsp70 plays a crucial role as a scaffolding protein that transfers the Akt signal to tuberin. PMID:19897899

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

    PubMed

    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

  14. 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

  15. Phosphorylated tyrosine in the flagellum filament protein of Pseudomonas aeruginosa

    SciTech Connect

    Kelly-Wintenberg, K.; Anderson, T.; Montie, T.C. )

    1990-09-01

    Purified flagella from two strains of {sup 32}P-labeled Pseudomonas aeruginosa were shown to be phosphorylated. This was confirmed by autoradiography of flagellin protein in polyacrylamide gels. Thin-layer electrophoresis and autoradiography of flagellin partial hydrolysates indicated that phosphotyrosine was the major phosphorylated amino acid. High-pressure liquid chromatographic analysis confirmed the presence of phosphotyrosine in flagellum filament protein. Preliminary data indicated that less than one tyrosine per subunit was phosphorylated. No evidence was found for phosphorylation of serine or threonine. A function related to tyrosine phosphorylation has not been determined.

  16. Phosphorylated tyrosine in the flagellum filament protein of Pseudomonas aeruginosa.

    PubMed Central

    Kelly-Wintenberg, K; Anderson, T; Montie, T C

    1990-01-01

    Purified flagella from two strains of 32P-labeled Pseudomonas aeruginosa were shown to be phosphorylated. This was confirmed by autoradiography of flagellin protein in polyacrylamide gels. Thin-layer electrophoresis and autoradiography of flagellin partial hydrolysates indicated that phosphotyrosine was the major phosphorylated amino acid. High-pressure liquid chromatographic analysis confirmed the presence of phosphotyrosine in flagellum filament protein. Preliminary data indicated that less than one tyrosine per subunit was phosphorylated. No evidence was found for phosphorylation of serine or threonine. A function related to tyrosine phosphorylation has not been determined. Images PMID:1697577

  17. Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo

    SciTech Connect

    Ray, L.B.; Sturgill, T.W. )

    1988-06-01

    Exposure of 3T3-L1 cells to insulin stimulates a soluble, serine(threonine)-specific protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) in vitro. The enzyme, termed MAP kinase, was isolated from insulin-treated or control cells radiolabeled with {sup 32}P{sub i}. A 40-kDa phosphoprotein was found to elute in exact correspondence with enzymatic activity during hydrophobic interaction and gel filtration chromatography of extracts from cells stimulated with insulin. Both MAP kinase activity and the phosphoprotein were absent in fractions prepared from untreated cells. The {sup 32}P incorporated into the 40-kDa protein was stable during treatment with alkali. Phospho amino acid analysis confirmed that the radiolabel was primarily incorporated into phosphotyrosine and to a lesser extend phosphothreonine. In addition, MAP kinase was incompletely but specifically adsorbed by antibodies to phosphotyrosine. The authors conclude, based on these data and additional studies from this laboratory, that MAP kinase is phosphorylated on tyrosine in vivo. The data are consistent with the possibility that MAP kinase may be a substrate for the insulin receptor or another insulin-regulated tyrosine kinase.

  18. Respiration, oxidative phosphorylation, and uncoupling protein in Candida albicans.

    PubMed

    Cavalheiro, R A; Fortes, F; Borecký, J; Faustinoni, V C; Schreiber, A Z; Vercesi, A E

    2004-10-01

    The respiration, membrane potential (Deltapsi), and oxidative phosphorylation of mitochondria in situ were determined in spheroplasts obtained from Candida albicans control strain ATCC 90028 by lyticase treatment. Mitochondria in situ were able to phosphorylate externally added ADP (200 microM) in the presence of 0.05% BSA. Mitochondria in situ generated and sustained stable mitochondrial Deltapsi respiring on 5 mM NAD-linked substrates, 5 mM succinate, or 100 microM N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride plus 1 mM ascorbate. Rotenone (4 microM) inhibited respiration by 30% and 2 micro M antimycin A or myxothiazole and 1 mM cyanide inhibited it by 85%. Cyanide-insensitive respiration was partially blocked by 2 mM benzohydroxamic acid, suggesting the presence of an alternative oxidase. Candida albicans mitochondria in situ presented a carboxyatractyloside-insensitive increase of Deltapsi induced by 5 mM ATP and 0.5% BSA, and Deltapsi decrease induced by 10 microM linoleic acid, both suggesting the existence of an uncoupling protein. The presence of this protein was subsequently confirmed by immunodetection and respiration experiments with isolated mitochondria. In conclusion, Candida albicans ATCC 90028 possesses an alternative electron transfer chain and alternative oxidase, both absent in animal cells. These pathways can be exceptional targets for the design of new chemotherapeutic agents. Blockage of these respiratory pathways together with inhibition of the uncoupling protein (another potential target for drug design) could lead to increased production of reactive oxygen species, dysfunction of Candida mitochondria, and possibly to oxidative cell death. PMID:15448865

  19. Solid polymer electrolyte from phosphorylated chitosan

    SciTech Connect

    Fauzi, Iqbal Arcana, I Made

    2014-03-24

    Recently, the need of secondary battery application continues to increase. The secondary battery which using a liquid electrolyte was indicated had some weakness. A solid polymer electrolyte is an alternative electrolytes membrane which developed in order to replace the liquid electrolyte type. In the present study, the effect of phosphorylation on to polymer electrolyte membrane which synthesized from chitosan and lithium perchlorate salts was investigated. The effect of the component’s composition respectively on the properties of polymer electrolyte, was carried out by analyzed of it’s characterization such as functional groups, ion conductivity, and thermal properties. The mechanical properties i.e tensile resistance and the morphology structure of membrane surface were determined. The phosphorylation processing of polymer electrolyte membrane of chitosan and lithium perchlorate was conducted by immersing with phosphoric acid for 2 hours, and then irradiated on a microwave for 60 seconds. The degree of deacetylation of chitosan derived from shrimp shells was obtained around 75.4%. Relative molecular mass of chitosan was obtained by viscometry method is 796,792 g/mol. The ionic conductivity of chitosan membrane was increase from 6.33 × 10{sup ?6} S/cm up to 6.01 × 10{sup ?4} S/cm after adding by 15 % solution of lithium perchlorate. After phosphorylation, the ionic conductivity of phosphorylated lithium chitosan membrane was observed 1.37 × 10{sup ?3} S/cm, while the tensile resistance of 40.2 MPa with a better thermal resistance. On the strength of electrolyte membrane properties, this polymer electrolyte membrane was suggested had one potential used for polymer electrolyte in field of lithium battery applications.

  20. 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.

  1. Enzymatic polymerization of dihydroquercetin using bilirubin oxidase.

    PubMed

    Khlupova, M E; Vasil'eva, I S; Shumakovich, G P; Morozova, O V; Chertkov, V A; Shestakova, A K; Kisin, A V; Yaropolov, A I

    2015-02-01

    Dihydroquercetin (or taxifolin) is one of the most famous flavonoids and is abundant in Siberian larch (Larix sibirica). The oxidative polymerization of dihydroquercetin (DHQ) using bilirubin oxidase as a biocatalyst was investigated and some physicochemical properties of the products were studied. DHQ oligomers (oligoDHQ) with molecular mass of 2800 and polydispersity of 8.6 were obtained by enzymatic reaction under optimal conditions. The oligomers appeared to be soluble in dimethylsulfoxide, dimethylformamide, and methanol. UV-visible spectra of oligoDHQ in dimethylsulfoxide indicated the presence of highly conjugated bonds. The synthesized oligoDHQ was also characterized by FTIR and (1)H and (13)C NMR spectroscopy. Comparison of NMR spectra of oligoDHQ with DHQ monomer and the parent flavonoids revealed irregular structure of a polymer formed via the enzymatic oxidation of DHQ followed by nonselective radical polymerization. As compared with the monomer, oligoDHQ demonstrated higher thermal stability and high antioxidant activity. PMID:25756538

  2. Production of MAG via enzymatic glycerolysis

    NASA Astrophysics Data System (ADS)

    Jamlus, Norul Naziraa Ahmad; Derawi, Darfizzi; Salimon, Jumat

    2015-09-01

    Enzymatic glycerolysis of a medium chain methyl ester, methyl laurate was performed using lipase Candida antarctica (Novozyme 435) for 6 hours at 55°C. The percentage of components mixture of product were determined by using gas chromatography technique. The enzymatic reaction was successfully produced monolaurin (45.9 %), dilaurin (47.1 %) and trilaurin (7.0 %) respectively. Thin layer chromatography (TLC) plate also showed a good separation of component spots. Fourier transformation infra-red (FTIR) spectrum showed the presence of ester carbonyl at wavenumber 1739.99 cm-1 and hydrogen bonded O-H at 3512.03 cm-1. The product is potentially to be used as emulsifier and additive in food industry, pharmaceutical, as well as antibacterial.

  3. Enzymatically Triggered Actuation of Miniaturized Tools

    PubMed Central

    Bassik, Noy; Brafman, Alla; Zarafshar, Aasiyeh M.; Jamal, Mustapha; Luvsanjav, Delgermaa; Selaru, Florin M.; Gracias, David H.

    2010-01-01

    We demonstrate a methodology that utilizes the specificity of enzyme-substrate biomolecular interactions to trigger miniaturized tools under biocompatible conditions. Miniaturized grippers were constructed using multilayer hinges that employed intrinsic strain energy and biopolymer triggers, as well as ferromagnetic elements. This composition obviated the need for external energy sources, and allowed for remote manipulation of the tools. Selective enzymatic degradation of biopolymer hinge components triggered closing of the grippers; subsequent reopening was achieved with an orthogonal enzyme. We highlight the utility of these enzymatically triggered tools by demonstrating the biopsy of liver tissue from a model organ system and gripping and releasing an alginate bead. This strategy suggests an approach for the development of smart materials and devices that autonomously reconfigure in response to extremely specific biological environments. PMID:20849106

  4. Biofunctional properties of enzymatic squid meat hydrolysate.

    PubMed

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

    2015-03-01

    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

  5. 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

  6. Function of platelet 47K protein phosphorylation

    SciTech Connect

    Imaoka, T.

    1987-05-01

    To provide insight into the biochemical pathway of platelet activation, they purified both unphosphorylated and phosphorylated P47 to homogeneity from human platelets. This study represents the first demonstration of a change of physiological action of P47 in response to phosphorylation in platelet activation. SVI labelled unphosphorylated P47 had an ability to bind with platelet membrane fraction in the presence of phosphatidylserine. Effect of diacylglycerol was inhibitory in this PS dependent P47 binding with membrane. Unphosphorylated P47 had an inhibitory activity in platelet actin polymerization. Molar ratio to inhibit actin polymerization was 1:8 (P47:actin). These activities were Ca independent. Purified TSP-labelled P47 lost the binding ability with membrane, also the inhibitory activity in actin polymerization. Therefore, they propose the hypothesis that unphosphorylated P47 may loosely bind with the inside of plasma membrane of platelet and inhibit actin polymerization as a modulator, when stimulated, protein Kinase C rapidly phosphorylate P47 and induce the activation of cytoskeletal network and subsequently release reaction.

  7. 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

  8. Transforming growth factor-{beta}-inducible phosphorylation of Smad3.

    PubMed

    Wang, Guannan; Matsuura, Isao; He, Dongming; Liu, Fang

    2009-04-10

    Smad proteins transduce the transforming growth factor-beta (TGF-beta) signal at the cell surface into gene regulation in the nucleus. Upon TGF-beta treatment, the highly homologous Smad2 and Smad3 are phosphorylated by the TGF-beta receptor at the SSXS motif in the C-terminal tail. Here we show that in addition to the C-tail, three (S/T)-P sites in the Smad3 linker region, Ser(208), Ser(204), and Thr(179) are phosphorylated in response to TGF-beta. The linker phosphorylation peaks at 1 h after TGF-beta treatment, behind the peak of the C-tail phosphorylation. We provide evidence suggesting that the C-tail phosphorylation by the TGF-beta receptor is necessary for the TGF-beta-induced linker phosphorylation. Although the TGF-beta receptor is necessary for the linker phosphorylation, the receptor itself does not phosphorylate these sites. We further show that ERK is not responsible for TGF-beta-dependent phosphorylation of these three sites. We show that GSK3 accounts for TGF-beta-inducible Ser(204) phosphorylation. Flavopiridol, a pan-CDK inhibitor, abolishes TGF-beta-induced phosphorylation of Thr(179) and Ser(208), suggesting that the CDK family is responsible for phosphorylation of Thr(179) and Ser(208) in response to TGF-beta. Mutation of the linker phosphorylation sites to nonphosphorylatable residues increases the ability of Smad3 to activate a TGF-beta/Smad-target gene as well as the growth-inhibitory function of Smad3. Thus, these observations suggest that TGF-beta-induced phosphorylation of Smad3 linker sites inhibits its antiproliferative activity. PMID:19218245

  9. Enzymatic transformation of nonfood biomass to starch.

    PubMed

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

    2013-04-30

    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

  10. 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

  11. StIKKing it to a death kinase: IKKs prevent TNF-?-induced cell death by phosphorylating RIPK1.

    PubMed

    Dillon, Christopher P; Balachandran, Siddharth

    2016-02-01

    Signaling pathways activated by the cytokine TNF-? are among the most intensively studied and well-understood in all mammalian biology. In a simplistic model, two primary signals emanate from the TNF-? receptor, one that activates cell survival via an NF-?B transcriptional response and a second that triggers cell death when cell survival signals are neutralized. The kinase RIPK1 participates in both these axes, and its poly-ubiquitylation was thought to represent the primary mechanism by which it toggles between survival versus death signaling. When RIPK1 is ubiquitylated, it acts non-enzymatically as an adaptor protein in IKK recruitment and subsequent NF-?B activation; when ubiquitylation of RIPK1 is prevented, it functions as a cell death kinase capable of triggering apoptosis or necroptosis. Bertrand and colleagues (Dondelinger et al., 2015) now demonstrate that phosphorylation of RIPK1 represents an additional mechanism by which this protein switches between its life and death duties. They show that both IKK-? and IKK-? phosphorylate RIPK1, dampening its capacity to assemble the death effectors FADD and caspase 8 into a functional pro-apoptotic signalsome. These IKKs also protect against RIPK1-mediated necroptosis. Importantly, IKK-?/? prevent RIPK1-driven cell death independently of NF-?B transcriptional responses. These findings identify phosphorylation of RIPK1 by IKKs as a new mechanism by which cell fate decisions downstream of TNFR1 are regulated. PMID:26630177

  12. Nanocelluloses and their phosphorylated derivatives for selective adsorption of Ag(+), Cu(2+) and Fe(3+) from industrial effluents.

    PubMed

    Liu, Peng; Borrell, Pere Ferrer; Boži?, Mojca; Kokol, Vanja; Oksman, Kristiina; Mathew, Aji P

    2015-08-30

    The potential of nanoscaled cellulose and enzymatically phosphorylated derivatives as bio-adsorbents to remove metal ions (Ag(+), Cu(2+) and Fe(3+)) from model water and industrial effluents is demonstrated. Introduction of phosphate groups onto nanocelluloses significantly improved the metal sorption velocity and sorption capacity. The removal efficiency was considered to be driven by the high surface area of these nanomaterials as well as the nature and density of functional groups on the nanocellulose surface. Generally, in the solutions containing only single types of metal ions, the metal ion selectivity was in the order Ag(+)>Cu(2+)>Fe(3+), while in the case of mixtures of ions, the order changed to Ag(+)>Fe(3+)>Cu(2+), irrespective of the surface functionality of the nanocellulose. In the case of industrial effluent from the mirror making industry, 99% removal of Cu(2+) and Fe(3+) by phosphorylated nanocellulose was observed. The study showed that phosphorylated nanocelluloses are highly efficient biomaterials for scavenging multiple metal ions, simultaneously, from industrial effluents. PMID:25867590

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

    PubMed

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

    2010-02-01

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

  14. Mass spectrometric characterization of phosphorylated peptides using MALDI in-source decay via redox reactions.

    PubMed

    Asakawa, Daiki; Takayama, Mitsuo

    2012-02-01

    Matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) has been used for characterization of a phosphorylated peptides and proteins because labile phosphate group is not lost during the MALDI-ISD process. The conventional MALDI-ISD is initiated by the hydrogen transfer from reducing matrix molecules to peptide backbone, leading to c'- and z'-series ions. In contrast, when an oxidizing chemical 5-nitrosalicylic acid (5-NSA) is served as the MALDI-ISD matrix, a- and x-series ions are specifically generated by hydrogen abstraction from peptide backbone to matrix molecule. The 5-NSA provides useful complementary information to the conventional MALDI-ISD for the analysis of amino acid sequencing and site localization of phosphorylation in peptides. The MALDI-ISD with reducing and oxidizing matrix could be a useful method for the de novo peptide sequencing. PMID:22359327

  15. Effects of permeant buffers on the initial time course of photophosphorylation and postillumination phosphorylation

    SciTech Connect

    Vinkler, C.; Avron, M.; Boyer, P.D.

    1980-03-25

    Permeant buffers (pyridine, imidazole, or phosphate) caused similar increases in the time required for onset of ATP synthesis in the light in the presence of valinomycin and K+; and in the illumination time required for postillumination phosphorylation with or without valinomycin and K+. Based on prior evidence, the minimum illumination time required for postillumination phosphorylation in thylakoid membranes is taken as a measure of the time required for formation of a transmembrane pH gradient sufficient to drive ATP synthesis. Our results are consistent with the view that, following illumination, as the transient transmembrane electric gradient decays, the establishment and maintenance of a pH gradient serve for energy transfer from the photosystems to the ATP synthase complex.

  16. Regulation of renal fibrosis by Smad3 Thr388 phosphorylation.

    PubMed

    Qu, Xinli; Li, Xueling; Zheng, Yaowu; Ren, Yi; Puelles, Victor G; Caruana, Georgina; Nikolic-Paterson, David J; Li, Jinhua

    2014-04-01

    Transforming growth factor-? (TGF-?) promotes tissue fibrosis via receptor-mediated phosphorylation of the receptor-activated Smad2/3, together with Smad4. Of these, Smad3 plays a major profibrotic role in mouse models of tissue fibrosis. Transcriptional activity of the Smad3 protein is regulated by phosphorylation of residues in the C-terminal domain and the linker region. Herein, we examined the role of a novel phosphorylation site within the MH2 domain (T388) in the regulation of Smad3 activity. Confocal microscopy using an Smad3 phosphorylated T388-specific antibody identified phosphorylation of Smad3 T388 in myofibroblasts and tubular epithelial cells in human focal and segmental glomerulosclerosis and mouse models of unilateral ureteric obstruction and diabetic nephropathy, whereas phosphorylated T388 was largely absent in normal kidney. In vitro, TGF-?1 induced phosphorylation of Smad3 T388 in a biphasic pattern. A point mutation of T388/V in an Smad3 construct demonstrated that phosphorylation of T388 promotes Smad3 binding to Smad4 and CDK8, but was not necessary for nuclear translocation. Furthermore, T388 phosphorylation was required for TGF-?-induced collagen I gene promoter activity and extracellular matrix production in cultured fibroblasts. In conclusion, our study identifies phosphorylation of T388 in the Smad3 MH2 domain as an important mechanism that regulates the profibrotic TGF-?/Smad3 signaling pathway, which has direct relevance to human and experimental fibrotic kidney disease. PMID:24485922

  17. 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

  18. Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation.

    PubMed

    Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan M F; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul M K

    2015-08-01

    Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser(65))--which lies within its ubiquitin-like domain (Ubl)--and indirectly through phosphorylation of ubiquitin at Ser(65). How Ser(65)-phosphorylated ubiquitin (ubiquitin(Phospho-Ser65)) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin(Phospho-Ser65) binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser(65) by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin(Phospho-Ser65), thereby promoting Parkin Ser(65) phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser(65) phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin(Phospho-Ser65) to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser(65). Finally, purified Parkin maximally phosphorylated at Ser(65) in vitro cannot be further activated by the addition of ubiquitin(Phospho-Ser65). Our results thus suggest that a major role of ubiquitin(Phospho-Ser65) is to promote PINK1-mediated phosphorylation of Parkin at Ser(65), leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser(65)-binding pocket on the surface of Parkin that is critical for the ubiquitin(Phospho-Ser65) interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin(Phospho-Ser65), which could aid in the development of Parkin activators that mimic the effect of ubiquitin(Phospho-Ser65). PMID:26116755

  19. Binding to serine 65-phosphorylated ubiquitin primes Parkin for optimal PINK1-dependent phosphorylation and activation

    PubMed Central

    Kazlauskaite, Agne; Martínez-Torres, R Julio; Wilkie, Scott; Kumar, Atul; Peltier, Julien; Gonzalez, Alba; Johnson, Clare; Zhang, Jinwei; Hope, Anthony G; Peggie, Mark; Trost, Matthias; van Aalten, Daan MF; Alessi, Dario R; Prescott, Alan R; Knebel, Axel; Walden, Helen; Muqit, Miratul MK

    2015-01-01

    Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser65)—which lies within its ubiquitin-like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of ubiquitinPhospho-Ser65. PMID:26116755

  20. Single-molecule spectroscopy exposes hidden states in an enzymatic electron relay

    PubMed Central

    Grossman, Iris; Yuval Aviram, Haim; Armony, Gad; Horovitz, Amnon; Hofmann, Hagen; Haran, Gilad; Fass, Deborah

    2015-01-01

    The ability to query enzyme molecules individually is transforming our view of catalytic mechanisms. Quiescin sulfhydryl oxidase (QSOX) is a multidomain catalyst of disulfide-bond formation that relays electrons from substrate cysteines through two redox-active sites to molecular oxygen. The chemical steps in electron transfer have been delineated, but the conformational changes accompanying these steps are poorly characterized. Here we use single-molecule Förster resonance energy transfer (smFRET) to probe QSOX conformation in resting and cycling enzyme populations. We report the discovery of unanticipated roles for conformational changes in QSOX beyond mediating electron transfer between redox-active sites. In particular, a state of the enzyme not previously postulated or experimentally detected is shown to gate, via a conformational transition, the entrance into a sub-cycle within an expanded QSOX kinetic scheme. By tightly constraining mechanistic models, smFRET data can reveal the coupling between conformational and chemical transitions in complex enzymatic cycles. PMID:26468675

  1. Single-molecule spectroscopy exposes hidden states in an enzymatic electron relay

    NASA Astrophysics Data System (ADS)

    Grossman, Iris; Yuval Aviram, Haim; Armony, Gad; Horovitz, Amnon; Hofmann, Hagen; Haran, Gilad; Fass, Deborah

    2015-10-01

    The ability to query enzyme molecules individually is transforming our view of catalytic mechanisms. Quiescin sulfhydryl oxidase (QSOX) is a multidomain catalyst of disulfide-bond formation that relays electrons from substrate cysteines through two redox-active sites to molecular oxygen. The chemical steps in electron transfer have been delineated, but the conformational changes accompanying these steps are poorly characterized. Here we use single-molecule Förster resonance energy transfer (smFRET) to probe QSOX conformation in resting and cycling enzyme populations. We report the discovery of unanticipated roles for conformational changes in QSOX beyond mediating electron transfer between redox-active sites. In particular, a state of the enzyme not previously postulated or experimentally detected is shown to gate, via a conformational transition, the entrance into a sub-cycle within an expanded QSOX kinetic scheme. By tightly constraining mechanistic models, smFRET data can reveal the coupling between conformational and chemical transitions in complex enzymatic cycles.

  2. 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

  3. GAL4 is phosphorylated as a consequence of transcriptional activation.

    PubMed Central

    Sadowski, I; Niedbala, D; Wood, K; Ptashne, M

    1991-01-01

    GAL4 protein isolated from yeast in which it is active is phosphorylated predominantly on two different serine residues. One of these was identified as Ser-837; substitution of this residue for alanine has no detectable effect on transcriptional activation by GAL4. Phosphorylation at Ser-837 requires that both the DNA binding and transcriptional activation functions be intact. We propose that some phosphorylations of GAL4, including that at Ser-837, occur concomitantly with activation of transcription. Images PMID:1961715

  4. Enzymatic Catalytic Beds For Oxidation Of Alcohols

    NASA Technical Reports Server (NTRS)

    Jolly, Clifford D.; Schussel, Leonard J.

    1993-01-01

    Modules containing beds of enzymatic material catalyzing oxidation of primary alcohols and some other organic compounds developed for use in wastewater-treatment systems of future spacecraft. Designed to be placed downstream of multifiltration modules, which contain filters and sorbent beds removing most of non-alcoholic contaminants but fail to remove significant amounts of low-molecular-weight, polar, nonionic compounds like alcohols. Catalytic modules also used on Earth to oxidize primary alcohols and other compounds in wastewater streams and industrial process streams.

  5. 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.

  6. Enzymatic processing of protein-based fibers.

    PubMed

    Fu, Jiajia; Su, Jing; Wang, Ping; Yu, Yuanyuan; Wang, Qiang; Cavaco-Paulo, Artur

    2015-12-01

    Wool and silk are major protein fiber materials used by the textile industry. Fiber protein structure-function relationships are briefly described here, and the major enzymatic processing routes for textiles and other novel applications are deeply reviewed. Fiber biomodification is described here with various classes of enzymes such as protease, transglutaminase, tyrosinase, and laccase. It is expected that the reader will get a perspective on the research done as a basis for new applications in other areas such as cosmetics and pharma. PMID:26428240

  7. A Crystallographic Snapshot of Tyrosine Trans-phosphorylation in Action

    SciTech Connect

    Chen, H.; Xu, C; Ma, J; Eliseenkova, A; Li, W; Pollock, P; Pitteloud, N; Miller, W; Neubert, T; Mohammadi, M

    2008-01-01

    Tyrosine trans-phosphorylation is a key event in receptor tyrosine kinase signaling, yet, the structural basis for this process has eluded definition. Here, we present the crystal structure of the FGF receptor 2 kinases caught in the act of trans-phosphorylation of Y769, the major C-terminal phosphorylation site. The structure reveals that enzyme- and substrate-acting kinases engage each other through elaborate and specific interactions not only in the immediate vicinity of Y769 and the enzyme active site, but also in regions that are as much of 18 {angstrom} away from D626, the catalytic base in the enzyme active site. These interactions lead to an unprecedented level of specificity and precision during the trans-phosphorylation on Y769. Time-resolved mass spectrometry analysis supports the observed mechanism of trans-phosphorylation. Our data provide a molecular framework for understanding the mechanism of action of Kallmann syndrome mutations and the order of trans-phosphorylation reactions in FGFRs. We propose that the salient mechanistic features of Y769 trans-phosphorylation are applicable to trans-phosphorylation of the equivalent major phosphorylation sites in many other RTKs.

  8. Tropomyosin Ser-283 pseudo-phosphorylation slows myofibril relaxation.

    PubMed

    Nixon, Benjamin R; Liu, Bin; Scellini, Beatrice; Tesi, Chiara; Piroddi, Nicoletta; Ogut, Ozgur; Solaro, R John; Ziolo, Mark T; Janssen, Paul M L; Davis, Jonathan P; Poggesi, Corrado; Biesiadecki, Brandon J

    2013-07-01

    Tropomyosin (Tm) is a central protein in the Ca(2+) regulation of striated muscle. The ?Tm isoform undergoes phosphorylation at serine residue 283. While the biochemical and steady-state muscle function of muscle purified Tm phosphorylation have been explored, the effects of Tm phosphorylation on the dynamic properties of muscle contraction and relaxation are unknown. To investigate the kinetic regulatory role of ?Tm phosphorylation we expressed and purified native N-terminal acetylated Ser-283 wild-type, S283A phosphorylation null and S283D pseudo-phosphorylation Tm mutants in insect cells. Purified Tm's regulate thin filaments similar to that reported for muscle purified Tm. Steady-state Ca(2+) binding to troponin C (TnC) in reconstituted thin filaments did not differ between the 3 Tm's, however disassociation of Ca(2+) from filaments containing pseudo-phosphorylated Tm was slowed compared to wild-type Tm. Replacement of pseudo-phosphorylated Tm into myofibrils similarly prolonged the slow phase of relaxation and decreased the rate of the fast phase without altering activation kinetics. These data demonstrate that Tm pseudo-phosphorylation slows deactivation of the thin filament and muscle force relaxation dynamics in the absence of dynamic and steady-state effects on muscle activation. This supports a role for Tm as a key protein in the regulation of muscle relaxation dynamics. PMID:23232082

  9. Regulation of acetylcholine receptor phosphorylation by calcium and calmodulin.

    PubMed Central

    Smilowitz, H; Hadjian, R A; Dwyer, J; Feinstein, M B

    1981-01-01

    Acetylcholine receptor-enriched membranes prepared from frozen electric organ of Torpedo californica by differential centrifugation and density step gradient centrifugation were assayed for endogenous phosphorylation in the absence and presence of calmodulin and calcium. Each of the membrane fractions exhibited a 3- to 6-fold stimulation of endogenous phosphorylation by calcium and calmodulin. Both calcium and calmodulin were needed for maximal stimulation although calcium alone afforded a small, reproducible stimulation of endogenous phosphorylation. In the presence of fluoride, a phosphatase inhibitor, the calmodulin plus calcium stimulation was increased an additional 3-fold. The phosphorylation reaction was rapid, and maximal phosphorylation was achieved in 2 min. Stimulation of phosphorylation by calcium and calmodulin was completely inhibited by 25 microM trifluoperazine; at 50 microM it inhibited basal phosphorylation by 60%, suggesting that most of the basal phosphorylation may be due to the endogenous calmodulin present in our membrane preparation. NaDodSO4/polyacrylamide gel electrophoresis revealed that at least three of the phosphorylated species (both in the presence and in the absence of calcium and calmodulin) correspond to subunits of the purified acetylcholine receptor from T. californica (i.e., 65,000, 58,000, and 50,000 daltons) which are the beta, gamma, and delta subunits of the receptor. Images PMID:6946419

  10. A Cytosolic Arabidopsis d-Xylulose Kinase Catalyzes the Phosphorylation of 1-Deoxy-d-Xylulose into a Precursor of the Plastidial Isoprenoid Pathway1

    PubMed Central

    Hemmerlin, Andréa; Tritsch, Denis; Hartmann, Michael; Pacaud, Karine; Hoeffler, Jean-François; van Dorsselaer, Alain; Rohmer, Michel; Bach, Thomas J.

    2006-01-01

    Plants are able to integrate exogenous 1-deoxy-d-xylulose (DX) into the 2C-methyl-d-erythritol 4-phosphate pathway, implicated in the biosynthesis of plastidial isoprenoids. Thus, the carbohydrate needs to be phosphorylated into 1-deoxy-d-xylulose 5-phosphate and translocated into plastids, or vice versa. An enzyme capable of phosphorylating DX was partially purified from a cell-free Arabidopsis (Arabidopsis thaliana) protein extract. It was identified by mass spectrometry as a cytosolic protein bearing d-xylulose kinase (XK) signatures, already suggesting that DX is phosphorylated within the cytosol prior to translocation into the plastids. The corresponding cDNA was isolated and enzymatic properties of a recombinant protein were determined. In Arabidopsis, xylulose kinases are encoded by a small gene family, in which only two genes are putatively annotated. The additional gene is coding for a protein targeted to plastids, as was proved by colocalization experiments using green fluorescent protein fusion constructs. Functional complementation assays in an Escherichia coli strain deleted in xk revealed that the cytosolic enzyme could exclusively phosphorylate xylulose in vivo, not the enzyme that is targeted to plastids. xk activities could not be detected in chloroplast protein extracts or in proteins isolated from its ancestral relative Synechocystis sp. PCC 6803. The gene encoding the plastidic protein annotated as “xylulose kinase” might in fact yield an enzyme having different phosphorylation specificities. The biochemical characterization and complementation experiments with DX of specific Arabidopsis knockout mutants seedlings treated with oxo-clomazone, an inhibitor of 1-deoxy-d-xylulose 5-phosphate synthase, further confirmed that the cytosolic protein is responsible for the phosphorylation of DX in planta. PMID:16920870

  11. 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

  12. Regulation of cardiac C-protein phosphorylation

    SciTech Connect

    Titus, F.L.

    1985-01-01

    Molecular mechanisms of cardiac sympathetic and parasympathetic responses were addressed by studying subcellular changes in protein phosphorylation, cAMP-dependent protein kinase activity and protein phosphatase activity in frog hearts. B-adrenergic agonists increased and muscarinic cholinergic agonists decreased (/sup 32/P)phosphate incorporation into C-protein, a thick filament component. Regulation of protein phosphatase activity by Iso and methacholine (MCh) was assayed using extracts of drug treated frog hearts and (/sup 32/P)phospho-C-protein as substrate. Total phosphatase activity decreased 21% in extracts from hearts perfused with 0.1 ..mu..M Iso and 17% in hearts exposed to Iso plus 1 ..mu..M methacholine. This decrease reflected decreased phosphatase-2A activity. No changes in total phosphatase activity were measurable in broken cells treated with Iso or MCh. The results suggest adrenergic stimulation changes contractile activity in frog hearts by activating cAMP-dependent protein kinase associated with particulate cellular elements and inactivating soluble protein phosphatase-2A. This is the first demonstration of coordinated regulation of these enzymes by B-adrenergic agonists favoring phosphorylation of effector proteins. Coordinated regulation by methacholine in the presence of Iso was not observed.

  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. Inhibition of rabbit muscle aldolase by phosphorylated aromatic compounds.

    PubMed Central

    Blonski, C; De Moissac, D; Périé, J; Sygusch, J

    1997-01-01

    The interactions of the phosphorylated derivatives of hydroquinone (HQN-P2), resorcinol (RSN-P2), 4-hydroxybenzaldehyde (HBA-P) and 2, 4-dihydroxybenzaldehyde (DHBA-P; phosphate group at position 4) with fructose bisphosphate aldolase were analysed by enzyme kinetics, UV/visible difference spectroscopy and site-directed mutagenesis. Enzyme activity was competitively inhibited in the presence of HQN-P2, RSN-P2 and HBA-P, whereas DHBA-P exhibited slow-binding inhibition. Inhibition by DHBA-P involved active-site Schiff-base formation and required a phenol group ortho to the aldehyde moiety. Rates of enzyme inactivation and of Schiff-base formation by DHBA-P were identical, and corresponded to 3.2-3.5 DHBA-P molecules covalently bound per aldolase tetramer at maximal inactivation. Site-directed mutagenesis of the active-site lysine residues at positions 107, 146 and 229 was found to be consistent with Schiff-base formation between DHBA-P and Lys-146, and this was promoted by Lys-229. Mutation of Glu-187, located vicinally between Lys-146 and Lys-229 in the active site, perturbed the rate of Schiff-base formation, suggesting a functional role for Glu-187 in Schiff-base formation and stabilization. The decreased cleavage activity of the active-site mutants towards fructose 1, 6-bisphosphate is consistent with a proton-transfer mechanism involving Lys-229, Glu-187 and Lys-146. PMID:9173904

  15. Enzymatic sulfation of mucus glycoprotein in gastric mucosa

    SciTech Connect

    Liau, Y.H.; Carter, S.R.; Gwozdzinski, K.; Nadziejko, C.; Slomiany, A.; Slomiany, B.L.

    1986-05-01

    Among the posttranslational modifications that mucus glycoprotein undergo prior to secretion into the gastric lumen is the process of sulfation of the carbohydrate chains. These sulfate groups impart strongly negative charge to nucus glycoprotein and are thought to play a major role in the maintenance of gastric mucosal integrity. The authors report here the presence and some properties of an enzyme involved in the sulfation of gastric mucus glycoprotein. The sulfotransferase activity which catalyzes the transfer of sulfate ester group from PAPS to mucus glycoprotein was located in the detergent extracts of the microsomal fraction of rat gastric mucosa. Optimum enzymatic activity for sulfation of gastric mucin was obtained using 0.5% Triton X-100 and 25mM NaF at a pH of 6.8. ATP, ADP, MgCl/sub 2/ and MnCl/sub 2/ at concentrations examined were inhibitory. Under optimal conditions, the rate of sulfate incorporation was proportional to the microsomal enzyme protein concentration up to 50..mu..g and remained constant with time of incubation for at least 1h. The apparent Km value of the enzyme for gastric mucus glycoprotein was 8.3 x 10/sup -6/M. The /sup 35/S-labeled product of the enzyme reaction cochromatographed on Bio-Gel A-50 with gastric mucin, and gave on CsCl equilibrium density gradient centrifugation a band at the density of 1.48 in which the /sup 35/S label coincided with the glycoprotein.

  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. The bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system: regulation by protein phosphorylation and phosphorylation-dependent protein-protein interactions.

    PubMed

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

    2014-06-01

    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

  18. Sensitive Electrochemical Detection of Enzymatically-generated Thiocholine at Carbon Nanotube Modified Glassy Carbon Electrode

    SciTech Connect

    Liu, Guodong; Riechers, Shawn L.; Mellen, Maria C.; Lin, Yuehe

    2005-11-01

    A carbon nanotube modified glassy-carbon (CNT/GC) electrode was used for enhancing the sensitivity of electrochemical measurements of enzymatically-generated thiocholine. Cyclic voltammetric and amperometric characteristics of thiocholine at CNT/GC, glassy carbon, carbon paste, and gold electrodes were compared. The CNT layer leads to a greatly improved anodic detection of enzymatically generated thiocholine product including lower oxidation overpotential (0.15 V) and higher sensitivity because of its electrocatalytic activity, fast electron transfer and large surface area. The sensor performance was optimized with respect to the operating conditions. Under the optimal batch conditions, a detection limit of 5 ?10 -6 mol/L was obtained with good precision (RSD = 5.2%, n=10). Furthermore, the attractive response of thiocholine on a CNT/GC electrode has allowed it to be used for constant-potential flow injection analysis. The detection limit was greatly improved to 0.3 ?10-6 mol/L. The high sensitivity electrochemical detection of enzymatically generated thiocholine with a CNT sensing platform holds great promise to prepare an acetylcholinesterase biosensor for monitoring organophosphate pesticides and nerve agents.

  19. Mapping the Reaction Coordinates of Enzymatic Defluorination

    SciTech Connect

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

    2011-09-28

    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 into how this formidable feat is achieved. Here, we present a series of high-resolution (1.15-1.80 {angstrom}) 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 also is finely tailored for the smaller fluorine halogen atom to establish selectivity toward 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.

  20. Enzymatic deconstruction of xylan for biofuel production

    PubMed Central

    DODD, DYLAN; CANN, ISAAC K. O.

    2010-01-01

    The combustion of fossil-derived fuels has a significant impact on atmospheric carbon dioxide (CO2) levels and correspondingly is an important contributor to anthropogenic global climate change. Plants have evolved photosynthetic mechanisms in which solar energy is used to fix CO2 into carbohydrates. Thus, combustion of biofuels, derived from plant biomass, can be considered a potentially carbon neutral process. One of the major limitations for efficient conversion of plant biomass to biofuels is the recalcitrant nature of the plant cell wall, which is composed mostly of lignocellulosic materials (lignin, cellulose, and hemicellulose). The heteropolymer xylan represents the most abundant hemicellulosic polysaccharide and is composed primarily of xylose, arabinose, and glucuronic acid. Microbes have evolved a plethora of enzymatic strategies for hydrolyzing xylan into its constituent sugars for subsequent fermentation to biofuels. Therefore, microorganisms are considered an important source of biocatalysts in the emerging biofuel industry. To produce an optimized enzymatic cocktail for xylan deconstruction, it will be valuable to gain insight at the molecular level of the chemical linkages and the mechanisms by which these enzymes recognize their substrates and catalyze their reactions. Recent advances in genomics, proteomics, and structural biology have revolutionized our understanding of the microbial xylanolytic enzymes. This review focuses on current understanding of the molecular basis for substrate specificity and catalysis by enzymes involved in xylan deconstruction. PMID:20431716

  1. Enzymatic and bacterial conversions during sourdough fermentation.

    PubMed

    Gänzle, Michael G

    2014-02-01

    Enzymatic and microbial conversion of flour components during bread making determines bread quality. Metabolism of sourdough microbiota and the activity of cereal enzymes are interdependent. Acidification, oxygen consumption, and thiols accumulation by microbial metabolism modulate the activity of cereal enzymes. In turn, cereal enzymes provide substrates for bacterial growth. This review highlights the role of cereal enzymes and the metabolism of lactic acid bacteria in conversion of carbohydrates, proteins, phenolic compounds and lipids. Heterofermentative lactic acid bacteria prevailing in wheat and rye sourdoughs preferentially metabolise sucrose and maltose; the latter is released by cereal enzymes during fermentation. Sucrose supports formation of acetate by heterofermentative lactobacilli, and the formation of exopolysaccharides. The release of maltose and glucose by cereal enzymes during fermentation determines the exopolysaccharide yield in sourdough fermentations. Proteolysis is dependent on cereal proteases. Peptidase activities of sourdough lactic acid bacteria determine the accumulation of (bioactive) peptides, amino acids, and amino acid metabolites in dough and bread. Enzymatic conversion and microbial metabolism of phenolic compounds is relevant in sorghum and millet containing high levels of phenolic compounds. The presence of phenolic compounds with antimicrobial activity in sorghum selects for fermentation microbiota that are resistant to the phenolic compounds. PMID:24230468

  2. Microfluidic enzymatic biosensing systems: A review.

    PubMed

    Mross, Stefan; Pierrat, Sebastien; Zimmermann, Tom; Kraft, Michael

    2015-08-15

    Microfluidic biosensing systems with enzyme-based detection have been extensively studied in the last years owing to features such as high specificity, a broad range of analytes and a high degree of automation. This review gives an overview of the most important factors associated with these systems. In the first part, frequently used immobilization protocols such as physisorption and covalent bonding and detection techniques such as amperometry and fluorescence measurements are discussed with respect to effort, lifetime and measurement range. The Michaelis-Menten model describing the kinetics of enzymatic reactions, the role of redox mediators and the limitations of the linear measurement range of enzymatic sensors are introduced. Several possibilities of extending the linear measurement range in microfluidic systems such as diffusion-limiting membranes and the flow injection setup are presented. Regarding the integration of enzymes into microfluidic systems during the fabrication process, the constraints imposed by the biomolecules due to the limited usage of high temperatures and solvents are addressed. In the second part, the most common forms of enzyme integration into microfluidic systems, i.e. in channels and on electrodes, on microparticles, on paper and thread and as injected enzyme solutions, are reviewed, focusing on fabrication, applications and performance. PMID:25841121

  3. 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

  4. 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

  5. Electronic Detection of the Enzymatic Degradation of Starch

    E-print Network

    Gruner, George

    Electronic Detection of the Enzymatic Degradation of Starch Alexander Star,*, Vikram Joshi, Tzong 90095 astar@nano.com Received March 6, 2004 ABSTRACT The enzymatic degradation of starch can in the removal of the starch from both the silicon surfaces and the side walls of the SWNTs in the FETs

  6. 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...

  7. Enzymatically Amplified Surface Plasmon Resonance Imaging Detection of DNA by

    E-print Network

    Enzymatically Amplified Surface Plasmon Resonance Imaging Detection of DNA by Exonuclease III utilizing the enzyme exonuclease III in conjunction with 3-terminated DNA microarrays for the amplifiedIII with double-stranded DNA as well as this new enzymatically amplified SPR imaging process with a 16-mer target

  8. RESEARCH Open Access Comparison of enzymatic reactivity of corn stover

    E-print Network

    California at Riverside, University of

    RESEARCH Open Access Comparison of enzymatic reactivity of corn stover solids prepared by dilute liquid (IL) thermochemical pretreatments of corn stover were followed over time for the same range. Results: Corn stover solids from pretreatment by DA, AFEX, and IL were enzymatically digested over a range

  9. Maximum oxidative phosphorylation capacity of the mammalian heart

    E-print Network

    Mootha, Vamsi K.

    Maximum oxidative phosphorylation capacity of the mammalian heart VAMSI K. MOOTHA, ANDREW E. ARAI, AND ROBERT S. BALABAN Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National. Maximum oxidative phosphorylation capacity of the mammalian heart. Am. J. Physiol. 272 (Heart Circ

  10. The importance of intrinsic disorder for protein phosphorylation

    E-print Network

    Radivojac, Predrag

    a major regulatory mechanism in eukaryotic cells. Due to the high variability of amino acid residues and transport, and signal transduction (1,2). A high percentage of cell-signaling and cancer-associated proteins phosphorylation (4). Protein phosphorylation represents an important regulatory mechanism in eukaryotic cells

  11. A Gi-independent mechanism mediating Akt phosphorylation in platelets

    PubMed Central

    Xiang, Binggang; Zhang, Guoying; Liu, Junling; Morris, Andrew J.; Smyth, Susan S.; Gartner, T. Kent; Li, Zhenyu

    2010-01-01

    Summary Background The serine-threonine kinase Akt plays an important role in regulating platelet activation. Stimulation of platelets with various agonists results in Akt activation as indicated by Akt phosphorylation. However, the mechanisms of Akt phosphorylation in platelets are not completely understood. Objectives and Methods We used P2Y12 knockout mice to address the role of P2Y12 in Akt phosphorylation in response to thrombin receptors in platelets. Results Thrombin or the PAR4 thrombin receptor peptide AYPGKF at high concentrations stimulated substantial phosphorylation of Akt residues thr308 and Ser473 in P2Y12 deficient platelets. AYPGKF-induced Akt phosphorylation is enhanced by expression of recombinant human PAR4 cDNA in Chinese hamster ovary (CHO) cells. P2Y12-independent Akt phosphorylation was not inhibited by integrin inhibitor peptide RGDS or integrin ?3 deficiency. Akt phosphorylation induced by thrombin or AYPGKF in P2Y12 deficient platelets was inhibited by the calcium chelator dimethyl-BAPTA, the Src family kinase inhibitor PP2, and PI3K inhibitors, respectively. Conclusions Our results reveal a novel P2Y12-independent signaling pathway mediating Akt phosphorylation in response to thrombin receptors. PMID:20586915

  12. A novel type of protein kinase phosphorylates actin in the actin-fragmin complex.

    PubMed Central

    Eichinger, L; Bomblies, L; Vandekerckhove, J; Schleicher, M; Gettemans, J

    1996-01-01

    Actin-fragmin kinase (AFK) from Physarum polycephalum specifically phosphorylates actin in the EGTA-resistant 1:1 actin-fragmin complex. The cDNA deduced amino acid sequence reveals two major domains of approximately 35 kDa each that are separated by a hinge-like proline/serine-rich segment of 50 residues. Whereas the N-terminal domain does not show any significant similarity to protein sequences from databases, there are six complete kelch repeats in the protein that comprise almost the entire C-terminal half of the molecule. To prove the intrinsic phosphorylation activity of AFK, full-length or partial cDNA fragments were expressed both in a reticulocyte lysate and in Escherichia coli. In both expression systems, we obtained specific actin phosphorylation and located the catalytic domain in the N-terminal half. Interestingly, this region did not contain any of the known protein kinase consensus sequences. The only known sequence motif present that could have been involved in nucleotide binding was a nearly perfect phosphate binding loop (P-loop). However, introduction of two different point mutations into this putative P-loop sequence did not alter the catalytic activity of the kinase, which indicates an as yet unknown mechanism for phosphate transfer. Our data suggest that AFK belongs to a new class of protein kinases and that this actin phosphorylation might be the first example of a widely distributed novel type of regulation of the actin cytoskeleton in non-muscle cells. Images PMID:8896448

  13. Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5

    PubMed Central

    2004-01-01

    Doublecortin (DCX) is a 40 kDa microtubule-associated protein required for normal neural migration and cortical layering during development. Mutations in the human DCX gene cause a disruption of cortical neuronal migration. Defects in cdk5 (cyclin-dependent kinase 5) also cause defects in neural migration and cortical layering. DCX is a substrate for cdk5 in vitro and in vivo and the major site of in vitro phosphorylation is Ser-297. We used a highly developed MS strategy to identify the cdk5 phosphorylation sites and determine the major and minor sites. Several phosphopeptides were identified from a tryptic digest of 32P-labelled, cdk5-phosphorylated DCX using a combination of off-line HPLC and matrix-assisted laser-desorption ionization-MS with alkaline phosphatase treatment. Tandem MS/MS enabled the identification of seven phosphorylation sites for cdk5. Monitoring of 32P label indicated that there was one major site, Ser-28, at the N-terminus, and a major site, Ser-339, in the serine/proline-rich domain at the C-terminus. Five other sites, Ser-287, Thr-289, Ser-297, Thr-326 and Ser-332, were also found in the tail. Site-directed mutagenesis largely supported these findings. Single mutation of Ser-28 reduced but did not abolish phosphorylation. Double, rather than single, mutation for Ser-332 and Ser-339 was required to reduce overall phosphorylation, suggesting an interaction between these sites. Truncations of the tail produced a significant reduction in cdk5 phosphorylation of DCX. These results do not support Ser-297 as the major cdk5 phosphorylation site in DCX, but indicate that DCX is subject to complex multisite phosphorylation. This illustrates the importance of a well-developed MS strategy to identify phosphorylation sites. PMID:15099191

  14. Characterization of Ser338 Phosphorylation for Raf-1 Activation*

    PubMed Central

    Zang, Mengwei; Gong, Jun; Luo, Lingqi; Zhou, Jing; Xiang, Xiaoqin; Huang, Wei; Huang, Qiren; Luo, Xixi; Olbrot, Martin; Peng, Yihong; Chen, Changyan; Luo, Zhijun

    2008-01-01

    Raf kinases are essential for regulating cell proliferation, survival, and tumorigenesis. However, the mechanisms by which Raf is activated are still incompletely understood. Phosphorylation plays a critical role in Raf activation in response to mitogens. The present study characterizes phosphorylation of Ser338, a crucial event for Raf-1 activation. Here we report that mutation of Lys375 to Met diminishes phosphorylation of Ser338 on both wild type Raf-1 in cells treated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and a constitutively active mutant in which Tyr340/Tyr341 are replaced by 2 aspartic acids, a conserved substitution present in natural B-Raf. The loss of Ser338 phosphorylation in these Raf mutants is not engendered by a mutation-induced conformational change, inasmuch as mutation of another site (Ser471 to Ala) in the activation segment also abolishes Ser338 phosphorylation, whereas both the kinase-dead mutants of Raf-1 are phosphorylated well by active Pak1. Furthermore, our data demonstrate that EGF-stimulated phosphorylation of Ser338 is inhibited by Sorafenib, a Raf kinase inhibitor, but not by the MEK inhibitor U0126. Interestingly, a kinase-dead mutation and Sorafenib also markedly reduce phosphorylation of Ser445 on B-Raf, a site equivalent to Raf-1 Ser338. Finally, our data reveal that Ser338 is phosphorylated on inactive Raf-1 by an active mutant of Raf-1 when they are dimerized in cells and that artificial dimerization of Raf-1 causes Ser338 phosphorylation, accompanied by activation of ERK1/2. Altogether, our data suggest that Ser338 on Raf-1 is autophosphorylated in response to mitogens. PMID:18775988

  15. Enzymatic Hydrogen Production from Starch and Water

    SciTech Connect

    Zhang, Y.-H. Percival; Evans, Barbara R; Mielenz, Jonathan R; Hopkins, Robert C.; Adams, Michael W. W.

    2007-01-01

    A novel enzymatic reaction was conducted for producing hydrogen from starch and water at 30oC. The overall reaction comprised of 13 enzymes, 1 cofactor (NADP+), and phosphate was driven by energy stored in carbohydrate starch according to the overall stoichiometry stoichiometric reaction of C6H10O5 (l) + 7 H2O (l) --> 12 H2 (g) + 6 CO2 (g). It is spontaneous and unidirectional because of negative Gibbs free energy and the removal of gaseous products from the aqueous reaction solution. With technology improvement and integration with fuel cells, this technology would be suitable for mobile applications and also solve the challenges associated with hydrogen storage, distribution, and infrastructure in a hydrogen economy.

  16. Enzymatic production of glycerol acetate from glycerol.

    PubMed

    Oh, Seokhyeon; Park, Chulhwan

    2015-02-01

    In this study, we report the enzymatic production of glycerol acetate from glycerol and methyl acetate. Lipases are essential for the catalysis of this reaction. To find the optimum conditions for glycerol acetate production, sequential experiments were designed. Type of lipase, lipase concentration, molar ratio of reactants, reaction temperature and solvents were investigated for the optimum conversion of glycerol to glycerol acetate. As the result of lipase screening, Novozym 435 (Immobilized Candida antarctica lipase B) was turned out to be the optimal lipase for the reaction. Under the optimal conditions (2.5 g/L of Novozym 435, 1:40 molar ratio of glycerol to methyl acetate, 40 °C and tert-butanol as the solvent), glycerol acetate production was achieved in 95.00% conversion. PMID:25640720

  17. Pretreatment and enzymatic hydrolysis of corn fiber

    SciTech Connect

    Grohmann, K.; Bothast, R.J.

    1996-10-01

    Corn fiber is a co-product of the corn wet milling industry which is usually marketed as a low value animal feed ingredient. Approximately 1.2 x 10{sup 6} dry tons of this material are produced annually in the United States. The fiber is composed of kernel cell wall fractions and a residual starch which can all be potentially hydrolyzed to a mixture of glucose, xylose, arabinose and galactose. We have investigated a sequential saccharification of polysaccharides in corn fiber by a treatment with dilute sulfuric acid at 100 to 160{degrees}C followed by partial neutralization and enzymatic hydrolysis with mixed cellulose and amyloglucosidase enzymes at 45{degrees}C. The sequential treatment achieved a high (approximately 85%) conversion of all polysaccharides in the corn fiber to monomeric sugars, which were in most cases fermentable to ethanol by the recombinant bacterium Escherichia coli KOll.

  18. 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.

  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. Kinetics of the enzymatic hydrolysis of cellulose

    SciTech Connect

    Wald, S.; Wilke, C.R.; Blanch, H.W.

    1984-01-01

    Enzymatic hydrolysis of cellulose for sugar production offers advantages of higher conversion, minimal by-product formation, low energy requirements, and mild operating conditions over other chemical conversions. The development of a kinetic model, based on observable, macroscopic properties of the overall system, is helpful in design and economic evaluation of processes for sugar conversion and ethanol production. A kinetic model is presented, incorporating enzyme adsorption, product inhibition, and considers a multiple enzyme and substrate system. This model was capable of simulating saccharification of a lignocellulosic material, rice straw, at high substrate (up to 333 g/L) and enzyme concentrations (up to 9.2 FPU/mL) that are common to proposed process designs. (Refs. 37).

  1. ?-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

  2. Some enzymatic activities associated with purified parapoxvirions.

    PubMed Central

    Caplen, H S; Holowczak, J A

    1983-01-01

    Purified virions of milker's nodule virus, a parapoxvirus, were shown to contain an RNA polymerase, a nucleotide phosphohydrolase, and a protein kinase associated with or encapsulated within the DNA-containing core of the virus. In vitro, the activated viral RNA polymerase transcribed only 7 to 8% of the genome, in the form of 8S to 14S polyadenylated RNA molecules which were complementary to sequences present in milker's nodule virus DNA but not vaccinia virus DNA or DNA prepared from the host cells in which the virus was propagated. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that in vitro, the activated viral protein kinase phosphorylated viral polypeptides of 95, 60, 33.5, 15, and 13.8 kilodaltons. Images PMID:6188861

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

    SciTech Connect

    Franke, Claudia; Matschl, Urte; Bruns, Michael . 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.

  4. Impact of mass transport on the enzymatic hydrolysis of rapeseed oil.

    PubMed

    Schröter, Sandra; Stahmann, Klaus-Peter; Schnitzlein, Klaus

    2015-01-01

    In order to assess the capillary segmented flow reactor as a potentially appropriate reactor device for the enzymatic hydrolysis of vegetable oils, a study was made to reveal the impact of incident mass transfer processes on the hydrolysis rate. As demonstrated by means of experiments performed in a modified Lewis-cell type contactor, which allows the independent adjustment of flow rates for both phases, the enzymatic hydrolysis rate of rapeseed oil is strongly governed by mass transport processes taking place in both phases. In the oil phase, any increase in convective mass transfer results in an enhancement of hydrolysis rate due to facilitated removal of fatty acids from interface layer which is known to inhibit the activity of the enzyme adsorbed at the interface. At asynchronous condition when solely the water phase is agitated, however, convective mass transport in the interface layer has an inverse effect on the hydrolysis rate due to the generation of considerable shear stress in the vicinity of the interface unfavorable for the performance of the enzymes. By operating at synchronous agitation conditions, the shear stress can considerably be reduced. Generally, the positive effect of mass transport in the oil phase compensates the negative one in the aqueous phase thus resulting in an overall increase in hydrolysis rate of 57% with increasing stirrer rates. The results can be applied to the operation of segmented-flow capillary reactors by choosing the oil phase as disperse phase and the water phase as continuous phase, respectively. PMID:25007743

  5. 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. PMID:26060532

  6. 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

  7. 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

  8. Temporal Regulation of Connexin Phosphorylation in Embryonic and Adult Tissues

    PubMed Central

    King, Timothy J.; Lampe, Paul D.

    2006-01-01

    Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells in tissues and are important in development, tissue/cellular homeostasis and carcinogenesis. Genome databases indicate that there are at least 20 connexins in the mouse and human. Connexin phosphorylation has been implicated in connexin assembly into gap junctions, gap junction turnover and cell signaling events that occur in response to tumor promoters and oncogenes. Connexin43 (Cx43), the most widely expressed and abundant gap junction protein, can be phosphorylated at several different serine and tyrosine residues. Here, we focus on the dynamic regulation of Cx43 phosphorylation in tissue and how these regulatory events are affected during development, wound healing and carcinogenesis. Activation of several kinases including protein kinase A, protein kinase C, p34cdc2/cyclin B kinase, casein kinase 1, mitogen-activated protein kinase and pp60src kinase can lead to phosphorylation of different residues in the C-terminal region of Cx43. The use of antibodies specific for phosphorylation at defined residues has allowed examination of specific phosphorylation events both in tissue culture and in vivo. These new antibody tools and those under development will allow us to correlate specific phosphorylation events with changes in connexin function. PMID:16137642

  9. Structural Impact of Tau Phosphorylation at Threonine 231.

    PubMed

    Schwalbe, Martin; Kadavath, Harindranath; Biernat, Jacek; Ozenne, Valery; Blackledge, Martin; Mandelkow, Eckhard; Zweckstetter, Markus

    2015-08-01

    Phosphorylation of the microtubule-associated protein Tau influences the assembly and stabilization of microtubules and is deregulated in several neurodegenerative diseases. The high flexibility of Tau, however, has prevented an atomic-level description of its phosphorylation-induced structural changes. Employing an extensive set of distance and orientational restraints together with a novel ensemble calculation approach, we determined conformational ensembles of Tau fragments in the non-phosphorylated state and, when phosphorylated at T231/S235 or T231/S235/S237/S238, four important sites of phosphorylation in Alzheimer disease. Comparison of the molecular ensembles showed that phosphorylation of the regulatory T231 does not perturb the backbone conformation of the proximal microtubule-binding (225)KVAVVR(230) motif. Instead, phosphorylated T231 selectively engages in a salt bridge with R230 that can compete with the formation of intermolecular salt bridges to tubulin. Our study provides an ensemble description which will be useful for the analysis of conformational transitions in Tau and other intrinsically disordered proteins. PMID:26165593

  10. Steroid hormone receptors and their regulation by phosphorylation.

    PubMed Central

    Weigel, N L

    1996-01-01

    The steroid/thyroid hormone receptor superfamily of ligand-activated transcription factors encompasses not only the receptors for steroids, thyroid hormone, retinoids and vitamin D, but also a large number of proteins whose functions and/or ligands are unknown and which are thus termed orphan receptors. Recent studies have highlighted the importance of phosphorylation in receptor function. Although most of the phosphorylation sites are serine and threonine residues, a few of the family members are also phosphorylated on tyrosine. Those steroid receptor family members that are bound to heat-shock proteins in the absence of ligand typically are basally phosphorylated and exhibit increases in phosphorylation upon ligand binding. Most of these sites contain Ser-Pro motifs, and there is evidence that cyclin-dependent kinases and MAP kinases (mitogen-activated protein kinases) phosphorylate subsets of these sites. In contrast, phosphorylation sites identified thus far in members of the family that bind to DNA in the absence of hormone typically do not contain Ser-Pro motifs and are frequently casein kinase II or protein kinase A sites. Phosphorylation has been implicated in DNA binding, transcriptional activation and stability of the receptors. The finding that some of the steroid receptor family members can be activated in the absence of ligand by growth factors or neurotransmitters that modulate kinase and/or phosphatase pathways underscores the role of phosphorylation in receptor function. Hence this family of transcription factors integrates signals from ligands as well as from signal transduction pathways, resulting in alterations in mRNA and protein expression that are unique to the complex signals received. PMID:8920964

  11. Regulation of AMPA receptor phosphorylation by the neuropeptide PACAP38.

    PubMed

    Toda, Alyssa M A; Huganir, Richard L

    2015-05-26

    Dynamic changes in synaptic strength are thought to be critical for higher brain function such as learning and memory. Alterations in synaptic strength can result from modulation of AMPA receptor (AMPAR) function and trafficking to synaptic sites. The phosphorylation state of AMPAR subunits is one mechanism by which cells regulate receptor function and trafficking. Receptor phosphorylation is in turn regulated by extracellular signals; these include neuronal activity, neuropeptides, and neuromodulators such as dopamine and norepinephrine (NE). Although numerous studies have reported that the neuropeptide pituitary adenylate cyclase activating polypeptide 38 (PACAP38) alters hippocampal CA1 synaptic strength and GluA1 synaptic localization, its effect on AMPAR phosphorylation state has not been explored. We determined that PACAP38 stimulation of hippocampal cultures increased phosphorylation of S845, and decreased phosphorylation of T840 on the GluA1 AMPAR subunit. Increases in GluA1 S845 phosphorylation primarily occurred via PAC1 and VPAC2 receptor activation, whereas a reduction in GluA1 T840 phosphorylation was largely driven by PAC1 receptor activation and to a lesser extent by VPAC1 and VPAC2 receptor activation. GluA1 S845 phosphorylation could be blocked by a PKA inhibitor, and GluA1 T840 dephosphorylation could be blocked by a protein phosphatase 1/2A (PP1/PP2A) inhibitor and was partly blocked by a NMDA receptor (NMDAR) antagonist. These results demonstrate that the neuropeptide PACAP38 inversely regulates the phosphorylation of two distinct sites on GluA1 and may play an important role modulating AMPAR function and synaptic plasticity in the brain. PMID:25964356

  12. GRK6 phosphorylates I?B? at Ser(32)/Ser(36) and enhances TNF-?-induced inflammation.

    PubMed

    Ohba, Yuki; Nakaya, Michio; Watari, Kenji; Nagasaka, Akiomi; Kurose, Hitoshi

    2015-05-29

    G protein-coupled receptor kinases (GRKs) comprise a family of seven serine/threonine kinases that phosphorylate agonist-activated G protein-coupled receptors (GPCRs). It has recently been reported that GRKs regulate GPCR-independent signaling through the phosphorylation of intracellular proteins. To date, several intracellular substrates for GRK2 and GRK5 have been reported. However, those for GRK6 are poorly understood. Here we identified I?B?, a negative regulator of NF-?B signaling, as a substrate for GRK6. GRK6 directly phosphorylated I?B? at Ser(32)/Ser(36), and the kinase activity of GRK6 was required for the promotion of NF-?B signaling after TNF-? stimulation. Knockout of GRK6 in peritoneal macrophages remarkably attenuated the transcription of inflammatory genes after TNF-? stimulation. In addition, we developed a bioluminescence resonance energy transfer (BRET) probe to monitor GRK6 activity. Using this probe, we revealed that the conformational change of GRK6 was induced by TNF-?. In summary, our study demonstrates that TNF-? induces GRK6 activation, and GRK6 promotes inflammatory responses through the phosphorylation of I?B?. PMID:25881508

  13. SCF(beta-TRCP) and phosphorylation dependent ubiquitinationof I kappa B alpha catalyzed by Ubc3 and Ubc4.

    PubMed

    Strack, P; Caligiuri, M; Pelletier, M; Boisclair, M; Theodoras, A; Beer-Romero, P; Glass, S; Parsons, T; Copeland, R A; Auger, K R; Benfield, P; Brizuela, L; Rolfe, M

    2000-07-20

    NF kappa B is an important transcriptional regulator of multiple pro-inflammatory genes. In non-stimulated cells NF kappa B is anchored in the cytoplasm via the inhibitory protein I kappa B alpha. Following exposure to diverse pro-inflammatory signals (e.g. TNF alpha, IL1, LPS) various signal transduction cascades are initiated converging on the I kappa B kinase (IKK). IKK phosphorylates I kappa B alpha on serines 32 and 36 signaling the inhibitory protein for ubiquitin-mediated degradation. The SCF beta-TRCP complex is the ubiquitin ligase responsible for mediating phosphorylation dependent ubiquitination of I kappa B alpha. Here we reconstitute phosphorylation dependent ubiquitination of I kappa B alpha using recombinant components. Our results suggest that the cullin specificity of the SCF complex may reflect its ability to associate with Rbx1. We demonstrate specific ubiquitination of I kappa B alpha by Ubc3 and Ubc4 in a phosphorylation and SCF beta-TRCP dependent manner and that both are capable of associating with the SCF beta-TRCP complex isolated from human cells. Finally, we show that Ubc4 is in excess to Ubc3 in THP.1 cells and 19 times more efficient in catalyzing the reaction, suggesting that Ubc4 is the preferentially used Ubc in this reaction in vivo. Our results also suggest that ubiquitin is transferred directly from the Ubc to phospho-I kappa B alpha in a SCF beta-TRCP dependent reaction. Oncogene (2000) 19, 3529 - 3536 PMID:10918611

  14. Correlation between changes in light energy distribution and changes in thylakoid membrane polypeptide phosphorylation in Chlamydomonas reinhardtii

    SciTech Connect

    Wollman, F.A.; Delepelaire, P.

    1984-01-01

    We have used a new method to extensively modify the redox state of the plastoquinone pool in Chlamydomonas reinhardtii intact cells. This was achieved by an anaerobic treatment that inhibits the chlororespiratory pathway recently described by P. Bennoun. A state I (plus 3,4-dichlorophenyl-1,1-dimethylurea) ..-->.. anaerobic state transition induced a decrease in the maximal fluorescence yield at room temperature and in the F/sub PSII//F/sub PSI/ ratio at 77/sup 0/K, which was three times larger than in a classical state I ..-->.. state II transition. The fluorescence changes observed in vivo were similar in amplitude to those observed in vitro upon transfer to the light of dark-adapted, broken chloroplasts incubated in the presence of ATP. We then compared the phosphorylation pattern of thylakoid polypeptides in C. reinhardtii in vitro and in vivo using ..gamma..-(/sup 32/P)ATP and (/sup 32/P)orthophosphate labeling, respectively. The same set of polypeptides, mainly light-harvesting complex polypeptides, was phosphorylated in both cases. We observed that this phosphorylation process is reversible and is mediated by the redox state of the plastoquinone pool in vivo as well as in vitro. Similar changes of even larger amplitude were observed with the F34 mutant intact cells lacking in photosystem II centers. The presence of the photosystem II centers is then not required for the occurrence of the plastoquinone-mediated phosphorylation of light-harvesting complex polypeptides.

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

    NASA Technical Reports Server (NTRS)

    Kowalczyk, S.; Bandurski, R. S.

    1990-01-01

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

  16. Effects of Coxiella burnetii on MAPKinases phosphorylation.

    PubMed

    Boucherit, Nicolas; Barry, Abdoulaye Oury; Mottola, Giovanna; Trouplin, Virginie; Capo, Christian; Mege, Jean-Louis; Ghigo, Eric

    2012-02-01

    Q fever is a disease caused by Coxiella burnetii, an obligate intracellular bacterium. Acute Q fever is characterized by efficient immune response, whereas chronic Q fever is characterized by dysregulated immune response as demonstrated by the lack of granulomas, the failure of C. burnetii to induce lymphoproliferation, and interferon-? production. The mitogen-activated protein kinase (MAPK) signaling pathway plays crucial roles in innate immune responses and control of bacterial infections. However, its role in Q fever has not been addressed. First, we investigated the activation of MAPKs p38, c-jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in murine macrophages stimulated with C. burnetii. Coxiella burnetii NM phase I (virulent) and NM phase II (avirulent) induced the activation of JNK and ERK1/2. Avirulent C. burnetii activate p38, whereas C. burnetii did not induce the phosphorylation of p38. Second, the level of p38 activation was studied in Q fever patients. We found that p38 was activated in monocyte-derived macrophages from healthy donors and patients with acute Q fever in response to a potent agonist such as lipopolysaccharide. Interestingly, p38 was not activated in patients with active chronic Q fever and was activated in patients with cured chronic Q fever. These results suggest that the determination of p38 activation may serve as a tool for measuring Q fever activity. PMID:22092597

  17. Enzyme adsorption, precipitation and crosslinking of glucose oxidase and laccase on polyaniline nanofibers for highly stable enzymatic biofuel cells.

    PubMed

    Kim, Ryang Eun; Hong, Sung-Gil; Ha, Su; Kim, Jungbae

    2014-11-01

    Enzymatic biofuel cells have many great features as a small power source for medical, environmental and military applications. Both glucose oxidase (GOx) and laccase (LAC) are widely used anode and cathode enzymes for enzymatic biofuel cells, respectively. In this paper, we employed three different approaches to immobilize GOx and LAC on polyaniline nanofibers (PANFs): enzyme adsorption (EA), enzyme adsorption and crosslinking (EAC) and enzyme adsorption, precipitation and crosslinking (EAPC) approaches. The activity of EAPC-LAC was 32 and 25 times higher than that of EA-LAC and EAC-LAC, respectively. The half-life of EAPC-LAC was 53 days, while those of EA-LAC and EAC-LAC were 6 and 21 days, respectively. Similar to LAC, EAPC-GOx also showed higher activity and stability than EA-GOx and EAC-GOx. For the biofuel cell application, EAPC-GOx and EAPC-LAC were applied over the carbon papers to form enzyme anode and cathode, respectively. In order to improve the power density output of enzymatic biofuel cell, 1,4-benzoquinone (BQ) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were introduced as the electron transfer mediators on the enzyme anode and enzyme cathode, respectively. BQ- and ABTS-mediated enzymatic biofuel cells fabricated by EAPC-GOx and EAPC-LAC showed the maximum power density output of 37.4 ?W/cm(2), while the power density output of 3.1 ?W/cm(2) was shown without mediators. Under room temperature and 4°C for 28 days, enzymatic biofuel cells maintained 54 and 70% of its initial power density, respectively. PMID:25248697

  18. Enzymatic conversion of sucrose to hydrogen

    SciTech Connect

    Woodward, J.; Orr, M.

    1998-11-01

    The enzymatic conversion of sugars to hydrogen could be a promising method for alternative fuel production. Maple tree sap is a source of environmental sugar (e.g., sucrose) that has the potential to be converted into hydrogen using the enzymes invertase, glucose dehydrogenase (GDH), hydrogenase, and glucose isomerase (GI) and the cofactor NADP{sup +}/NADPH. The kinetics of hydrogen production have been studied, and optimal conditions for hydrogen production are described. At low initial sucrose concentrations, in the absence of glucose isomerase, stoichiometric yields of mol of H{sub 2}/mol of sucrose were achieved. At higher sucrose concentrations, the yield of hydrogen declined so that at an initial sucrose concentration of 292 mM only 7% yield of hydrogen was obtained. The reason for this low yield was studied and shown not to be caused by enzyme inactivation or a pH drop during the reaction but due to an instability of the cofactor NADP{sup +}. Although gluconic and inhibited both NADPH production and oxidation of GDH and hydrogenase, respectively, it was not the major cause of NADP{sup +} instability. Fructose was also shown to be converted to hydrogen if GI was present in the reaction mixture. Also, by starting with sucrose, 1.34 mol of H{sub 2}/mol of sucrose was obtained if GI was present in the reaction mixture.

  19. New enzymatic assay of iron in serum.

    PubMed

    Fujita, T; Hamasaki, H; Furukata, C; Nonobe, M

    1994-05-01

    A new enzymatic method for assaying iron in serum samples, suitable for automated analyzers, is reported. Three reagent mixtures are used: dilution buffer (pH 3.0; ascorbate), reagent 1 (pH 6.7; apoaconitase), and reagent 2 (pH 7.7; citrate, magnesium, and isocitrate dehydrogenase). Sera are diluted with dilution buffer. Fe3+ is liberated from transferrin in sera under acidic conditions, and then reduced by ascorbate. Reagent 1 is added to diluted specimens, and apoaconitase is reactivated by Fe2+ at neutral pH. The resulting solutions are mixed with reagent 2, so that holoaconitase hydrolyzes citrate to isocitrate and the isocitrate and NADP+ are converted to 2-oxoglutarate, NADPH, and CO2. Serum iron is determined linearly up to 70 mumol/L, with within-run CVs < or = 2.4% and day-to-day CVs < or = 2.9%. This method (y) gives results correlating with those of a Reference Method (x) proposed by the International Committee for Standardization in Haematology: y = 0.98x + 0.38 mumol/L (n = 72, r = 0.996, Sylx = 0.63 mumol/L). The mean (+/- SD) serum iron concentrations measured by our method were 18.5 +/- 5.4 and 15.2 +/- 6.0 mumol/L for 63 males and 166 females, respectively. PMID:8174249

  20. Enzymatic production of hydrogen from glucose

    SciTech Connect

    Woodward, J.; Mattingly, S.M.

    1995-06-01

    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 (H{sub 2}ase) which catalyzes the evolution of H{sub 2}. Thus hydrogen production from glucose was achieved using calf liver GDH and Pyrococcus furiosus H{sub 2}ase 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 stable enzymes are underway.

  1. 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

  2. Associate editor: K.W. Roche Synaptic plasticity and phosphorylation

    E-print Network

    Lee, Hey-Kyoung

    Associate editor: K.W. Roche Synaptic plasticity and phosphorylation Hey-Kyoung Lee Department, United States Abstract A number of neuronal functions, including synaptic plasticity, depend on proper kinase and protein phosphatase activity. Recent understanding of synaptic plasticity mechanisms

  3. Phosphorylation of a neuronal-specific beta-tubulin isotype

    SciTech Connect

    Diaz-Nido, J.; Serrano, L.; Lopez-Otin, C.; Vandekerckhove, J.; Avila, J. )

    1990-08-15

    Adult rats were intracraneally injected with ({sup 32}P) phosphate and brain microtubules isolated. The electrophoretically purified, in vivo phospholabeled, beta-tubulin was digested with the V8-protease and the labeled peptide purified by reversed-phase liquid chromatography. Its amino acid sequence corresponds to the COOH-terminal sequence of a minor neuronal beta 3-tubulin isoform from chicken and human. The phosphorylation site was at serine 444. A synthetic peptide with sequence EMYEDDEEESESQGPK, corresponding to that of the COOH terminus of beta 3-tubulin, was efficiently phosphorylated in vitro by casein kinase II at the same serine 444. The functional meaning of tubulin phosphorylation is still unclear. However, the modification of the protein takes place after microtubule assembly, and phosphorylated tubulin is mainly present in the assembled microtubule protein fraction.

  4. Phosphorylation of Mad controls competition between wingless and BMP signaling.

    PubMed

    Eivers, Edward; Demagny, Hadrien; Choi, Renee H; De Robertis, Edward M

    2011-01-01

    Bone morphogenetic proteins (BMPs) and Wnts are growth factors that provide essential patterning signals for cell proliferation and differentiation. Here, we describe a molecular mechanism by which the phosphorylation state of the Drosophila transcription factor Mad determines its ability to transduce either BMP or Wingless (Wg) signals. Previously, Mad was thought to function in gene transcription only when phosphorylated by BMP receptors. We found that the unphosphorylated form of Mad was required for canonical Wg signaling by interacting with the Pangolin-Armadillo transcriptional complex. Phosphorylation of the carboxyl terminus of Mad by BMP receptor directed Mad toward BMP signaling, thereby preventing Mad from functioning in the Wg pathway. The results show that Mad has distinct signal transduction roles in the BMP and Wnt pathways depending on its phosphorylation state. PMID:21990430

  5. Phosphoproteomics reveals extensive in vivo phosphorylation of Arabidopsis proteins involved

    E-print Network

    Hirt, Heribert

    oxide affinity chromatography (2) or TiO2 (3) coupled to mass spectrometric analysis. IMAC has been used most success- fully for the large-scale identification of phosphorylation sites, although TiO2 has been

  6. Abiotic regioselective phosphorylation of adenosine with borate in formamide.

    PubMed

    Furukawa, Yoshihiro; Kim, Hyo-Joong; Hutter, Daniel; Benner, Steven A

    2015-04-01

    Nearly 40 years ago, Schoffstall and his coworkers used formamide as a solvent to permit the phosphorylation of nucleosides by inorganic phosphate to give nucleoside phosphates, which (due to their thermodynamic instability with respect to hydrolysis) cannot be easily created in water by an analogous phosphorylation (the "water problem" in prebiotic chemistry). More recently, we showed that borate could stabilize certain carbohydrates against degradation (the "asphalt problem"). Here, we combine the two concepts to show that borate can work in formamide to guide the reactivity of nucleosides under conditions where they are phosphorylated. Specifically, reaction of adenosine in formamide with inorganic phosphate and pyrophosphate in the presence of borate gives adenosine-5'-phosphate as the only detectable phosphorylated product, with formylation (as opposed to hydrolysis) being the competing reaction. PMID:25826074

  7. Methods for generating phosphorylation site-specific immunological reagents

    DOEpatents

    Anderson, Carl W. (Stony Brook, NY); Appella, Ettore (Montgomery, MD); Sakaguchi, Kazuyasu (Montgomery, MD)

    2001-01-01

    The present invention provides methods for generating phosphorylation site-specific immunological reagents. More specifically, a phosphopeptide mimetic is incorporated into a polypeptide in place of a phosphorylated amino acid. The polypeptide is used as antigen by standard methods to generate either monoclonal or polyclonal antibodies which cross-react with the naturally phosphorylated polypeptide. The phosphopeptide mimetic preferably contains a non-hydrolyzable linkage from the appropriate carbon atom of the amino acid residue to a phosphate group. A preferred linkage is a CF.sub.2 group. Such a linkage is used to generate the phosphoserine mimetic F.sub.2 Pab, which is incorporated into a polypeptide sequence derived from p53 to produce antibodies which recognize a specific phosphorylation state of p53. A CF.sub.2 group linkage is also used to produce the phosphothreonine mimetic F.sub.2 Pmb, and to produce the phosphotyrosine mimetic, F.sub.2 Pmp.

  8. Phosphorylation of Mad Controls Competition Between Wingless and BMP Signaling

    PubMed Central

    Eivers, Edward; Demagny, Hadrien; Choi, Renee H.; De Robertis, Edward M.

    2011-01-01

    Bone morphogenetic proteins (BMPs) and Wnts are growth factors that provide essential patterning signals for cell proliferation and differentiation. Here, we describe a molecular mechanism by which the phosphorylation state of the Drosophila transcription factor Mad determines its ability to transduce either BMP or Wingless (Wg) signals. Previously, Mad was thought to function in gene transcription only when phosphorylated by BMP receptors. We found that the unphosphorylated form of Mad was required for canonical Wg signaling by interacting with the Pangolin-Armadillo transcriptional complex. Phosphorylation of the carboxyl terminus of Mad by BMP receptor directed Mad toward BMP signaling, thereby preventing Mad from functioning in the Wg pathway. The results show that Mad has distinct signal transduction roles in the BMP and Wnt pathways depending on its phosphorylation state. PMID:21990430

  9. Resources for Assignment of Phosphorylation Sites on Peptides and Proteins.

    PubMed

    Ravikumar, Vaishnavi; Macek, Boris; Mijakovic, Ivan

    2016-01-01

    Reversible protein phosphorylation is a key regulatory posttranslational modification that plays a significant role in major cellular signaling processes. Phosphorylation events can be systematically identified, quantified, and localized on protein sequence using publicly available bioinformatic tools. Here we present the software tools commonly used by the phosphoproteomics community, discuss their underlying principles of operation, and provide a protocol for large-scale phosphoproteome data analysis using the MaxQuant software suite. PMID:26584934

  10. Contractile protein phosphorylation predicts human heart disease phenotypes

    PubMed Central

    Fullerton, David A.; Buttrick, Peter M.

    2013-01-01

    Human heart failure has been associated with a low level of thin-filament protein phosphorylation and an increase in calcium sensitivity of contraction relative to both “control” human heart tissue and tissue from small animal models. However, diverse strategies of human tissue procurement and the reliance on tissue obtained from subjects with end-stage heart failure suggest this may be an incomplete characterization. Therefore, we evaluated cardiac left ventricular (LV) biopsy samples from patients with aortic stenosis undergoing valve replacement who presented either with LV hypertrophy and preserved systolic function (Hyp) or with LV dilation and reduced ejection fraction (Dil). In Hyp, total troponin I (TnI) phosphorylation was markedly increased and myosin light chain 2 (MLC2) phosphorylation was unchanged relative to a control group of patients with normal LV function. Conversely, in Dil, total TnI phosphorylation was significantly reduced compared with control subjects and MLC2 phosphorylation was increased. Site-specific analysis of TnI phosphorylation revealed phenotype-specific differences such that Hyp samples demonstrated significant increases in phosphorylation at serine 22/23 and Dil samples had significant decreases at serine 43. The ratio of phosphorylation at the two sites was biased toward serine 22/23 in Hyp and toward serine 43/45 in Dil. Western blot analysis showed that protein phosphatase-1 was reduced in Hyp and protein phosphatase-2 was reduced in Dil. These data suggest that posttranslational modifications of sarcomeric proteins, both singly and in combination, are stage specific. Defining these changes in progressive heart disease may provide important diagnostic and treatment information. PMID:23564307

  11. Taking electrons out of bioelectronics: bioprotonic memories and enzymatic logic gates (Presentation Recording)

    NASA Astrophysics Data System (ADS)

    Rolandi, Marco

    2015-10-01

    In living systems, protonic and ionic currents are the basis for all information processing. As such, artificial devices based on protonic and ionic currents offer an exciting opportunity for bioelectronics. Proton transport in nature is important for ATP oxidative phosphorylation, the HCVN1 voltage gated proton channel, light activated proton pumping in bacteriorhodopsin, and the proton conducting single water file of the antibiotic gramicidin. In these systems, protons move along hydrogen bond networks formed by water and the hydrated biomolecules (proton wires). We have previously demonstrated complementary H+- and OH-- FETs with acid and base doped biopolymer proton wires and PdHx proton conducting contacts. Here, I will discuss proton-conducting devices based oh highly conductive proton wires that emulate brain synapses, display memristive behaviour, and are connected to form shift registries. Furthermore, I will present the integration of these devices with enzymatic logic gates for integrated biotic-abiotic protonic information processing. Preliminary results on using these devices to affect biological function will be discussed.

  12. Structural insights into the enzymatic mechanism of the pathogenic MAPK phosphothreonine lyase.

    PubMed

    Zhu, Yongqun; Li, Hongtao; Long, Chengzu; Hu, Liyan; Xu, Hao; Liu, Liping; Chen, She; Wang, Da-Cheng; Shao, Feng

    2007-12-14

    The OspF family of phosphothreonine lyase, including SpvC from Salmonella, irreversibly inactivates the dual-phosphorylated host MAPKs (pT-X-pY) through beta elimination. We determined crystal structures of SpvC and its complex with a phosphopeptide substrate. SpvC adopts a unique fold of alpha/beta type. The disordered N terminus harbors a canonical D motif for MAPK substrate docking. The enzyme-substrate complex structure indicates that recognition of the phosphotyrosine followed by insertion of the threonine phosphate into an arginine pocket places the phosphothreonine into the enzyme active site. This requires the conformational flexibility of pT-X-pY, which suggests that p38 (pT-G-pY) is likely the preferred physiological substrate. Structure-based biochemical and enzymatic analysis allows us to propose a general acid/base mechanism for beta elimination reaction catalyzed by the phosphothreonine lyase. The mechanism described here provides a structural understanding of MAPK inactivation by a family of pathogenic effectors conserved in plant and animal systems and may also open a new route for biological catalysis. PMID:18060821

  13. Nicotine Enhances the Antiapoptotic Function of Mcl-1 through Phosphorylation*

    PubMed Central

    Zhao, Jinfeng; Xin, Meiguo; Wang, Ton; Zhang, Yangde; Deng, Xingming

    2009-01-01

    Lung cancer has a strong etiological association with cigarette smoking. Nicotine, a major component in tobacco smoke, functions as a survival agonist that inhibits apoptosis following various stresses. However, the mechanism of action remains elusive. Mcl-1, a major antiapoptotic protein of the Bcl2 family, is extensively expressed in both small cell (SCLC) and non-small cell lung cancer (NSCLC) cells, suggesting that Mcl-1 may be a therapeutic target of patients with lung cancer. Here we found that nicotine induces Mcl-1 phosphorylation through activation of ERK1/2 in association with increased chemoresistance of human lung cancer cells. Since nicotine stimulates Mcl-1 phosphorylation and survival in cells expressing WT but has no such effects in cells expressing T163A Mcl-1 mutant, this indicates that nicotine induces Mcl-1 phosphorylation exclusively at the T 163 site and that phosphorylation of Mcl-1 at T163 is required for nicotine-induced survival. Mechanistically, nicotine-induced Mcl-1 phosphorylation significantly enhances the half-life of Mcl-1, which renders Mcl-1 a long-term survival activity. Specific depletion of Mcl-1 by RNA interferenceblocks nicotine-stimulated survival and enhances apoptotic cell death. Thus, nicotine-enhanced survival of lung cancer cells may occur through activation of Mcl-1 by phosphorylation at T163 site, which may contribute to development of human lung cancer and/or chemoresistance. PMID:19903766

  14. Tyrosine phosphorylation of clathrin heavy chain under oxidative stress.

    PubMed

    Ihara, Yoshito; Yasuoka, Chie; Kageyama, Kan; Wada, Yoshinao; Kondo, Takahito

    2002-09-20

    In mouse pancreatic insulin-producing betaTC cells, oxidative stress due to H(2)O(2) causes tyrosine phosphorylation in various proteins. To identify proteins bearing phosphotyrosine under stress, the proteins were affinity purified using an anti-phosphotyrosine antibody-conjugated agarose column. A protein of 180kDa was identified as clathrin heavy chain (CHC) by electrophoresis and mass spectrometry. Immunoprecipitated CHC showed tyrosine phosphorylation upon H(2)O(2) treatment and the phosphorylation was suppressed by the Src kinase inhibitor, PP2. The phosphorylation status of CHC affected the intracellular localization of CHC and the clathrin-dependent endocytosis of transferrin under oxidative stress. In conclusion, CHC is a protein that is phosphorylated at tyrosine by H(2)O(2) and this phosphorylation status is implicated in the intracellular localization and functions of CHC under oxidative stress. The present study demonstrates that oxidative stress affects intracellular vesicular trafficking via the alteration of clathrin-dependent vesicular trafficking. PMID:12237126

  15. Multisite phosphorylation of spinach leaf sucrose-phosphate synthase

    SciTech Connect

    Huber, J.L.; Huber, S.C. )

    1990-05-01

    Spinach leaf sucrose-phosphate synthase is phosphorylated both in vivo and in vitro on serine residues. Phosphorylation of SPS in vivo yields twelve major phosphopeptides after a tryptic digest and two dimensional mapping. The in vivo labeling of three of these SPS P-peptides is reduced in illuminated leaves where the extracted enzyme is activated relative to that of dark leaves. Two of these inhibitory sites are phosphorylated as well when SPS is inactivated in vitro using ({sup 32}P)ATP. In vivo phosphorylation of two other sites is enhanced during mannose feeding of the leaves (in light or dark) which produces the highest activation state of SPS. Overall, the results confirm that light-dark regulation of SPS activity occurs as a result of regulatory seryl-phosphorylation and involves a balance between phosphorylation of sites which inhibit or stimulate activity. Regulation of the SPS protein kinase that inhibits activity is relatively unaffected by phosphate but inhibited by G1c 6-P (IC{sub 50}{approx}5 mM), which may explain the control of SPS activation state by light-dark signals.

  16. Platelet tyrosine-specific protein phosphorylation is regulated by thrombin.

    PubMed Central

    Ferrell, J E; Martin, G S

    1988-01-01

    Intact human platelets, terminally differentiated cells with no growth potential, were found to possess unusually high levels of tyrosine-specific protein phosphorylation. The physiological platelet activator thrombin transiently elevated platelet phosphotyrosine content, apparently through stimulation of one or more tyrosine-specific protein kinases. Immunoblotting with antiphosphotyrosine antiserum showed that thrombin caused dramatic changes in the tyrosine phosphorylation of a number of individual protein bands and that these changes occurred in three distinct temporal waves. Most but not all of the protein bands phosphorylated at tyrosine in response to thrombin were also tyrosine phosphorylated in response to chilling or the combination of ionophore A23187 and tetradecanoylphorbol acetate. Thrombin stimulated the phosphorylation of the tyrosine kinase pp60c-src, primarily at Ser-12 and Tyr-527, although the effects of these phosphorylations on platelet pp60c-src function were not apparent. Together, these results suggest that tyrosine-specific protein kinases of uncertain identity are involved in signal transduction in platelets. Images PMID:2464741

  17. Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats

    SciTech Connect

    Deaciuc, I.V.; Spitzer, J.A. )

    1989-11-01

    The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with (32P)H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis.

  18. Functionalized Graphene Oxide as a Nanocarrier in a Multienzyme Labeling Amplification Strategy for Ultrasensitive Electrochemical Immunoassay of Phosphorylated p53 (S392)

    SciTech Connect

    Du, Dan; Wang, Limin; Shao, Yuyan; Wang, Jun; Engelhard, Mark H.; Lin, Yuehe

    2011-01-06

    P53 phosphorylation plays an important role in many biological processes and might be used as a potential biomarker in clinical diagnoses. We report a new electrochemical immunosensor for ultrasensitive detection of phosphorylated p53 at Ser392 (phospho-p53-392) based on graphene oxide (GO) as a nanocarrier in multienzymes amplification strategy. Greatly enhanced sensitivity was achieved by using the bioconjugates featuring horseradish peroxidase (HRP) and p53392 signal antibody (p53Ab2) linked to functionalized GO (HRP-p53Ab2-GO) at high ratio of HRP/p53Ab2. After a sandwich immunoreaction, the HRP-p53Ab2-GO captured onto the electrode surface produced an amplified electrocatalytic response by the reduction of enzymatically oxidized thionine in the presence of hydrogen peroxide. The increase of response current was proportional to the phospho-p53 concentration in the range of 0.02 to 2 nM with the detection limit of 0.01 nM, which was 10-fold lower than that of traditional sandwich electrochemical measurement for p53. The amplified immunoassay developed in this work shows acceptable stability and reproducibility and the assay results for phospho-p53 spiked in human plasma also show good recovery (92%~103.8%). This simple and low-cost immunosensor shows great promise for detection of other phosphorylated proteins and clinical applications.

  19. Threonine 308 phosphorylated form of Akt translocates to the nucleus of PC12 cells under nerve growth factor stimulation and associates with the nuclear matrix protein nucleolin.

    PubMed

    Borgatti, Paola; Martelli, Alberto M; Tabellini, Giovanna; Bellacosa, Alfonso; Capitani, Silvano; Neri, Luca M

    2003-07-01

    We have examined the issue of whether or not in PC12 cells it may be observed a nerve growth factor (NGF) nuclear translocation of an active (phosphorylated) Akt. Western blot analysis with antibodies to either total or phosphorylated Akt showed a maximal nuclear translocation after 15 min of NGF stimulation. NGF increased rapidly and transiently the enzymatic activity of immunoprecipitable nuclear Akt and after 45 min the values returned to a level close to the basal one. Enzyme translocation was blocked by the selective phosphoinositide 3-kinase inhibitor, LY294002. Confocal microscopy of samples stained with antibody to Akt showed an evident increase in immunostaining intensity in the nuclear interior after NGF treatment. Treatment of cells with inhibitors of protein phosphatase PP2A, calyculin A, or okadaic acid, maintained the phosphorylation levels of nuclear Akt. Immunoprecipitation experiments revealed an association between Akt and PP2A that was maximal when nuclear Akt activity was decreased. Both total and active Akt associated with the nuclear matrix and, in particular, with the protein nucleolin, with which Akt co-immunoprecipitated. These findings strongly suggest that the intranuclear translocation of active Akt is an important step in the signaling pathways elicited by the neurotrophin NGF and that the intranuclear control of Akt is achieved through the action of PP2A. PMID:12767043

  20. Anaplastic lymphoma kinase protein expression, genetic abnormalities, and phosphorylation in soft tissue tumors: Phosphorylation is associated with recurrent metastasis.

    PubMed

    Ishibashi, Yukinao; Miyoshi, Hiroaki; Hiraoka, Koji; Arakawa, Fumiko; Haraguchi, Toshiaki; Nakashima, Shinji; Hashiguchi, Toshihiro; Shoda, Takanori; Hamada, Tetsuya; Okawa, Takahiro; Higuchi, Fujio; Shiba, Naoto; Nagata, Kensei; Ohshima, Koichi

    2015-04-01

    Gene and protein abnormalities of anaplastic lymphoma kinase (ALK) play an important role in the pathogenesis of various cancers and serve as important therapeutic targets. We investigated ALK protein expression, phosphorylation, and genetic aberrations using fluorescence in situ hybridization (FISH) in 81 soft tissue tumor samples: inflammatory myofibroblastic tumor, n=1; alveolar soft part sarcoma, n=2; leiomyosarcoma, n=10; well-differentiated liposarcoma, n=7; pleomorphic liposarcoma, n=2; extraskeletal osteosarcoma, n=1; epithelioid sarcoma, n=1; synovial sarcoma, n=4; malignant peripheral nerve sheath tumor, n=4; undifferentiated pleomorphic sarcoma, n=19; rhabdomyosarcoma, n=6; myxofibrosarcoma, n=8; myxoid liposarcoma, n=11; fibrosarcoma, n=4; and desmoid-type fibromatosis, n=1. ALK protein expression, gene signal gain (without translocation), and phosphorylation were observed in 33/81 (40.7%), 55/81 (67.9%), and 30/81 (37.0%) tumor samples, respectively. ALK protein expression was statistically associated with phosphorylation, but not with gene signal gain. ALK phosphorylation-positive cases showed a statistically worse metastasis-free survival compared with phosphorylation-negative cases (P=0.0215). Particularly, metastasis of myxoid liposarcoma was associated with ALK phosphorylation (P=0.0019), but not with ALK protein expression or gene signal gain. However, the prognosis had no association with ALK protein expression, gene signal gain, or phosphorylation. ALK protein expression and phosphorylation play an important role in tumor biology and provide potential therapeutic targets for soft tissue tumors. Future research should focus on the oncogenic role and the efficacy of potential inhibitors of ALK. PMID:25683346

  1. Neuronal uptake and propagation of a rare phosphorylated high-molecular-weight tau derived from Alzheimer's disease brain

    PubMed Central

    Takeda, Shuko; Wegmann, Susanne; Cho, Hansang; DeVos, Sarah L.; Commins, Caitlin; Roe, Allyson D.; Nicholls, Samantha B.; Carlson, George A.; Pitstick, Rose; Nobuhara, Chloe K.; Costantino, Isabel; Frosch, Matthew P.; Müller, Daniel J.; Irimia, Daniel; Hyman, Bradley T.

    2015-01-01

    Tau pathology is known to spread in a hierarchical pattern in Alzheimer's disease (AD) brain during disease progression, likely by trans-synaptic tau transfer between neurons. However, the tau species involved in inter-neuron propagation remains unclear. To identify tau species responsible for propagation, we examined uptake and propagation properties of different tau species derived from postmortem cortical extracts and brain interstitial fluid of tau-transgenic mice, as well as human AD cortices. Here we show that PBS-soluble phosphorylated high-molecular-weight (HMW) tau, though very low in abundance, is taken up, axonally transported, and passed on to synaptically connected neurons. Our findings suggest that a rare species of soluble phosphorylated HMW tau is the endogenous form of tau involved in propagation and could be a target for therapeutic intervention and biomarker development. PMID:26458742

  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. Nanocrystal Bioassembly: Asymmetry, Proximity, and Enzymatic Manipulation

    SciTech Connect

    Claridge, Shelley A

    2008-05-01

    Research at the interface between biomolecules and inorganic nanocrystals has resulted in a great number of new discoveries. In part this arises from the synergistic duality of the system: biomolecules may act as self-assembly agents for organizing inorganic nanocrystals into functional materials; alternatively, nanocrystals may act as microscopic or spectroscopic labels for elucidating the behavior of complex biomolecular systems. However, success in either of these functions relies heavily uponthe ability to control the conjugation and assembly processes.In the work presented here, we first design a branched DNA scaffold which allows hybridization of DNA-nanocrystal monoconjugates to form discrete assemblies. Importantly, the asymmetry of the branched scaffold allows the formation of asymmetric2assemblies of nanocrystals. In the context of a self-assembled device, this can be considered a step toward the ability to engineer functionally distinct inputs and outputs.Next we develop an anion-exchange high performance liquid chromatography purification method which allows large gold nanocrystals attached to single strands of very short DNA to be purified. When two such complementary conjugates are hybridized, the large nanocrystals are brought into close proximity, allowing their plasmon resonances to couple. Such plasmon-coupled constructs are of interest both as optical interconnects for nanoscale devices and as `plasmon ruler? biomolecular probes.We then present an enzymatic ligation strategy for creating multi-nanoparticle building blocks for self-assembly. In constructing a nanoscale device, such a strategy would allow pre-assembly and purification of components; these constructs can also act as multi-label probes of single-stranded DNA conformational dynamics. Finally we demonstrate a simple proof-of-concept of a nanoparticle analog of the polymerase chain reaction.

  4. Enzymatic reduction of azo and indigoid compounds.

    PubMed

    Pricelius, S; Held, C; Murkovic, M; Bozic, M; Kokol, V; Cavaco-Paulo, A; Guebitz, G M

    2007-11-01

    A customer- and environment-friendly method for the decolorization azo dyes was developed. Azoreductases could be used both to bleach hair dyed with azo dyes and to reduce dyes in vat dyeing of textiles. A new reduced nicotinamide adenine dinucleotide-dependent azoreductase of Bacillus cereus, which showed high potential for reduction of these dyes, was purified using a combination of ammonium sulfate precipitation and chromatography and had a molecular mass of 21.5 kDa. The optimum pH of the azoreductase depended on the substrate and was within the range of pH 6 to 7, while the maximum temperature was reached at 40 degrees C. Oxygen was shown to be an alternative electron acceptor to azo compounds and must therefore be excluded during enzymatic dye reduction. Biotransformation of the azo dyes Flame Orange and Ruby Red was studied in more detail using UV-visible spectroscopy, high-performance liquid chromatography, and mass spectrometry (MS). Reduction of the azo bonds leads to cleavage of the dyes resulting in the cleavage product 2-amino-1,3 dimethylimidazolium and N approximately 1 approximately ,N approximately 1 approximately -dimethyl-1,4-benzenediamine for Ruby Red, while only the first was detected for Flame Orange because of MS instability of the expected 1,4-benzenediamine. The azoreductase was also found to reduce vat dyes like Indigo Carmine (C.I. Acid Blue 74). Hydrogen peroxide (H(2)O(2)) as an oxidizing agent was used to reoxidize the dye into the initial form. The reduction and oxidation mechanism of Indigo Carmine was studied using UV-visible spectroscopy. PMID:17891390

  5. Phosphorylation in sealed rod outer segments: effects of cyclic nucleotides.

    PubMed

    Shuster, T A; Farber, D B

    1984-01-31

    Rod outer segments (ROS) from rat were purified on Percoll gradients. These ROS had intact plasma membranes since they were impermeable to small molecules. Protein phosphorylation in the purified ROS was studied after the plasma membrane was disrupted by freeze/thawing. [gamma-32P]ATP was used as phosphate donor. ATP concentration, time, temperature, and light or dark adaptation were varied in the assays. The 32P-labeled proteins were separated by polyacrylamide gel electrophoresis and autoradiographed. Rhodopsin was the dominant phosphorylated protein, and the addition of adenosine cyclic 3',5'-phosphate (cAMP) or guanosine cyclic 3',5'-phosphate (cGMP) (10(-4) M) did not qualitatively alter the ROS phosphorylation pattern. The only cyclic nucleotide effect we could establish in these experiments was the inhibition of rhodopsin phosphorylation by cGMP. This inhibition did not appear to be competitive with ATP since cAMP was much less inhibitory than cGMP and the phosphorylation in the presence of cGMP reached a plateau at a much lower level than in control conditions. Hypotheses implying an involvement of protein phosphorylation/dephosphorylation in dark adaptation have been formulated [Miller, J. A., & Paulsen, R. (1975) J. Biol. Chem. 250, 4427-4432; Kuhn, H., McDowell, J. H., Leser, K. H., & Bader, S. (1977) Biophys. Struct. Mech. 3, 175-180]; we suggest that cGMP may control this process through the modulation of the extent of inhibition of phosphorylation of the visual pigment. PMID:6322841

  6. Phosphorylation of Rpt6 regulates synaptic strength in hippocampal neurons

    PubMed Central

    Djakovic, Stevan N.; Marquez-Lona, Esther M.; Jakawich, Sonya K.; Wright, Rebecca; Chu, Carissa; Sutton, Michael A.; Patrick, Gentry N.

    2012-01-01

    It has become increasingly evident that protein degradation via the ubiquitin proteasome system plays a fundamental role in the development, maintenance and remodeling of synaptic connections in the central nervous system. We and others have recently described the activity-dependent regulation of proteasome activity (Djakovic et al., 2009) and recruitment of proteasomes into spine compartments (Bingol and Schuman, 2006) involving the phosphorylation of the 19S ATPase subunit, Rpt6, by the plasticity kinase Ca2+/calmodulin-dependent protein kinases II alpha CaMKII?) (Bingol et al., 2010). Here, we investigated the role of Rpt6 phosphorylation on proteasome function and synaptic strength. Utilizing a phospho-specific antibody we verified that Rpt6 is phosphorylated at Serine 120 (S120) by CaMKII?. In addition, we found that Rpt6 is phosphorylated by CaMKII? in an activity-dependent manner. In addition, we showed that a serine 120 to aspartic acid phospho-mimetic mutant of Rpt6 (S120D) increases its resistance to detergent extraction in rat hippocampal dendrites, indicating phosphorylated Rpt6 may promote the tethering of proteasomes to scaffolds and cytoskeletal components. Interestingly, expression of Rpt6 S120D decreased miniature excitatory postsynaptic current (mEPSC) amplitude, while expression of a phospho-dead mutant (S120A) increased mEPSC amplitude. Surprisingly, homeostatic scaling of mEPSC amplitude produced by chronic application of bicuculline or tetrodotoxin is both mimicked and occluded by altered Rpt6 phosphorylation. Together these data suggest that CaMKII-dependent phosphorylation of Rpt6 at S120 may be an important regulatory mechanism for proteasome-dependent control of synaptic remodeling in slow homeostatic plasticity. PMID:22496558

  7. Effects of Phosphorylation in Chlamydomonas Centrin Ser 167.

    PubMed

    Sanoguet, Zuleika; Campbell, Muriel; Ramos, Sindia; Seda, Christina; Moreno, Luis Pérez; Pastrana-Rios, Belinda

    2006-01-01

    Centrin is a conserved calcium binding protein belonging to the EF-hand superfamily with two independent structural domains. This protein is found to be phosphorylated near the carboxyl terminal end. Our goal was to perform a novel comparative study of phosphorylated and unphosphorylated centrin by Fourier transform infrared (FT-IR) spectroscopy, two-dimensional correlation spectroscopy (2D-COS) analysis and differential scanning calorimetry (DSC). To achieve this goal, we have bacterially expressed, isolated, purified and phosphorylated centrin. We verified the extent of phosphorylation to be >97% for centrin by MALDI MS analysis and determined the absence of aggregated protein. The thermal denaturation temperature and ?Cp were determined to be T(m) = 112.1 °C (?Cp = 7.8 Kcal/mole/?C) and T(m) = 111.0°C (?Cp = 5.0 Kcal/mole/°C) for holo-centrin and phosphorylated centrin, respectively. We have also described the molecular dynamics leading up to the thermal denaturation of the protein: for holo-centrin the vibrational modes associated with the calcium binding sites aspartates and glutamates, loops then the arginines, followed by the structured backbone vibrational modes the ?-helix at 1635 cm(-1) then ?-sheet and finally the more exposed ?-helix at 1650 cm(-1); while for phosphorylated centrin aspartate, glutamate and arginine, followed by the backbone associated vibrational modes ?-helix (1650 cm(-1)), loop then the ?-sheet (1633 cm(-1)) and finally the ?-helix (1637 cm(-1)). Therefore, the effect on domain stability due to phosphorylation at Ser(167) was observed in the loops as well as the ?-helix at 1650 cm(-1). PMID:22162668

  8. Real-time imaging elucidates the role of H2O2 in regulating kinetics of epidermal growth factor-induced and Src-mediated tyrosine phosphorylation signaling

    NASA Astrophysics Data System (ADS)

    Su, Ting; Li, Xiangyong; Liu, Nisha; Pan, Shaotao; Lu, Jinling; Yang, Jie; Zhang, Zhihong

    2012-07-01

    Reversible oxidation is emerging as an important regulatory mechanism in protein tyrosine phosphorylation. Generation of hydrogen peroxide (H2O2), upon growth factor stimulation, is hypothesized to inhibit activity of protein tyrosine phosphatases (PTPs). This ensures that protein tyrosine kinases can elevate the steady-state level of protein tyrosine phosphorylation, which then allows propagation of the tyrosine phosphorylation signal. However, the effects of H2O2 on the kinetics of tyrosine phosphorylation signaling remain poorly understood, especially in living cells. Therefore, we used a genetically encoded Src kinase-specific biosensor based on fluorescence resonance energy transfer (FRET) to image the kinetics of the Src-mediated tyrosine phosphorylation signaling (Src signaling) induced by epidermal growth factor (EGF). We examined the kinetics under increased and decreased H2O2 levels. Through a straightforward, quantitative analysis method which characterized the signaling kinetics, we demonstrated that H2O2 modulated the amplitude and duration of the signal by inhibiting PTPs' activity. Our evidence also suggested the effect of H2O2 on Src activation is mediated by H2O2-dependent inhibition of PTPs. Furthermore, we provide evidence showing global elevation of intracellular H2O2 level attenuates EGF-induced Src signaling.

  9. 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.

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

    PubMed Central

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

    2014-01-01

    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. PMID:25056908

  11. Mechanisms for enzymatic cleavage of the N-glycosidic bond in DNA.

    PubMed

    Drohat, Alexander C; Maiti, Atanu

    2014-11-14

    DNA glycosylases remove damaged or enzymatically modified nucleobases from DNA, thereby initiating the base excision repair (BER) pathway, which is found in all forms of life. These ubiquitous enzymes promote genomic integrity by initiating repair of mutagenic and/or cytotoxic lesions that arise continuously due to alkylation, deamination, or oxidation of the normal bases in DNA. Glycosylases also perform essential roles in epigenetic regulation of gene expression, by targeting enzymatically-modified forms of the canonical DNA bases. Monofunctional DNA glycosylases hydrolyze the N-glycosidic bond to liberate the target base, while bifunctional glycosylases mediate glycosyl transfer using an amine group of the enzyme, generating a Schiff base intermediate that facilitates their second activity, cleavage of the DNA backbone. Here we review recent advances in understanding the chemical mechanism of monofunctional DNA glycosylases, with an emphasis on how the reactions are influenced by the properties of the nucleobase leaving-group, the moiety that varies across the vast range of substrates targeted by these enzymes. PMID:25181003

  12. 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

  13. Reciprocal Phosphorylation and Palmitoylation Control Dopamine Transporter Kinetics.

    PubMed

    Moritz, Amy E; Rastedt, Danielle E; Stanislowski, Daniel J; Shetty, Madhur; Smith, Margaret A; Vaughan, Roxanne A; Foster, James D

    2015-11-27

    The dopamine transporter is a neuronal protein that drives the presynaptic reuptake of dopamine (DA) and is the major determinant of transmitter availability in the brain. Dopamine transporter function is regulated by protein kinase C (PKC) and other signaling pathways through mechanisms that are complex and poorly understood. Here we investigate the role of Ser-7 phosphorylation and Cys-580 palmitoylation in mediating steady-state transport kinetics and PKC-stimulated transport down-regulation. Using both mutational and pharmacological approaches, we demonstrate that these post-translational modifications are reciprocally regulated, leading to transporter populations that display high phosphorylation-low palmitoylation or low phosphorylation-high palmitoylation. The balance between the modifications dictates transport capacity, as conditions that promote high phosphorylation or low palmitoylation reduce transport Vmax and enhance PKC-stimulated down-regulation, whereas conditions that promote low phosphorylation or high palmitoylation increase transport Vmax and suppress PKC-stimulated down-regulation. Transitions between these functional states occur when endocytosis is blocked or undetectable, indicating that the modifications kinetically regulate the velocity of surface transporters. These findings reveal a novel mechanism for control of DA reuptake that may represent a point of dysregulation in DA imbalance disorders. PMID:26424792

  14. Tyrosine Phosphorylation in Toll-Like Receptor Signaling

    PubMed Central

    Chattopadhyay, Saurabh; Sen, Ganes C.

    2014-01-01

    There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge on the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways. PMID:25022196

  15. Phosphorylation of Ubc9 by Cdk1 enhances SUMOylation activity.

    PubMed

    Su, Yee-Fun; Yang, Tsunghan; Huang, Hoting; Liu, Leroy F; Hwang, Jaulang

    2012-01-01

    Increasing evidence has pointed to an important role of SUMOylation in cell cycle regulation, especially for M phase. In the current studies, we have obtained evidence through in vitro studies that the master M phase regulator CDK1/cyclin B kinase phosphorylates the SUMOylation machinery component Ubc9, leading to its enhanced SUMOylation activity. First, we show that CDK1/cyclin B, but not many other cell cycle kinases such as CDK2/cyclin E, ERK1, ERK2, PKA and JNK2/SAPK1, specifically enhances SUMOylation activity. Second, CDK1/cyclin B phosphorylates the SUMOylation machinery component Ubc9, but not SAE1/SAE2 or SUMO1. Third, CDK1/cyclin B-phosphorylated Ubc9 exhibits increased SUMOylation activity and elevated accumulation of the Ubc9-SUMO1 thioester conjugate. Fourth, CDK1/cyclin B enhances SUMOylation activity through phosphorylation of Ubc9 at serine 71. These studies demonstrate for the first time that the cell cycle-specific kinase CDK1/cyclin B phosphorylates a SUMOylation machinery component to increase its overall SUMOylation activity, suggesting that SUMOylation is part of the cell cycle program orchestrated by CDK1 through Ubc9. PMID:22509284

  16. Phosphorylation of NBR1 by GSK3 modulates protein aggregation

    PubMed Central

    Nicot, Anne-Sophie; Lo Verso, Francesca; Ratti, Francesca; Pilot-Storck, Fanny; Streichenberger, Nathalie; Sandri, Marco; Schaeffer, Laurent; Goillot, Evelyne

    2014-01-01

    The autophagy receptor NBR1 (neighbor of BRCA1 gene 1) binds UB/ubiquitin and the autophagosome-conjugated MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) proteins, thereby ensuring ubiquitinated protein degradation. Numerous neurodegenerative and neuromuscular diseases are associated with inappropriate aggregation of ubiquitinated proteins and GSK3 (glycogen synthase kinase 3) activity is involved in several of these proteinopathies. Here we show that NBR1 is a substrate of GSK3. NBR1 phosphorylation by GSK3 at Thr586 prevents the aggregation of ubiquitinated proteins and their selective autophagic degradation. Indeed, NBR1 phosphorylation decreases protein aggregation induced by puromycin or by the DES/desmin N342D mutant found in desminopathy patients and stabilizes ubiquitinated proteins. Importantly, decrease of protein aggregates is due to an inhibition of their formation and not to their autophagic degradation as confirmed by data on Atg7 knockout mice. The relevance of NBR1 phosphorylation in human pathology was investigated. Analysis of muscle biopsies of sporadic inclusion body myositis (sIBM) patients revealed a strong decrease of NBR1 phosphorylation in muscles of sIBM patients that directly correlated with the severity of protein aggregation. We propose that phosphorylation of NBR1 by GSK3 modulates the formation of protein aggregates and that this regulation mechanism is defective in a human muscle proteinopathy. PMID:24879152

  17. The effects of connexin phosphorylation on gap junctional communication

    PubMed Central

    Lampe, Paul D.; Lau, Alan F.

    2010-01-01

    Gap junctions are specialized membrane domains composed of collections of channels that directly connect neighboring cells providing for the cell-to-cell diffusion of small molecules, including ions, amino acids, nucleotides, and second messengers. Vertebrate gap junctions are composed of proteins encoded by the “connexin” gene family. In most cases examined, connexins are modified post-translationally by phosphorylation. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the connexin “lifecycle”, such as the trafficking, assembly/disassembly, degradation, as well as, the gating of gap junction channels. Since connexin43 (Cx43) is widely expressed in tissues and cell lines, we understand the most about how it is regulated, and thus, connexin43 phosphorylation is a major focus of this review. Recent reports utilizing new methodologies combined with the latest genome information have shown that activation of several kinases including protein kinase A, protein kinase C, p34cdc2/cyclin B kinase, casein kinase 1, mitogen-activated protein (MAP) kinase and pp60src kinase can lead to phosphorylation at 12 of the 21 serine and two of the six tyrosine residues in the C-terminal region of connexin43. In several cases, use of site-directed mutants of these sites have shown that these specific phosphorylation events can be linked to changes in gap junctional communication. PMID:15109565

  18. Graphene-based immunosensor for electrochemical quantification of phosphorylated p53 (S15)

    SciTech Connect

    Xie, Yunying; Chen, Aiqiong; Du, Dan; Lin, Yuehe

    2011-08-01

    We reported a graphene-based immunosensor for electrochemical quantification of phosphorylated p53 on serine 15 (phospho-p53/15), a potential biomarker of gamma-radiation exposure. The principle is based on sandwich immunoassay and the resulting immunocomplex is formed among phospho-p53 capture antibody, phospho-p53/15 antigen, biotinylated phospho-p5315 detection antibody and horseradish peroxidase (HRP)-labeled streptavidin. The introduced HRP results in an electrocatalytic response to reduction of hydrogen peroxide in the presence of thionine. Graphene served as sensor platform not only promotes electron transfer, but also increases the surface area to introduce a large amount of capture antibody, thus increasing the detection sensitivity. The experimental conditions including blocking agent, immunoreaction time and substrate concentration have been optimized. Under the optimum conditions, the increase of response current is proportional to the phospho-p53/15 concentration in the range of 0.2–10 ng mL?1, with the detection limit of 0.1 ng mL?1. The developed immunosensor exhibits acceptable stability and reproducibility and the assay results for phospho-p53/15 are in good correlation with the known values. This easily fabricated immunosensor provides a new promising tool for analysis of phospho-p53/15 and other phosphorylated proteins.

  19. Interhelical interaction and receptor phosphorylation regulate the activation kinetics of different human ?1-adrenoceptor variants.

    PubMed

    Ahles, Andrea; Rodewald, Fabian; Rochais, Francesca; Bünemann, Moritz; Engelhardt, Stefan

    2015-01-16

    G protein-coupled receptors represent the largest class of drug targets, but genetic variation within G protein-coupled receptors leads to variable drug responses and, thereby, compromises their therapeutic application. One of the most intensely studied examples is a hyperfunctional variant of the human ?1-adrenoceptor that carries an arginine at position 389 in helix 8 (Arg-389-ADRB1). However, the mechanism underlying the higher efficacy of the Arg-389 variant remained unclear to date. Despite its hyperfunctionality, we found the Arg-389 variant of ADRB1 to be hyperphosphorylated upon continuous stimulation with norepinephrine compared with the Gly-389 variant. Using ADRB1 sensors to monitor activation kinetics by fluorescence resonance energy transfer, Arg-389-ADRB1 exerted faster activation speed and arrestin recruitment than the Gly-389 variant. Both activation speed and arrestin recruitment depended on phosphorylation of the receptor, as shown by knockdown of G protein-coupled receptor kinases and phosphorylation-deficient ADRB1 mutants. Structural modeling of the human ?1-adrenoceptor suggested interaction of the side chain of Arg-389 with opposing amino acid residues in helix 1. Site-directed mutagenesis of Lys-85 and Thr-86 in helix 1 revealed that this interaction indeed determined ADRB1 activation kinetics. Taken together, these findings indicate that differences in interhelical interaction regulate the different activation speed and efficacy of ADRB1 variants. PMID:25451930

  20. 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

  1. Highly efficient production of phosphorylated hepatitis B core particles in yeast Pichia pastoris.

    PubMed

    Freivalds, Janis; Dislers, Andris; Ose, Velta; Pumpens, Paul; Tars, Kaspars; Kazaks, Andris

    2011-02-01

    Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanomaterials). Despite numerous reports exploiting different expression systems, a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking. Here, we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris. The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P. pastoris genome via homologous recombination. A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein. Fermentation conditions were established ensuring biomass accumulation of 163g/L. A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs, with a yield of about 3.0mg per 1g of wet cells. Purification is performed within a day and may be easily scaled up if necessary. The quality of HBc VLPs was verified by electron microscopy. Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites. To our knowledge, this is the first report of HBc phosphorylation in yeast. PMID:20854910

  2. Office of Technology Transfer Material Transfer Agreements

    E-print Network

    Tullos, Desiree

    Office of Technology Transfer · Material Transfer Agreements · Confidentiality Agreements · Copyright / Patent Licensing The Office of Technology Transfer facilitates the transfer of innovations out of the university for public benefit TOOLS #12;Office of Technology Transfer Facilitating transfer of innovations

  3. Increased iPLA2 activity and levels of phosphorylated GSK3B in platelets are associated with donepezil treatment in Alzheimer's disease patients.

    PubMed

    Talib, L L; Hototian, S R; Joaquim, H P G; Forlenza, O V; Gattaz, W F

    2015-12-01

    Reduced phospholipase A2 (PLA2) activity and increased phosphorylation of glycogen synthase kinase 3B (GSK3B) participate in the production of beta-amyloid plaques and of neurofibrillary tangles, which are two neuropathological hallmarks of Alzheimer's disease (AD). Experimental evidences suggest a neuroprotective effect of the cholinesterase inhibitor donepezil in the treatment the disease. The aims of the present study were to evaluate in AD patients the effects of treatment with donepezil on PLA2 activity and GSK3B level. Thirty patients with AD were treated during 6 months with 10 mg daily of donepezil. Radio-enzymatic assays were used to measure PLA2 activity and Elisa assays for GSK3B level, both in platelets. Before treatment and after 3 and 6 months on donepezil, AD patients underwent a cognitive assessment and platelet samples were collected. Values were compared to a healthy control group of 42 sex- and age-matched elderly individuals. Before treatment, iPLA2 activity was lower in patients with AD as compared to controls (p < 0.001). At baseline, no differences were found in GSK3B level between both groups. After 3 and 6 months of treatment, we found a significant increase in iPLA2 activity (p = 0.015 and p < 0.001, respectively). iPLA2 increment was related to the cognitive improvement during treatment (p = 0.037). After 6 months, we found an increase in phosphorylated GSK3B (p = 0.02). The present findings suggest two possible mechanisms by which donepezil delays the progression of AD. The increment of iPLA2 activity may reduce the production of beta-amyloid plaques, whereas the phosphorylation of GSK3B inactivates the enzyme, reducing thus the phosphorylation of tau protein. PMID:25920742

  4. High volumetric power density, non-enzymatic, glucose fuel cells

    PubMed Central

    Oncescu, Vlad; Erickson, David

    2013-01-01

    The development of new implantable medical devices has been limited in the past by slow advances in lithium battery technology. Non-enzymatic glucose fuel cells are promising replacement candidates for lithium batteries because of good long-term stability and adequate power density. The devices developed to date however use an “oxygen depletion design” whereby the electrodes are stacked on top of each other leading to low volumetric power density and complicated fabrication protocols. Here we have developed a novel single-layer fuel cell with good performance (2??W cm?2) and stability that can be integrated directly as a coating layer on large implantable devices, or stacked to obtain a high volumetric power density (over 16??W cm?3). This represents the first demonstration of a low volume non-enzymatic fuel cell stack with high power density, greatly increasing the range of applications for non-enzymatic glucose fuel cells. PMID:23390576

  5. 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.

  6. 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

  7. Investigations in sono-enzymatic degradation of ibuprofen.

    PubMed

    Chakma, Sankar; Moholkar, Vijayanand S

    2016-03-01

    The drug ibuprofen (IBP) appears frequently in the wastewater discharge from pharmaceutical industries. This paper reports studies in degradation of IBP employing hybrid technique of sono-enzymatic treatment. This paper also establishes synergy between individual mechanisms of enzyme and sonolysis for IBP degradation by identification of degradation intermediates, and Arrhenius & thermodynamic analysis of the experimental data. Positive synergy between sonolysis and enzyme treatment is attributed to formation of hydrophilic intermediates during degradation. These intermediates form due to hydroxylation and oxidation reactions induced by radicals formed during transient cavitation. Activation energy and enthalpy change in sono-enzymatic treatment are lower as compared to enzyme treatment, while frequency factor and entropy change are higher as compared to sonolysis. Degradation of IBP in sono-enzymatic treatment is revealed to be comparable with other hybrid techniques like photo-Fenton, sono-photocatalysis, and sono-Fenton. PMID:26552749

  8. High volumetric power density, non-enzymatic, glucose fuel cells.

    PubMed

    Oncescu, Vlad; Erickson, David

    2013-01-01

    The development of new implantable medical devices has been limited in the past by slow advances in lithium battery technology. Non-enzymatic glucose fuel cells are promising replacement candidates for lithium batteries because of good long-term stability and adequate power density. The devices developed to date however use an "oxygen depletion design" whereby the electrodes are stacked on top of each other leading to low volumetric power density and complicated fabrication protocols. Here we have developed a novel single-layer fuel cell with good performance (2??W cm?²) and stability that can be integrated directly as a coating layer on large implantable devices, or stacked to obtain a high volumetric power density (over 16??W cm?³). This represents the first demonstration of a low volume non-enzymatic fuel cell stack with high power density, greatly increasing the range of applications for non-enzymatic glucose fuel cells. PMID:23390576

  9. Engineering aspects of enzymatic signal transduction: photoreceptors in the retina.

    PubMed Central

    Detwiler, P B; Ramanathan, S; Sengupta, A; Shraiman, B I

    2000-01-01

    Identifying the basic module of enzymatic amplification as an irreversible cycle of messenger activation/deactivation by a "push-pull" pair of opposing enzymes, we analyze it in terms of gain, bandwidth, noise, and power consumption. The enzymatic signal transduction cascade is viewed as an information channel, the design of which is governed by the statistical properties of the input and the noise and dynamic range constraints of the output. With the example of vertebrate phototransduction cascade we demonstrate that all of the relevant engineering parameters are controlled by enzyme concentrations and, from functional considerations, derive bounds on the required protein numbers. Conversely, the ability of enzymatic networks to change their response characteristics by varying only the abundance of different enzymes illustrates how functional diversity may be built from nearly conserved molecular components. PMID:11106590

  10. Cascade catalysis in membranes with enzyme immobilization for multi-enzymatic conversion of CO2 to methanol.

    PubMed

    Luo, Jianquan; Meyer, Anne S; Mateiu, R V; Pinelo, Manuel

    2015-05-25

    Facile co-immobilization of enzymes is highly desirable for bioconversion methods involving multi-enzymatic cascade reactions. Here we show for the first time that three enzymes can be immobilized in flat-sheet polymeric membranes simultaneously or separately by simple pressure-driven filtration (i.e. by directing membrane fouling formation), without any addition of organic solvent. Such co-immobilization and sequential immobilization systems were examined for the production of methanol from CO2 with formate dehydrogenase (FDH), formaldehyde dehydrogenase (FaldDH) and alcohol dehydrogenase (ADH). Enzyme activity was fully retained by this non-covalent immobilization strategy. The two immobilization systems had similar catalytic efficiencies because the second reaction (formic acid?formaldehyde) catalyzed by FaldDH was found to be the cascade bottleneck (a threshold substrate concentration was required). Moreover, the trade-off between the mitigation of product inhibition and low substrate concentration for the adjacent enzymes probably made the co-immobilization meaningless. Thus, sequential immobilization could be used for multi-enzymatic cascade reactions, as it allowed the operational conditions for each single step to be optimized, not only during the enzyme immobilization but also during the reaction process, and the pressure-driven mass transfer (flow-through mode) could overcome the diffusion resistance between enzymes. This study not only offers a green and facile immobilization method for multi-enzymatic cascade systems, but also reveals the reaction bottleneck and provides possible solutions for the bioconversion of CO2 to methanol. PMID:25698375

  11. CONNEXIN 43 PHOSPHORYLATION – STRUCTURAL CHANGES AND BIOLOGICAL EFFECTS

    PubMed Central

    Solan, Joell L.; Lampe, Paul D.

    2009-01-01

    SYNOPSIS Vertebrate gap junctions, composed of proteins from the connexin gene family, play critical roles in embryonic development, coordinated contraction of excitable cells, tissue homeostasis, normal cell growth and differentiation. Phosphorylation of connexin43, the most abundant and ubiquitously expressed connexin, has been implicated in the regulation of gap junctional communication at several stages of the connexin “life cycle” including hemichannel oligomerization, export of the protein to the plasma membrane, hemichannel activity, gap junction assembly, gap junction channel gating and connexin degradation. Consistent with a short (1?5 h) protein half-life, connexin43 phosphorylation is dynamic and changes in response to activation of many different kinases. This review assesses our current understanding of the effects of phosphorylation on connexin43 structure and function that in turn regulate gap junction biology with an emphasis on events occurring in heart and skin. PMID:19309313

  12. Phosphorylation of Izumo1 and its role in male infertility

    PubMed Central

    Young, Samantha AM; Aitken, John; Baker, Mark A

    2015-01-01

    Izumo1 is a testis-specific gene product, whose function is essential for sperm-egg fusion. Throughout its lifespan, Izumo1 is posttranslationally modified, being both N-linked glycosylated on its extracellular domain and phosphorylated on the intracellular C-terminal tail. Within the caput regions of the rat epididymis, two phosphorylation events have been documented. However, as sperm pass through the epididymis, this cytoplasmic portion of Izumo1 has been shown to contain up to seven phosphorylation sites. Remarkably, in the rat, in correlation with these events, Izumo1 undergoes sub-cellular re-location, moving from the head/tail regions of the spermatozoa, to a predominantly equatorial segment location once they have reached the caudal end of the epididymis. PMID:25994654

  13. Phosphorylation and the Cajal body: Modification in search of function

    PubMed Central

    Hebert, Michael D.

    2010-01-01

    The Cajal body (CB) is a subnuclear domain that contains proteins and factors involved in a diverse range of activities including ribonucleoprotein maturation, histone gene transcription and telomerase assembly. Among these activities, the CBs’ role in small nuclear ribonucleoprotein (snRNP) biogenesis is best characterized. Although CBs are found in plants, flies and mammals, not all cell types contain CBs. Rather, CBs are most prominent in transcriptionally active cells, such as cancer and neuronal cells. Many CB components, including the CB marker protein coilin, are phosphorylated in humans. The functional consequence of phosphorylation on CB assembly, activity and disassembly is largely unknown. Also unknown are the signaling pathways, kinases and phosphatases that act upon proteins which localize in the CB. The goal of this review is to demonstrate the need for a concerted effort towards elucidating the functional consequence of phosphorylation on CB formation and activity. PMID:20193656

  14. 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

  15. Phosphorylation of unique domains of Src family kinases

    PubMed Central

    Amata, Irene; Maffei, Mariano; Pons, Miquel

    2014-01-01

    Members of the Src family of kinases (SFKs) are non-receptor tyrosine kinases involved in numerous signal transduction pathways. The catalytic, SH3 and SH2 domains are attached to the membrane-anchoring SH4 domain through the intrinsically disordered “Unique” domains, which exhibit strong sequence divergence among SFK members. In the last decade, structural and biochemical studies have begun to uncover the crucial role of the Unique domain in the regulation of SFK activity. This mini-review discusses what is known about the phosphorylation events taking place on the SFK Unique domains, and their biological relevance. The modulation by phosphorylation of biologically relevant inter- and intra- molecular interactions of Src, as well as the existence of complex phosphorylation/dephosphorylation patterns observed for the Unique domain of Src, reinforces the important functional role of the Unique domain in the regulation mechanisms of the Src kinases and, in a wider context, of intrinsically disordered regions in cellular processes. PMID:25071818

  16. 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.

  17. Partial high-resolution structure of phosphorylated and non-phosphorylated leucine-rich amelogenin protein adsorbed to hydroxyapatite

    SciTech Connect

    Masica, David L.; Gray, Jeffrey J.; Shaw, Wendy J.

    2011-07-21

    The formation of biogenic materials requires the interaction of organic molecules with the mineral phase. In forming enamel, the amelogenin proteins contribute to the mineralization of hydroxyapatite (HAp). Leucine-rich amelogenin protein (LRAP) is a naturally occurring splice variant of amelogenin that comprises amelogenin’s predicted HAp binding domains. We determined the partial structure of phosphorylated and non-phosphorylated LRAP variants bound to HAp using combined solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. The ssNMR measurements indicate a largely extended structure for both variants, though some measurements are consistent with a partially helical N-terminal segment. Structure prediction was biased using 21 ssNMR measurements at five HAp crystal faces. The predicted fold of LRAP is similar at all HAp faces studied, regardless of phosphorylation. LRAP’s predicted structure is relatively extended with a helix-turn-helix motif in the N-terminal domain and some helix in the C-terminal domain. The N-terminal domain of the phosphorylated variant binds HAp more tightly than the N-terminal domain of the non-phosphorylated variant. Both variants are predicted to preferentially bind the {010} HAp crystal face providing further evidence that amelogenins block crystal growth on the a and b faces to allow elongated crystals in the c-axis. Pacific Northwest National Laboratory is operated by Battelle for the US Department of Energy.

  18. 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

  19. Zidovudine Inhibits Thymidine Phosphorylation in the Isolated Perfused Rat Heart?

    PubMed Central

    Susan-Resiga, Delia; Bentley, Alice T.; Lynx, Matthew D.; LaClair, Darcy D.; McKee, Edward E.

    2007-01-01

    Zidovudine (AZT; 3?-azido-3?-deoxythymidine), a thymidine analog, has been a staple of highly active antiretroviral therapy. It is phosphorylated in the host to the triphosphate and functions by inhibiting the viral reverse transcriptase. However, long-term use of AZT is linked to various tissue toxicities, including cardiomyopathy. These toxicities are associated with mitochondrial DNA depletion, which is hypothesized to be caused by AZT triphosphate inhibition of mitochondrial DNA polymerase ?. In previous work with isolated heart mitochondria, we demonstrated that AZT phosphorylation beyond the monophosphate was not detected and that AZT itself was a potent inhibitor of thymidine phosphorylation. This suggests an alternative hypothesis in which depletion of the TTP pool may limit mitochondrial DNA replication. The present work extends these studies to the whole cell by investigating the metabolism of thymidine and AZT in the intact isolated perfused rat heart. [3H]thymidine is converted to [3H]TTP in a time- and concentration-dependent manner. The level of [3H]TMP is low, suggesting that the reaction catalyzed by thymidine kinase is the rate-limiting step in phosphorylation. [3H]AZT is converted in a time- and concentration-dependent manner to AZT monophosphate, the only phosphorylated product detected after 3 h of perfusion. Both compounds display negative cooperativity, similar to the observations with cloned and purified mitochondrial thymidine kinase 2. The presence of AZT in the perfusate inhibits the phosphorylation of [3H]thymidine with a 50% inhibitory concentration of 24 ± 4 ?M. These data support the hypothesis that AZT-induced mitochondrial cardiotoxicity may be caused by a limiting pool of TTP that lowers mitochondrial DNA replication. PMID:17220403

  20. Phosphorylation and regulation of glutamate receptors by CaMKII

    PubMed Central

    MAO, Li-Min; JIN, Dao-Zhong; XUE, Bing; CHU, Xiang-Ping; WANG, John Q

    2015-01-01

    Ca2+/calmodulin-dependent protein kinase II (CaMKII) is the most abundant kinase within excitatory synapses in the mammalian brain. It interacts with and phosphorylates a large number of synaptic proteins, including major ionotropic glutamate receptors (iGluRs) and group I metabotropic glutamate receptors (mGluRs), to constitutively and/or activity-dependently regulate trafficking, subsynaptic localization, and function of the receptors. Among iGluRs, the N-methyl-D-aspartate receptor (NMDAR) is a direct target of CaMKII. By directly binding to an intracellular C-terminal (CT) region of NMDAR GluN2B subunits, CaMKII phosphorylates a serine residue (S1303) in the GluN2B CT. CaMKII also phosphorylates a serine site (S831) in the CT of ?-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors. This phosphorylation enhances channel conductance and is critical for synaptic plasticity. In addition to iGluRs, CaMKII binds to the proximal CT region of mGluR1a, which enables the kinase to phosphorylate threonine 871. Agonist stimulation of mGluR1a triggers a CaMKII-mediated negative feedback to facilitate endocytosis and desensitization of the receptor. CaMKII also binds to the mGluR5 CT. This binding seems to anchor and accumulate inactive CaMKII at synaptic sites. Active CaMKII dissociates from mGluR5 and may then bind to adjacent GluN2B to mediate the mGluR5-NMDAR coupling. Together, glutamate receptors serve as direct substrates of CaMKII. By phosphorylating these receptors, CaMKII plays a central role in controlling the number and activity of the modified receptors and determining the strength of excitatory synaptic transmission. PMID:24964855

  1. Technology transfer

    NASA Technical Reports Server (NTRS)

    Handley, Thomas

    1992-01-01

    The requirements for a successful technology transfer program and what such a program would look like are discussed. In particular, the issues associated with technology transfer in general, and within the Jet Propulsion Laboratory (JPL) environment specifically are addressed. The section on background sets the stage, identifies the barriers to successful technology transfer, and suggests actions to address the barriers either generally or specifically. The section on technology transfer presents a process with its supporting management plan that is required to ensure a smooth transfer process. Viewgraphs are also included.

  2. Excited singlet molecular O2 (1?g) is generated enzymatically from excited carbonyls in the dark

    PubMed Central

    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 [18O]-labeled triplet, ground state molecular oxygen [18O2 (3?g-)], chemical trapping of 18O2 (1?g) with disodium salt of anthracene-9,10-diyldiethane-2,1-diyl disulfate yielding the corresponding double-[18O]-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 18O2 (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

  3. Enzymatic biofuel cells: 30 years of critical advancements.

    PubMed

    Rasmussen, Michelle; Abdellaoui, Sofiene; Minteer, Shelley D

    2016-02-15

    Enzymatic biofuel cells are bioelectronic devices that utilize oxidoreductase enzymes to catalyze the conversion of chemical energy into electrical energy. This review details the advancements in the field of enzymatic biofuel cells over the last 30 years. These advancements include strategies for improving operational stability and electrochemical performance, as well as device fabrication for a variety of applications, including implantable biofuel cells and self-powered sensors. It also discusses the current scientific and engineering challenges in the field that will need to be addressed in the future for commercial viability of the technology. PMID:26163747

  4. 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.

  5. 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

  6. Protein microchips : use for immunoassay and enzymatic reactions.

    SciTech Connect

    Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloschuk, S.; Chupeeva, V.; Mirzabekov, A.; Biochip Technology Center; Russian Academy of Sciences

    2000-02-15

    Different proteins such as antibodies, antigens, and enzymes were immobilized within the 100 x 100 x 20-{mu}m gel pads of protein microchips. A modified polyacrylamide gel has been developed to accommodate proteins of a size up to 400,000 daltons. Electrophoresis in the microchip reaction chamber speeded up antigen-antibody interactions within the gel. Protein microchips were used in immunoassays for detection of antigens or antibodies, as well as to carry out enzymatic reactions and to measure their kinetics in the absence or presence of an inhibitor. A protein microchip can be used several times in different immunoassays and enzymatic kinetic measurements.

  7. [New enzymatic strategies for enantiomers of lactams and amino acids].

    PubMed

    Forró, Eniko

    2011-01-01

    To fulfil the requirements of modern go-ahead research, new direct and indirect enzymatic strategies and new techniques have been devised for the preparation of enantiopure beta- and gamma-lactams and beta- and gamma-amino acids, and some of them have been scaled up. For example, a formal total synthesis of enantiopure Anatoxina, a neurotoxic alkaloid, but a potent and stereospecific agonist at nicotinic acetylcholine receptors has been introduced. An efficient and very simple method has been developed for the synthesis of the antibacterial cispentacin [(1R,2S)-2-amino-1-cyclopentanecarboxylic acid] and 8 new derivatives. A highly efficient enzymatic procedure has been elaborated for the synthesis of the blockbuster drug Abacavir and Carbovir intermediate (1S,4R)-4-aminocyclopent-2-ene-1-carboxylic acid. The first enzymatic method has been devised for the synthesis of (R)-3-amino-4-(2,4,5-trifluorophenyl)butanoic acid, the intermediate for the new antidiabetic drug Sitagliptine. Direct enzymatic strategies have been reported for the synthesis of (2R,3S)-3-phenylisoserine, a key intermediate of the side-chain of the antitumor product Taxol. A new enzymatic method has been developed for the total synthesis of crispine A enantiomers with antitumor activity. As amino acids are among the main products in the above-mentioned enzymatic methods, a new gas-chromatographic method has been acquired for the enantioseparation of acyclic and carbocyclic cis- and trans-beta-amino acids via a rapid double derivatization technique (esterification followed by N-acylation). APPLICABILITY: Through the utilization of enzymes, efficient enantioselective procedures in organic media have been developed and applied for the preparation of enantiopure, biologically active (beta- and gamma-lactams and beta- and gamma-amino acids (Scheme 15). Two of our recently elaborated enzymatic methods for the synthesis of beta- and gamma-amino acids have been patented. Acros Organics and BioBlocks Inc. serve as the sales companies of more than 20 enantiopure products that we have prepared by enzymatic methods. PMID:22165415

  8. Palladium-Catalyzed Phosphorylation of Aryl Mesylates and Tosylates.

    PubMed

    Fu, Wai Chung; So, Chau Ming; Kwong, Fuk Yee

    2015-12-01

    The first general palladium catalyst for the phosphorylation of aryl mesylates and tosylates is reported. The newly developed system exhibits excellent functional group compatibility. For instance, free amino, keto, ester, and amido groups, as well as heterocycles, remain intact during the course of reaction. The mesylated derivatives of biologically active compounds such as 17?-estradiol and 6-hydroxyflavone are also shown to be applicable substrates. A one-pot phosphorylation-amination sequence is described for the facile synthesis of potential pharmacophores. PMID:26574778

  9. 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

  10. Nanostructured enzymatic biosensor based on fullerene and gold nanoparticles: Preparation, characterization and analytical applications

    E-print Network

    Tuscia, Università Degli Studi Della

    Nanostructured enzymatic biosensor based on fullerene and gold nanoparticles: Preparation 2013 Available online 25 December 2013 Keywords: Enzymatic biosensor Nanostructured materials Electron was finalized to the setup of a laccase biosensor based on a multilayer material consisting in Au

  11. Oxidative enzymatic response of white-rot fungi to single-walled carbon nanotubes

    E-print Network

    Blanchette, Robert A.

    : Saprotrophic fungi Enzymatics Carbon nanomaterials a b s t r a c t Although carbon nanomaterials such as single. This study investigates the peroxidase and laccase enzymatic response of the saprotrophic white-rot fungi

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

    SciTech Connect

    Chen, Steven C.; Kennedy, Brian K.; Lampe, Paul D.

    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.

  13. Observation of Phosphorylation Site-Specific Dissociation of Singly

    E-print Network

    Kim, Myung Soo

    Observation of Phosphorylation Site-Specific Dissociation of Singly Protonated Phosphopeptides dissociation such as in post-source decay (PSD) [6]. Dis- sociation can be further enhanced by using ion activation techniques such as collisionally activated dissociation (CAD) [3, 7], ultraviolet

  14. Phosphorylation of histone H3: a balancing act between chromosome

    E-print Network

    Corces, Victor G.

    condensation and cell-cycle progression during mitosis and meiosis. In addition, this modification is important properly during mitosis and is sufficiently malleable and modifiable to enable access to genetic H2A H2B Acetylation Phosphorylation Methylation H3 Transcription Mitosis Silencing N-terminal tail

  15. AMPK phosphorylation of raptor mediates a metabolic checkpoint

    PubMed Central

    Gwinn, Dana M.; Shackelford, David B.; Egan, Daniel F.; Mihaylova, Maria M.; Mery, Annabelle; Vasquez, Debbie S.; Turk, Benjamin E.; Shaw, Reuben J.

    2009-01-01

    Summary AMPK is a highly conserved sensor of cellular energy status that is activated under conditions of low intracellular ATP. AMPK responds to energy stress by suppressing cell growth and biosynthetic processes, in part through its inhibition of the rapamycin-sensitive mTOR (mTORC1) pathway. AMPK phosphorylation of the TSC2 tumor suppressor contributes to suppression of mTORC1, however TSC2-deficient cells remain responsive to energy stress. Using a proteomic and bioinformatics approach, we sought to identify additional substrates of AMPK that mediate its effects on growth control. We report here that AMPK directly phosphorylates the mTOR binding partner raptor on two well-conserved serine residues, and this phosphorylation induces 14-3-3 binding to raptor. The phosphorylation of raptor by AMPK is required for the inhibition of mTORC1 and cell cycle arrest induced by energy stress. These findings uncover a novel conserved effector of AMPK that mediates its role as a metabolic checkpoint coordinating cell growth with energy status. PMID:18439900

  16. Regulation of Yin Yang 1 by Tyrosine Phosphorylation.

    PubMed

    Wang, Gary Z; Goff, Stephen P

    2015-09-01

    Yin Yang 1 (YY1) is a member of the GLI-Krüppel class of DNA and RNA binding transcription factors that can either activate or repress gene expression during cell growth, differentiation, and embryogenesis. Although much is known about YY1 interacting proteins and the target promoters regulated by YY1, much less is known about YY1 regulation through post-translational modifications. In this study we show that YY1 is tyrosine-phosphorylated in multiple cell types. Using a combination of pharmacological inhibition, kinase overexpression, and kinase knock-out studies, we demonstrate that YY1 is a target of multiple Src family kinases in vitro and in vivo. Moreover, we have identified multiple sites of YY1 phosphorylation and analyzed the effect of phosphorylation on the activity of YY1-responsive retroviral and cellular promoters. Phosphorylation of tyrosine 383 interferes with DNA and RNA binding, leading to the down-regulation of YY1 activity. Finally, we provide the first evidence that YY1 is a downstream target of epidermal growth factor receptor signaling in vivo. Taken together, the identification of YY1 as a target of Src family kinases provide key insights into the inhibitory role of tyrosine kinases in modulating YY1 activity. PMID:26198631

  17. Phosphorylated Mesoporous Carbon as a Solid Acid Catalyst

    SciTech Connect

    Dai, Sheng; Mayes, Richard T; Fulvio, Pasquale F; Ma, Zhen

    2011-01-01

    Mesoporous carbon catalyst supports are attractive due to their wide chemical stability while potentially increasing masstransport through and providing a path for larger molecules to access catalytic sites. Herein we report the synthesis of a 10 phosphorylated mesoporous carbon solid-acid catalyst characterized by NH3-TPD and isopropanol dehydration.

  18. Stem rust spores elicit rapid RPG1 phosphorylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stem rust threatens cereal production worldwide. Understanding the mechanism by which durable resistance genes, such as Rpg1, function is critical. We show that the RPG1 protein is phosphorylated within 5 min by exposure to spores from avirulent but not virulent races of stem rust. Transgenic mutant...

  19. ATF-2 contains a phosphorylation-dependent transcriptional activation domain.

    PubMed Central

    Livingstone, C; Patel, G; Jones, N

    1995-01-01

    The ATF-2 transcription factor can mediate adenovirus E1A-inducible transcriptional activation. Deletion analysis has indicated that the N-terminal region of ATF-2 is essential for this response. Furthermore, the N-terminus can activate transcription in the absence of E1A when fused to a heterologous DNA binding domain. However, in the intact protein this activation domain is masked. In this report we show that residues in the N-terminus required for activation are also required for mediating E1A stimulation. In particular two threonine residues at positions 69 and 71 are essential. These residues are phosphorylated in vivo and can be efficiently phosphorylated in vitro by the JNK/SAPK subgroup of the MAPK family. ATF-2 can bind to a UV-inducible kinase through a region in the N-terminus that is distinct from the sites of phosphorylation; this binding region is both necessary for phosphorylation by JNK/SAPK in vitro and for transcriptional activation in vivo. The activity of the N-terminus is stimulated by UV irradiation which stimulates the signalling pathway leading to JNK/SAPK. Finally, although ATF-2 binds to the E1A protein, the N-terminal activation domain is not required for this interaction. The results show that ATF-2, like other members of the ATF/CREB family of DNA binding proteins is regulated by specific signalling pathways. Images PMID:7737129

  20. Phosphorylation of proteins in Dictyostelium discoideum during development

    SciTech Connect

    Coffman, D.S.

    1982-01-01

    The phosphoproteins in D. discoideum were studied with respect to their formation, metabolic stability, cellular and subcellular distribution. Special emphasis was on the role of cAMP on the pattern of phosphorylation. Amoebae were metabolically labeled with /sup 32/P/sub i/; subsequently proteins of the total lysate, nuclei and membranes were resolved by SDS-polyacrylamide gel electrophoresis and subjected to autoradiography. Numerous changes in the profile of phosphoproteins were observed during development. Functions were assigned to four membranal phosphoproteins; only one protein, the heavy chain of myosin, was susceptible to phosphorylation in vitro when purified membranes and /sup 32/P-ATP were used. A comparison between the time of protein synthesis and phosphorylation, as examined in vivo using /sup 35/S-methionine and /sup 32/P/sub i/ labeling of amoebae and two-dimensional gel electrophoresis, indicated that phosphorylation is concurrent with synthesis. It appears then that there are two classes of membranal phosphoproteins in D. discoideum which differ with respect to the stability of the phosphate moiety. It is evident that the turnover of the phosphate moiety in myosin heavy chain plays a crucial role in the function of myosin; a role for the metabolically inert phosphate of other membranal proteins remains to be established. The G protein which couples occupancy of hormone receptor to stimulation of adenylate cyclase in higher multicellular eukaryotes was detected in D. discoideum. The G protein is present in approximately equal amounts in vegetative and in developing amoebae.

  1. 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...

  2. Stress Induces Pain Transition by Potentiation of AMPA Receptor Phosphorylation

    PubMed Central

    Li, Changsheng; Yang, Ya; Liu, Sufang; Fang, Huaqiang; Zhang, Yong; Furmanski, Orion; Skinner, John; Xing, Ying; Johns, Roger A.

    2014-01-01

    Chronic postsurgical pain is a serious issue in clinical practice. After surgery, patients experience ongoing pain or become sensitive to incident, normally nonpainful stimulation. The intensity and duration of postsurgical pain vary. However, it is unclear how the transition from acute to chronic pain occurs. Here we showed that social defeat stress enhanced plantar incision-induced AMPA receptor GluA1 phosphorylation at the Ser831 site in the spinal cord and greatly prolonged plantar incision-induced pain. Interestingly, targeted mutation of the GluA1 phosphorylation site Ser831 significantly inhibited stress-induced prolongation of incisional pain. In addition, stress hormones enhanced GluA1 phosphorylation and AMPA receptor-mediated electrical activity in the spinal cord. Subthreshold stimulation induced spinal long-term potentiation in GluA1 phosphomimetic mutant mice, but not in wild-type mice. Therefore, spinal AMPA receptor phosphorylation contributes to the mechanisms underlying stress-induced pain transition. PMID:25297100

  3. Phosphorylated ubiquitin chain is the genuine Parkin receptor

    PubMed Central

    Okatsu, Kei; Koyano, Fumika; Kimura, Mayumi; Kosako, Hidetaka; Saeki, Yasushi

    2015-01-01

    PINK1 selectively recruits Parkin to depolarized mitochondria for quarantine and removal of damaged mitochondria via ubiquitylation. Dysfunction of this process predisposes development of familial recessive Parkinson’s disease. Although various models for the recruitment process have been proposed, none of them adequately explain the accumulated data, and thus the molecular basis for PINK1 recruitment of Parkin remains to be fully elucidated. In this study, we show that a linear ubiquitin chain of phosphomimetic tetra-ubiquitin(S65D) recruits Parkin to energized mitochondria in the absence of PINK1, whereas a wild-type tetra-ubiquitin chain does not. Under more physiologically relevant conditions, a lysosomal phosphorylated polyubiquitin chain recruited phosphomimetic Parkin to the lysosome. A cellular ubiquitin replacement system confirmed that ubiquitin phosphorylation is indeed essential for Parkin translocation. Furthermore, physical interactions between phosphomimetic Parkin and phosphorylated polyubiquitin chain were detected by immunoprecipitation from cells and in vitro reconstitution using recombinant proteins. We thus propose that the phosphorylated ubiquitin chain functions as the genuine Parkin receptor for recruitment to depolarized mitochondria. PMID:25847540

  4. Phosphorylation of intact erythrocytes in human muscular dystrophy

    SciTech Connect

    Johnson, R.M.; Nigro, M.

    1986-04-01

    The uptake of exogenous /sup 32/Pi into the membrane proteins of intact erythrocytes was measured in 8 patients with Duchenne muscular dystrophy. No abnormalities were noted after autoradiographic analysis. This contrasts with earlier results obtained when isolated membranes were phosphorylated with gamma-(/sup 32/P)ATP, and suggests a possible reinterpretation of those experiments.

  5. 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. PMID:26114094

  6. 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...

  7. CGGBP1 phosphorylation constitutes a telomere-protection signal

    PubMed Central

    Singh, Umashankar; Maturi, Varun; Jones, Rhiannon E; Paulsson, Ylva; Baird, Duncan M; Westermark, Bengt

    2014-01-01

    The shelterin proteins are required for telomere integrity. Shelterin dysfunction can lead to initiation of unwarranted DNA damage and repair pathways at chromosomal termini. Interestingly, many shelterin accessory proteins are involved in DNA damage signaling and repair. We demonstrate here that in normal human fibroblasts, telomeric ends are protected by phosphorylation of CGG triplet repeat-binding protein 1 (CGGBP1) at serine 164 (S164). We show that serine 164 is a major phosphorylation site on CGGBP1 with important functions. We provide evidence that one of the kinases that can phosphorylate S164 CGGBP1 is ATR. Overexpression of S164A phospho-deficient CGGBP1 exerted a dominant-negative effect, causing telomeric dysfunction, accelerated telomere shortening, enhanced fusion of telomeres, and crisis. However, overexpression of wild-type or phospho-mimicking S164E CGGBP1 did not cause these effects. This telomere damage was associated with reduced binding of the shelterin protein POT1 to telomeric DNA. Our results suggest that CGGBP1 phosphorylation at S164 is a novel telomere protection signal, which can affect telomere-protective function of the shelterin complex. PMID:24196442

  8. Computational studies of protein regulation by post-translational phosphorylation.

    PubMed

    Narayanan, Arjun; Jacobson, Matthew P

    2009-04-01

    Post-translational modifications of amino acids in proteins are important in regulating many cellular functions. Phosphorylation is the most well-studied post-translational modification, both experimentally and computationally. The introduction of a phosphate group, generally carrying a -2 charge at neutral pH, is a large electrostatic perturbation that alters the free energy landscape underlying protein structure and interactions. Much progress has been made in studying the structural basis of protein regulation by phosphorylation, and atomistic computational simulations are increasingly being used to complement and drive experiment. We focus on three areas: (1) protein kinases, where computational simulations have helped to provide a better understanding of the structural consequences of activation loop and glycine-rich loop phosphorylation; (2) peptide systems, for which molecular dynamics has enabled understanding of structural ordering induced by phosphorylation, and (3) phosphoregulation of membrane proteins. As the use of computation to elucidate principles of phosphoregulation is in its infancy, we also discuss areas for future progress. PMID:19339172

  9. Protein phosphorylation in Bradyrhizobium japonicum bacteroids and cultures.

    PubMed Central

    Karr, D B; Emerich, D W

    1989-01-01

    Protein phosphorylation was demonstrated in Bradyrhizobium japonicum bacteroids in vivo and in cultures in vivo and in vitro. Comparison of in vivo-labeled phosphoproteins of bacteroids and of cultured cells showed differences in both the pattern and intensity of labeling. In cultured cells, comparison of the labeling patterns and intensities of in vivo- and in vitro-labeled phosphoproteins showed a number of similarities; however, several phosphoproteins were found only after one of the two labeling conditions. The labeling intensity was time dependent in both in vivo and in vitro assays and was dependent on the presence of magnesium in in vitro assays. Differences in the rates of phosphorylation and dephosphorylation were noted for a number of proteins. The level of incorporation of 32P into protein was only 2% or less of the total phosphate accumulated during the in vivo labeling period. Several isolation and sample preparation procedures resulted in differences in labeling patterns. Phosphatase inhibitors and several potential metabolic effectors had negligible effects on the phosphorylation pattern. There were no significant changes in the phosphorylation patterns of cells cultured on mannitol, acetate, and succinate, although the intensity of the labeling did vary with the carbon source. Images PMID:2498290

  10. Ventricular Myosin Modifies In Vitro Step-Size When Phosphorylated

    PubMed Central

    Wang, Yihua; Ajtai, Katalin; Burghardt, Thomas P.

    2014-01-01

    Cardiac and skeletal muscle myosins have the central role in contraction transducing ATP free energy into the mechanical work of moving actin. Myosin has a motor domain containing ATP and actin binding sites and a lever-arm that undergoes rotation impelling bound actin. The lever-arm converts torque generated in the motor into the linear displacement known as step-size. The myosin lever-arm is stabilized by bound essential and regulatory light chains (ELC and RLC). RLC phosphorylation at S15 is linked to modified lever-arm mechanical characteristics contributing to myosin filament based contraction regulation and to the response of the muscle to disease. Myosin step-size was measured using a novel quantum dot (Qdot) assay that previously confirmed a 5 nm step-size for fast skeletal myosin and multiple unitary steps, most frequently 5 and 8 nm, and a rare 3 nm displacement for ? cardiac myosin (?Mys). S15 phosphorylation in ?Mys is now shown to change step-size distribution by advancing the 8 nm step frequency. After phosphorylation, the 8 nm step is the dominant myosin step-size resulting in significant gain in the average step-size. An increase in myosin step-size will increase the amount of work produced per ATPase cycle. The results indicate that RLC phosphorylation modulates work production per ATPase cycle suggesting the mechanism for contraction regulation by the myosin filament. PMID:24726887

  11. Enzymatic activity of rodents acclimated to cold and long scotophase

    NASA Astrophysics Data System (ADS)

    Fourie, F. Le R.; Haim, A.

    1980-09-01

    Rodents representative of a diurnal species ( Rhabdomys pumilio) as well as a nocturnal species ( Praomys natalensis) were acclimated to cold (Ta = 8°C) at a photoperiod of LD 12:12 and a long scotophase (LD 8; 16) at a temperature of 25° C(Ta). Control groups were kept for both species at Ta = 25° C and LD 12:12 and winter acclimated individuals were obtained during July and August to serve as further reference. Blood samples obtained from the tail were analysed for enzymes representative of three major biochemical pathways. The enzymatic activity of LDH (glycolytic pathway), MDH (Krebs cycle) and G6PDH (hexose monophosphate shunt, as an indicator of gonadal activity) were monitored to represent metabolic activity of the respective cycles. Cold acclimated as well as winter acclimatized mice revealed similar enzymatic patterns for both species and significant increases in LDH and MDH were recorded with a concurrent decrease in G6PDH activity. Specimens exposed to long scotophase exhibited similar enzymatic patterns for both species studied, but enzymatic activity was higher than those of cold acclimated individuals. From these results it is concluded that cold as well as long scotophase induce metabolic adaptations through biochemical activity in the experimental animals. The effect of long scotophase is assumed to be an important factor in the induction of winter acclimatization.

  12. 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

  13. UNCORRECTED Novel chemo-enzymatic oligomers of cinnamic acids

    E-print Network

    Desai, Umesh R

    UNCORRECTED PROOF Novel chemo-enzymatic oligomers of cinnamic acids as direct and indirect to study a carbox- ylic acid-based scaffold, which displayed major acceleration in the inhibition oligomers of 4-hydroxycinnamic acids, DHPs, which display inter- esting anticoagulant properties. Oligomers

  14. Enzymatic corn wet milling: engineering process and cost model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymatic Corn Wet Milling (E-Milling) is a proposed alternative process to conventional wet milling for the recovery and purification of starch and coproducts using proteases to eliminate the need for sulfites and to decrease the steeping time. In 2005, the total starch production in USA by conven...

  15. 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...

  16. 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 ...

  17. Ultrasound Enhancement of Enzymatic Hydrolysis of Cellulose Plant Matter

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The work reported here is based on acceleration of enzymatic hydrolysis of plant biomass substrate by introduction of low intensity, uniform ultrasound field into a reaction chamber (bio-reactor). This method may serve as improvement of rates in the hydrolysis of cellulosic materials to sugars, whi...

  18. 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...

  19. Enzymatic monitoring of lignin and lignin derivatives biooxidation.

    PubMed

    Ibrahim, Victor; Mamo, Gashaw

    2016-01-01

    Lignin oxidation was enzymatically monitored by measuring methanol released during the reaction. The methanol was oxidized to formaldehyde and hydrogen peroxide, and the latter used to oxidize ABTS to a product measured spectrophotometrically. The efficiency was comparable to the commonly used gas chromatography method. The assay was fast and inexpensive. PMID:26632344

  20. Enzymatic cell disruption of microalgae biomass in biorefinery processes.

    PubMed

    Demuez, Marie; Mahdy, Ahmed; Tomás-Pejó, Elia; González-Fernández, Cristina; Ballesteros, Mercedes

    2015-10-01

    When employing biotechnological processes for the procurement of biofuels and bio-products from microalgae, one of the most critical steps affecting economy and yields is the "cell disruption" stage. Currently, enzymatic cell disruption has delivered effective and cost competitive results when compared to mechanical and chemical cell disruption methods. However, the introduction of enzymes implies additional associated cost within the overall process. In order to reduce this cost, autolysis of microalgae is proposed as alternative enzymatic cell disruption method. This review aims to provide the state of the art of enzymatic cell disruption treatments employed in biorefinery processes and highlights the use of endopeptidases. During the enzymatic processes of microalgae life cycle, some lytic enzymes involved in cell division and programmed cell death have been proven useful in performing cell lysis. In this context, the role of endopeptidases is emphasized. Mirroring these natural events, an alternative cell disruption approach is proposed and described with the potential to induce the autolysis process using intrinsic cell enzymes. Integrating induced autolysis within biofuel production processes offers a promising approach to reduce overall global costs and energetic input associated with those of current cell disruption methods. A number of options for further inquiry are also discussed. PMID:25976593

  1. Coupled chemo(enzymatic) reactions in continuous flow

    PubMed Central

    Yuryev, Ruslan; Strompen, Simon

    2011-01-01

    Summary This review highlights the state of the art in the field of coupled chemo(enzymatic) reactions in continuous flow. Three different approaches to such reaction systems are presented herein and discussed in view of their advantages and disadvantages as well as trends for their future development. PMID:22238518

  2. MICROCHIP ENZYMATIC ASSAY OF ORGANOPHOSPHATE NERVE AGENTS. (R830900)

    EPA Science Inventory

    An on-chip enzymatic assay for screening organophosphate (OP) nerve agents, based on a pre-column reaction of organophosphorus hydrolase (OPH), electrophoretic separation of the phosphonic acid products, and their contactless-conductivity detection, is described. Factors affec...

  3. Enzymatic hydrolysis of organophosphate insecticides, a possible pesticide disposal method.

    PubMed Central

    Munnecke, D M

    1976-01-01

    A crude cell extract from a mixed bacterial culture growing on parathion, an organophosphate insecticide, hydrolyzed parathion (21 C) at a rate of 416 nmol/min per mg of protein. This rate of enzymatic hydrolysis, when compared with chemical hydrolysis by 0.1 N sodium hydroxide at 40 C, was 2, 450 times faster. Eight of 12 commonly used organophosphate insecticides were enzymatically hydrolyzed with this enzyme preparation at rates ranging from 12 to 1,360 nmol/min per mg of protein. Seven pesticides were hydrolyzed at rates significantly higher (40 to 1,005 times faster) than chemical hydrolysis. The pH optimum for enzymatic hydrolysis of the eight pesticides ranged from 8.5 to 9.5, with less than 50% of maximal activity expressed at pH 7.0. Maximal enzyme activity occurred at 35 C. The crude extract lost its activity at the rate of only 0.75%/day when stored at 6 C. Eight organic solvents, ranging from methanol to hexane, at low concentrations stimulated enzymatic hydrolysis by 3 to 20%, whereas at higher concentrations (1,000 mg/liter) they inhibited the reaction (9 to 50%). Parathion metabolites p-nitrophenol, hydroquinone, and diethylthiophosphoric acid, at up to 100-mg/liter concentrations, did not significantly influence enzyme activity. PMID:9901

  4. 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...

  5. ACID AND ENZYMATIC HYDROLYSIS OF SALINE BIOMASS FOR SUGAR PRODUCTION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Saline crops were evaluated for their potential to be used as feedstock for fermentable sugar production via dilute acid pretreatment and enzymatic hydrolysis. The saline crops included two woods, Athel (Tamarix aphylla L) and Eucalyptus (Eucalyptus camaldulensis), and two grasses, Jose Tall Wheatgr...

  6. Synthesis of poly(aminoamides)via enzymatic means

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Poly(aminoamides) constitute a subclass of polyamides that are water-soluble and useful for several applications. Commercially they are made via chemical reaction pathways. A review is made in this work of the enzymatic approaches towards their syntheses. Lipases and esterases have been found to ...

  7. Enzymatic hydrolysis of steryl ferulates and steryl glycosides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Steryl ferulates and steryl glycosides are phytosterol conjugates found characteristically in cereals. Their properties in enzymatic hydrolysis are, however, not yet well known. Steryl ferulates and steryl glycosides were extracted and purified from rye and wheat bran. Their rates of hydrolysis with...

  8. The Preparation and Enzymatic Hydrolysis of a Library of Esters

    ERIC Educational Resources Information Center

    Sanford, Elizabeth M.; Smith, Traci L.

    2008-01-01

    An investigative case study involving the preparation of a library of esters using Fischer esterification and alcoholysis of acid chlorides and their subsequent enzymatic hydrolysis by pig liver esterase and orange peel esterase is described. Students work collaboratively to prepare and characterize the library of esters and complete and evaluate…

  9. ERK Nuclear Translocation Is Dimerization-independent but Controlled by the Rate of Phosphorylation*S

    E-print Network

    Rieger, Bernd

    embryo fibroblast ERK1-knock-out cell line expressing green fluorescent protein (GFP)-tagged ERK1 that dimerization of ERK1 is not required for nuclear entry and retention. Consistent with this finding, energy migration Fo¨rster resonance energy transfer and fluorescence correlation spectros- copy measurements

  10. 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

  11. Intensification of Enzymatic Bio-Processing of Cotton by Low Intensity Uniform Ultrasound Field

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Use of enzymatic processing in textile applications is becoming increasingly popular, primarily because of rapid introduction of a new variety of highly efficient enzymes. In general, enzymatic bio-processing generates less toxic and readily biodegradable wastewater effluents. However, enzymatic bio...

  12. Enzymatic Resolution and Separation of Secondary Alcohols Based on Fatty Esters as Acylating Agents

    ERIC Educational Resources Information Center

    Monteiro, Carlos M.; Afonso, Carlos A. M.; Lourenco, Nuno M. T.

    2010-01-01

    The enzymatic resolution of "rac"-1-phenylethanol using ethyl myristate as acylating agent and solvent and "Candida antarctica" lipase B (CAL-B) as biocatalyst was demonstrated with catalyst and medium reuse. Both enantiomers of 1-phenylethanol were isolated by sequential enzymatic reactions and product distillations. From the first enzymatic

  13. Phosphorylation activity of the response regulator of the two-component signal transduction system AtoS-AtoC in E. coli.

    PubMed

    Lioliou, Efthimia E; Mimitou, Eleni P; Grigoroudis, Asterios I; Panagiotidis, Cynthia H; Panagiotidis, Christos A; Kyriakidis, Dimitrios A

    2005-10-10

    Antizyme, long known to be a non-competitive inhibitor of ornithine decarboxylase, is encoded by the atoC gene in Escherichia coli. The present study reveals another role for AtoC, that of a response regulator of the AtoS-AtoC two component system regulating the expression of the atoDAEB operon upon acetoacetate induction. This operon encodes enzymes involved in short-chain fatty acid catabolism in E. coli. Evidence is presented to show that AtoS is a sensor kinase that together with AtoC constitutes a two-component signal transduction system. AtoS is a membrane protein which can autophosphorylate and then transfer that phosphoryl group to AtoC. This process can also be reproduced in vitro. AtoC contains in its amino acid sequence a conserved aspartic acid (D55), which is the putative phosphorylation site, as well as an unexpected "H box" consensus sequence (SHETRTPV), common to histidine kinases, with the histidine contained therein (H73) being a second potential target for phosphorylation. Substitution of either D55 or H73 in His10-AtoC diminished but did not abrogate AtoC phosphorylation suggesting that either both residues can be phosphorylated independently or that the phosphate group can be transferred between them. However, the D55 mutation in comparison to H73 had a more pronounced effect in vivo, on the activation of atoDAEB promoter after acetoacetate induction, although it was the presence of both mutations that rendered AtoC totally unresponsive to induction. These data provide evidence that the gene products of atoS and atoC constitute a two-component signal transduction system, with some unusual properties, involved in the regulation of the atoDAEB operon. PMID:16153782

  14. Phosphorylation induces subtle structural changes in SpoIIAA, a key regulator of sporulation.

    PubMed Central

    Clarkson, Joanna; Campbell, Iain D; Yudkin, Michael D

    2003-01-01

    The phosphorylation state of SpoIIAA is a key factor in the regulation of sporulation in Bacillus subtilis. Previous crystallographic studies had led to the conclusion that phosphorylation alters the binding affinity of SpoIIAA for its partner proteins solely through the additional charge and bulk of the phosphoryl group: small structural changes observed elsewhere in the protein were considered to be random fluctuations rather than the result of phosphorylation. The results presented in the present paper show that NMR studies detect the same subtle structural changes in solution as those seen in the crystal, strongly implying that they are the direct result of phosphorylation. These subtle structural changes are similar to those that occur in a non-phosphorylated mutant that is defective in binding to one of its partner proteins. We propose that the structural changes which occur in SpoIIAA on phosphorylation act in concert with the phosphoryl group to alter its binding properties. PMID:12585962

  15. 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

  16. STUDIES OF HUNTINGTON'S DISEASE ASSOCIATED MOTOR DOMAIN PHOSPHORYLATION OF KINESIN-1

    E-print Network

    Lee, Tonghun

    STUDIES OF HUNTINGTON'S DISEASE ASSOCIATED MOTOR DOMAIN PHOSPHORYLATION OF KINESIN-1 BY HANNAH transport associated with Huntington's disease. Phosphorylation adds both negative charge and bulk. Disruptions in transport are associated with a variety of neurodegenerative diseases. Recent studies implicate

  17. Age-Dependent Changes in Protein Phosphorylation: Differential Studies in Rat Tissue

    E-print Network

    Gerstenecker, Gary Steven

    2009-12-16

    Protein phosphorylation is a reversible process that is involved in cellular growth, division and signaling. The mechanisms for protein phosphorylation can be significantly altered during the aging process. These alterations ...

  18. 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 ...

  19. Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding.

    PubMed

    Ting, Pamela Y; Johnson, Christian W; Fang, Cong; Cao, Xiaoqing; Graeber, Thomas G; Mattos, Carla; Colicelli, John

    2015-09-01

    RAS proteins are signal transduction gatekeepers that mediate cell growth, survival, and differentiation through interactions with multiple effector proteins. The RAS effector RAS- and RAB-interacting protein 1 (RIN1) activates its own downstream effectors, the small GTPase RAB5 and the tyrosine kinase Abelson tyrosine-protein kinase (ABL), to modulate endocytosis and cytoskeleton remodeling. To identify ABL substrates downstream of RAS-to-RIN1 signaling, we examined human HEK293T cells overexpressing components of this pathway. Proteomic analysis revealed several novel phosphotyrosine peptides, including Harvey rat sarcoma oncogene (HRAS)-pTyr(137). Here we report that ABL phosphorylates tyrosine 137 of H-, K-, and NRAS. Increased RIN1 levels enhanced HRAS-Tyr(137) phosphorylation by nearly 5-fold, suggesting that RAS-stimulated RIN1 can drive ABL-mediated RAS modification in a feedback circuit. Tyr(137) is well conserved among RAS orthologs and is part of a transprotein H-bond network. Crystal structures of HRAS(Y137F) and HRAS(Y137E) revealed conformation changes radiating from the mutated residue. Although consistent with Tyr(137) participation in allosteric control of HRAS function, the mutations did not alter intrinsic GTP hydrolysis rates in vitro. HRAS-Tyr(137) phosphorylation enhanced HRAS signaling capacity in cells, however, as reflected by a 4-fold increase in the association of phosphorylated HRAS(G12V) with its effector protein RAF proto-oncogene serine/threonine protein kinase 1 (RAF1). These data suggest that RAS phosphorylation at Tyr(137) allosterically alters protein conformation and effector binding, providing a mechanism for effector-initiated modulation of RAS signaling. PMID:25999467

  20. Phosphorylation of membrane proteins in erythrocytes treated with lead.

    PubMed Central

    Belloni-Olivi, L; Annadata, M; Goldstein, G W; Bressler, J P

    1996-01-01

    In immature rat microvessels, endothelial cells and glioma cells, exposure to lead results in an increase in the level of protein kinase C in membranes. In this paper we have extended these studies to human erythrocytes and, in addition, studied the phosphorylation of membrane proteins. A significant increase in the phosphorylation of membrane cytoskeletal proteins of molecular mass 120, 80, 52 and 45 kDa was observed in human erythrocytes treated for 60 min with lead acetate at concentrations greater than 100 nM. These same proteins were phosphorylated when erythrocytes were treated for 10 min with 50 nM phorbol 12-myristate 13-acetate (PMA). Similarly, protein kinase C activity was elevated and an increase in the amount of protein kinase C-alpha was observed in membranes from erythrocytes exposed to concentrations of lead acetate above 100 nM. No changes, however, in the activities of cAMP-dependent protein kinase, protein phosphatases I and IIA or casein kinase were observed. Phosphorylation of these membrane proteins stimulated by lead acetate or by PMA was not observed in erythrocytes depleted of protein kinase C by a 72-h treatment with 500 nM phorbol 12,13-dibutyrate. Finally, no changes in the levels of calcium or diacylglycerol were observed in erythrocytes stimulated with 100 nM lead acetate. These results indicate that, in erythrocytes, lead acetate stimulates the phosphorylation of membrane cytoskeletal proteins by a mechanism dependent on protein kinase C. Since levels of calcium or diacylglycerols did not increase, it appears that lead may activate the enzyme by a direct interaction. PMID:8615806

  1. Calcium-phospholipid enhanced protein phosphorylation in human placenta

    SciTech Connect

    Moore, J.J.; Moore, R.; Cardaman, R.C.

    1986-07-01

    Calcium-activated, phospholipid-dependent protein phosphorylation has not been studied in placenta. Human placental cytosol was subjected to an endogenous protein phosphorylation assay using (..gamma..-/sup 32/P)ATP in the presence of calcium and phosphatidylserine. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When compared to basal levels, calcium (10/sup -6/ M) in combination with phosphatidylserine (50 ..mu..g/ml) significantly enhanced (P < 100) /sup 32/P incorporation into phosphoproteins having mol wt 47,000, 43,000, and 37,000. Half-maximal /sup 22/P incorporation was observed with 3.5 x 10/sup -7/ M Ca/sup 2 +/ in the presence of phosphatidylserine (50 ..mu..g/ml). The effect of phosphatidylserine was biphasic. In the presence of Ca 10/sup -6/ M, /sup 32/P incorporation increased to a maximum at 70 /sup +/g/ml of phosphatidylserine. The increase was suppressed at 150 ..mu..g/ml. Tetracaine caused a dose-dependent inhibition of calcium-activated, phospholipid-dependent enhancement of the three phosphoproteins. Calcium in the absence of phospholipid enhanced the phosphorylation of a protein of 98,000 mol wt. Phosphatidylserine suppressed this enhancement. Calmodulin (10/sup -6/ M) had no detectable effect upon phosphorylation beyond that of calcium alone, but the calmodulin inhibitor R-24571 specifically inhibited the calcium-stimulated 98,000 mol wt phosphoprotein. Calcium-activated, phospholipid-dependent phospholipid-dependent phosphoproteins are present in human placental cytosol; whether calcium-activated, calmodulin-dependent phosphoproteins also are present remains a question.

  2. Ultrasound assisted enzymatic depolymerization of aqueous guar gum solution.

    PubMed

    Prajapat, Amrutlal L; Subhedar, Preeti B; Gogate, Parag R

    2016-03-01

    The present work investigates the effectiveness of application of low intensity ultrasonic irradiation for the intensification of enzymatic depolymerization of aqueous guar gum solution. The extent of depolymerization of guar gum has been analyzed in terms of intrinsic viscosity reduction. The effect of ultrasonic irradiation on the kinetic and thermodynamic parameters related to the enzyme activity as well as the intrinsic viscosity reduction of guar gum using enzymatic approach has been evaluated. The kinetic rate constant has been found to increase with an increase in the temperature and cellulase loading. It has been observed that application of ultrasound not only enhances the extent of depolymerization but also reduces the time of depolymerization as compared to conventional enzymatic degradation technique. In the presence of cellulase enzyme, the maximum extent of depolymerization of guar gum has been observed at 60W of ultrasonic rated power and ultrasonic treatment time of 30min. The effect of ultrasound on the kinetic and thermodynamic parameters as well as the molecular structure of cellulase enzyme was evaluated with the help of the chemical reaction kinetics model and fluorescence spectroscopy. Application of ultrasound resulted in a reduction in the thermodynamic parameters of activation energy (Ea), enthalpy (?H), entropy (?S) and free energy (?G) by 47%, 50%, 65% and 1.97%, respectively. The changes in the chemical structure of guar gum treated using ultrasound assisted enzymatic approach in comparison to the native guar gum were also characterized by FTIR. The results revealed that enzymatic depolymerization of guar gum resulted in a polysaccharide with low degree of polymerization, viscosity and consistency index without any change in the core chemical structure which could make it useful for incorporation in food products. PMID:26584988

  3. 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

    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.

  4. 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

  5. Calcium and protein phosphorylation in the transduction of gravity signal in corn roots

    NASA Technical Reports Server (NTRS)

    Friedmann, M.; Poovaiah, B. W.

    1991-01-01

    The involvement of calcium and protein phosphorylation in the transduction of gravity signal was studied using corn roots of a light-insensitive variety (Zea mays L., cv. Patriot). The gravitropic response was calcium-dependent. Horizontal placement of roots preloaded with 32P for three minutes resulted in changes in protein phosphorylation of polypeptides of 32 and 35 kD. Calcium depletion resulted in decreased phosphorylation of these phosphoproteins and replenishment of calcium restored the phosphorylation.

  6. Rapid changes in protein phosphorylation associated with light-induced gravity perception in corn roots

    NASA Technical Reports Server (NTRS)

    McFadden, J. J.; Poovaiah, B. W.

    1988-01-01

    The effect of light and calcium depletion on in vivo protein phosphorylation was tested using dark-grown roots of Merit corn. Light caused rapid and specific promotion of phosphorylation of three polypeptides. Pretreatment of roots with ethylene glycol bis N,N,N',N' tetraacetic acid and A23187 prevented light-induced changes in protein phosphorylation. We postulate that these changes in protein phosphorylation are involved in the light-induced gravity response.

  7. 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.

  8. Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth

    E-print Network

    Thomas, David D.

    Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth structural changes within the regulatory light chain (RLC) of smooth muscle myosin upon phosphorylation. Smooth muscle contraction is activated by phosphorylation of S19 on RLC, but the structural basis

  9. Modulation of tau phosphorylation within its microtubule-binding domain by cellular thiols.

    PubMed

    Jenkins, S M; Johnson, G V

    1999-11-01

    Tau is a microtubule-stabilizing protein that is functionally modulated by alterations in its phosphorylation state. Because phosphorylation regulates both normal and pathological tau functioning, it is of importance to identify the signaling pathways that regulate tau phosphorylation in vivo. The present study examined changes in tau phosphorylation and function in response to modulation of cellular thiol content. Treatment of cells with phenylarsine oxide, which reacts with vicinal thiols, selectively increased tau phosphorylation within its microtubule-binding domain, at the non-Ser/Thr-Pro sites Ser262/356, while decreasing tau phosphorylation at Ser/ Thr-Pro sites outside this region. This increase in tau phosphorylation correlated with a decrease in the amount of tau associated with the cytoskeleton and decreased microtubule stability. Phenylarsine oxide-induced tau phosphorylation was inhibited by oxidants and by the protein kinase inhibitor staurosporine. Although staurosporine completely eliminated the increase in tau phosphorylation at Ser262/356, as detected by immunostaining with 12E8, it had a comparatively minor effect on the changes in tau localization induced by phenylarsine oxide. The results suggest that regulation of cellular thiols is important for modulating tau phosphorylation and function in situ. Additionally, although phosphorylation of Ser262/356 decreases tau's interaction with the cytoskeleton, phosphorylation of these residues alone is not sufficient for the phenylarsine oxide-induced changes in tau localization. PMID:10537042

  10. Technology transfer

    NASA Technical Reports Server (NTRS)

    Penaranda, Frank E.

    1992-01-01

    The topics are presented in viewgraph form and include the following: international comparison of R&D expenditures in 1989; NASA Technology Transfer Program; NASA Technology Utilization Program thrusts for FY 1992 and FY 1993; National Technology Transfer Network; and NTTC roles.

  11. 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

  12. Phosphorylation of Human CTP Synthetase 1 by Protein Kinase A: IDENTIFICATION OF Thr455 AS A MAJOR SITE OF PHOSPHORYLATION*

    PubMed Central

    Choi, Mal-Gi; Carman, George M.

    2007-01-01

    CTP synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work, we examined the phosphorylation of the human CTPS1-encoded CTP synthetase 1 by protein kinase A. CTP synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7? ura8? double mutant that lacks CTP synthetase activity. Using purified CTP synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr455 was a substrate for protein kinase A. A Thr455 to Ala (T455A) mutation in CTP synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7? ura8? mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation, and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine. PMID:17189248

  13. Active Inhibitor-1 Maintains Protein Hyper-Phosphorylation in Aging Hearts and Halts Remodeling in Failing Hearts

    PubMed Central

    Haghighi, Kobra; Anjak, Ahmad; Cai, Wenfeng; Jiang, Min; Nicolaou, Persoulla; Pylar, George; Karakikes, Ioannis; Rapti, Kleopatra; Rubinstein, Jack; Hajjar, Roger J.; Kranias, Evangelia G.

    2013-01-01

    Impaired sarcoplasmic reticulum calcium cycling and depressed contractility are key characteristics in heart failure. Defects in sarcoplasmic reticulum function are characterized by decreased SERCA2a Ca-transport that is partially attributable to dephosphorylation of its regulator phospholamban by increased protein phosphatase 1 activity. Inhibition of protein phosphatase 1 through activation of its endogenous inhibitor-1 has been shown to enhance cardiac Ca-handling and contractility as well as protect from pathological stress remodeling in young mice. In this study, we assessed the long-term effects of inducible expression of constitutively active inhibitor-1 in the adult heart and followed function and remodeling through the aging process, up to 20 months. Mice with inhibitor-1 had normal survival and similar function to WTs. There was no overt remodeling as evidenced by measures of left ventricular end-systolic and diastolic diameters and posterior wall dimensions, heart weight to tibia length ratio, and histology. Higher phosphorylation of phospholamban at both Ser16 and Thr17 was maintained in aged hearts with active inhibitor-1, potentially offsetting the effects of elevated Ser2815-phosphorylation in ryanodine receptor, as there were no increases in arrhythmias under stress conditions in 20-month old mice. Furthermore, long-term expression of active inhibitor-1 via recombinant adeno-associated virus type 9 gene transfer in rats with pressure-overload induced heart failure improved function and prevented remodeling, associated with increased phosphorylation of phospholamban at Ser16 and Thr17. Thus, chronic inhibition of protein phosphatase 1, through increases in active inhibitor-1, does not accelerate age-related cardiomyopathy and gene transfer of this molecule in vivo improves function and halts remodeling in the long term. PMID:24312496

  14. Phosphorylation reaction in cAPK protein kinase-free energy quantum mechanical/molecular mechanics simulations.

    PubMed

    Valiev, Marat; Yang, Jie; Adams, Joseph A; Taylor, Susan S; Weare, John H

    2007-11-29

    We present results of a theoretical analysis of the phosphorylation reaction in cAMP-dependent protein kinase using a combined quantum mechanical and molecular mechanics (QM/MM) approach. Detailed analysis of the reaction pathway is provided using a novel QM/MM implementation of the nudged elastic band method, finite temperature fluctuations of the protein environment are taken into account using free energy calculations, and an analysis of hydrogen bond interactions is performed on the basis of calculated frequency shifts. The late transfer of the substrate proton to the conserved aspartate (D166), the activation free energy of 15 kcal/mol, and the slight exothermic (-3 kcal/mol) character of the reaction are all consistent with the experimental data. The near attack conformation of D166 in the reactant state is maintained by interactions with threonine-201, asparagine-177, and most notably by a conserved water molecule serving as a strong structural link between the primary metal ion and the D166. The secondary Mg ion acts as a Lewis acid, attacking the beta-gamma bridging oxygen of ATP. This interaction, along with a strong hydrogen bond between the D166 and the substrate, contributes to the stabilization of the transition state. Lys-168 maintains a hydrogen bond to a transferring phosphoryl group throughout a reaction process. This interaction increases in the product state and contributes to its stabilization. PMID:17983217

  15. PPAR?1 phosphorylation enhances proliferation and drug resistance in human fibrosarcoma cells

    SciTech Connect

    Pang, Xiaojuan; Shu, Yuxin; Niu, Zhiyuan; Zheng, Wei; Wu, Haochen; Lu, Yan; Shen, Pingping

    2014-03-10

    Post-translational regulation plays a critical role in the control of cell growth and proliferation. The phosphorylation of peroxisome proliferator-activated receptor ? (PPAR?) is the most important post-translational modification. The function of PPAR? phosphorylation has been studied extensively in the past. However, the relationship between phosphorylated PPAR?1 and tumors remains unclear. Here we investigated the role of PPAR?1 phosphorylation in human fibrosarcoma HT1080 cell line. Using the nonphosphorylation (Ser84 to alanine, S84A) and phosphorylation (Ser84 to aspartic acid, S84D) mutant of PPAR?1, the results suggested that phosphorylation attenuated PPAR?1 transcriptional activity. Meanwhile, we demonstrated that phosphorylated PPAR?1 promoted HT1080 cell proliferation and this effect was dependent on the regulation of cell cycle arrest. The mRNA levels of cyclin-dependent kinase inhibitor (CKI) p21{sup Waf1/Cip1} and p27{sup Kip1} descended in PPAR?1{sup S84D} stable HT1080 cell, whereas the expression of p18{sup INK4C} was not changed. Moreover, compared to the PPAR?1{sup S84A}, PPAR?1{sup S84D} up-regulated the expression levels of cyclin D1 and cyclin A. Finally, PPAR?1 phosphorylation reduced sensitivity to agonist rosiglitazone and increased resistance to anticancer drug 5-fluorouracil (5-FU) in HT1080 cell. Our findings establish PPAR?1 phosphorylation as a critical event in human fibrosarcoma growth. These findings raise the possibility that chemical compounds that prevent the phosphorylation of PPAR?1 could act as anticancer drugs. - Highlights: • Phosphorylation attenuates PPAR?1 transcriptional activity. • Phosphorylated PPAR?1 promotes HT1080 cells proliferation. • PPAR?1 phosphorylation regulates cell cycle by mediating expression of cell cycle regulators. • PPAR?1 phosphorylation reduces sensitivity to agonist and anticancer drug. • Our findings establish PPAR?1 phosphorylation as a critical event in HT1080 cells growth.

  16. Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection.

    PubMed

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

    2013-01-15

    A rapid assay operable under isothermal or nonisothermal conditions is described, where the sensitivity of a typical molecular beacon (MB) system is improved by using thermostable RNase H to enzymatically cleave an MB composed of a DNA stem and an RNA loop (R/D-MB). On hybridization of the R/D-MB to target DNA, there was a modest increase in fluorescence intensity (~5.7× above background) due to an opening of the probe and a concomitant reduction in the Förster resonance energy transfer efficiency. The 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 (12.9× above background), resulting in an approximately 2- to 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 polymerase chain reactions by measuring enhancement of donor fluorescence on 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

  17. Phosphorylation-Independent Activity of the Response Regulators AlgB and AlgR in Promoting Alginate Biosynthesis in Mucoid Pseudomonas aeruginosa

    PubMed Central

    Ma, Sheng; Selvaraj, Uma; Ohman, Dennis E.; Quarless, Ryan; Hassett, Daniel J.; Wozniak, Daniel J.

    1998-01-01

    Overproduction of the capsular polysaccharide alginate appears to confer a selective advantage for Pseudomonas aeruginosa in the lungs of cystic fibrosis patients. The regulators AlgB and AlgR, which are both required as positive activators in alginate overproduction, have homology with the regulator class of two-component environmental responsive proteins which coordinate gene expression through signal transduction mechanisms. Signal transduction in this class of proteins generally occurs via autophosphorylation of the sensor kinase protein and phosphotransfer from the sensor to a conserved aspartate residue, which is present in the amino terminus of the response regulator. Recently, kinB was identified downstream of algB and was shown to encode the cognate histidine protein kinase that efficiently phosphorylates AlgB. However, we show here that a null mutation in kinB in a mucoid cystic fibrosis isolate, P. aeruginosa FRD1, did not block alginate production. The role of the conserved aspartate residue in the phosphorylation of AlgB was examined. The predicted phosphorylation site of AlgB (D59) was mutated to asparagine (N), and a derivative of an AlgB lacking the entire amino-terminal phosphorylation domain (AlgB?1-145) was constructed. A hexahistidine tag was included at the amino terminus of the wild-type (H-AlgB), H-AlgB?1-145, and mutant (H-AlgB.59N) AlgB proteins. These derivatives were purified by Ni2+ affinity chromatography and examined for in vitro phosphorylation by the purified sensor kinase protein, KinB. The results indicated that while KinB efficiently phosphorylated H-AlgB, no phosphorylation of H-AlgB?1-145 or H-AlgB.D59N was apparent. An allelic exchange system was developed to transfer mutant algB alleles onto the chromosome of a P. aeruginosa algB mutant to examine the effect on alginate production. Despite the defect in AlgB phosphorylation, P. aeruginosa strains expressing AlgB.D59N or H-AlgB?1-145 remained mucoid. The roles of the conserved aspartate residues in the phosphorylation of AlgR were also examined. As seen with AlgB, mutations in the predicted phosphorylation site of AlgR (AlgR.D54N and AlgR.D85N) did not affect alginate production. These results indicate that in vivo phosphorylation of AlgB and AlgR are not required for their roles in alginate production. Thus, the mechanism by which these response regulators activate alginate genes in mucoid P. aeruginosa appears not to be mediated by conventional phosphorylation-dependent signal transduction. PMID:9473053

  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. 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

  20. 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.

  1. Allovalency revisited: an analysis of multisite phosphorylation and substrate rebinding

    E-print Network

    Jason W. Locasale

    2008-01-09

    The utilization of multiple phosphorylation sites in regulating a biological response is ubiquitous in cell signaling. If each site contributes an additional, equivalent binding site, then one consequence of an increase in the number of phosphorylations may be to increase the probability that, upon disassociation, a ligand immediately rebinds to its receptor. How such effects may influence cell signaling systems has been less studied. Here, a self-consistent integral equation formalism for ligand rebinding, in conjunction with Monte Carlo simulations, is employed to further investigate the effects of multiple, equivalent binding sites on shaping biological responses. Multiple regimes that characterize qualitatively different physics due to the differential prevalence of rebinding effects are predicted. Calculations suggest that when ligand rebinding contributes significantly to the dose response, a purely allovalent model can influence the binding curves nonlinearly. The model also predicts that ligand rebinding in itself appears insufficient to generative a highly cooperative biological response.

  2. Tangeretin inhibits extracellular-signal-regulated kinase (ERK) phosphorylation.

    PubMed

    Van Slambrouck, Séverine; Parmar, Virinder S; Sharma, Sunil K; De Bondt, Bart; Foré, Fleur; Coopman, Peter; Vanhoecke, Barbara W; Boterberg, Tom; Depypere, Herman T; Leclercq, Guy; Bracke, Marc E

    2005-03-14

    Tangeretin is a methoxyflavone from citrus fruits, which inhibits growth of human mammary cancer cells and cytolysis by natural killer cells. Attempting to unravel the flavonoid's action mechanism, we found that it inhibited extracellular-signal-regulated kinases 1/2 (ERK1/2) phosphorylation in a dose- and time-dependent way. In human T47D mammary cancer cells this inhibition was optimally observed after priming with estradiol. The spectrum of the intracellular signalling kinase inhibition was narrow and comparison of structural congeners showed that inhibition of ERK phosphorylation was not unique for tangeretin. Our data add tangeretin to the list of small kinase inhibitors with a restricted intracellular inhibition profile. PMID:15757658

  3. The dynamical nature of enzymatic catalysis.

    PubMed

    Callender, Robert; Dyer, R Brian

    2015-02-17

    CONSPECTUS: As is well-known, enzymes are proteins designed to accelerate specific life essential chemical reactions by many orders of magnitude. A folded protein is a highly dynamical entity, best described as a hierarchy or ensemble of interconverting conformations on all time scales from femtoseconds to minutes. We are just beginning to learn what role these dynamics play in the mechanism of chemical catalysis by enzymes due to extraordinary difficulties in characterizing the conformational space, that is, the energy landscape, of a folded protein. It seems clear now that their role is crucially important. Here we discuss approaches, based on vibrational spectroscopies of various sorts, that can reveal the energy landscape of an enzyme-substrate (Michaelis) complex and decipher which part of the typically very complicated landscape is relevant to catalysis. Vibrational spectroscopy is quite sensitive to small changes in bond order and bond length, with a resolution of 0.01 Å or less. It is this sensitivity that is crucial to its ability to discern bond reactivity. Using isotope edited IR approaches, we have studied in detail the role of conformational heterogeneity and dynamics in the catalysis of hydride transfer by LDH (lactate dehydrogenase). Upon the binding of substrate, the LDH·substrate system undergoes a search through conformational space to find a range of reactive conformations over the microsecond to millisecond time scale. The ligand is shuttled to the active site via first forming a weakly bound enzyme·ligand complex, probably consisting of several heterogeneous structures. This complex undergoes numerous conformational changes spread throughout the protein that shuttle the enzyme·substrate complex to a range of conformations where the substrate is tightly bound. This ensemble of conformations all have a propensity toward chemistry, but some are much more facile for carrying out chemistry than others. The search for these tightly bound states is clearly directed by the forces that the protein can bring to bear, very much akin to the folding process to form native protein in the first place. In fact, the conformational subspace of reactive conformations of the Michaelis complex can be described as a "collapse" of reactive substates compared with that found in solution, toward a much smaller and much more reactive set. These studies reveal how dynamic disorder in the protein structure can modulate the on-enzyme reactivity. It is very difficult to account for how the dynamical nature of the ground state of the Michaelis complex modulates function by transition state concepts since dynamical disorder is not a starting feature of the theory. We find that dynamical disorder may well play a larger or similar sized role in the measured Gibbs free energy of a reaction compared with the actual energy barrier involved in the chemical event. Our findings are broadly compatible with qualitative concepts of evolutionary adaptation of function such as adaptation to varying thermal environments. Our work suggests a methodology to determine the important dynamics of the Michaelis complex. PMID:25539144

  4. Enzymatic synthesis of oligo- and polysaccharide fatty acid esters.

    PubMed

    van den Broek, Lambertus A M; Boeriu, Carmen G

    2013-03-01

    Amphiphilic oligo- and polysaccharides (e.g. polysaccharide alkyl or alkyl-aryl esters) form a new class of polymers with exceptional properties. They function as polymeric surfactants, whilst maintaining most of the properties of the starting polymeric material such as emulsifying, gelling, and film forming properties combined with partial water solubility or permeability. At present carbohydrate fatty acid esters are generally obtained by chemical methods using toxic solvents and organic and inorganic catalysts that leave residual traces in the final products. Enzymatic reactions offer an attractive alternative route for the synthesis of polysaccharide esters. In this review the state of the art of enzymatic synthesis of oligo- and polysaccharides fatty esters has been described. PMID:23465902

  5. Rational design of functional and tunable oscillating enzymatic networks

    NASA Astrophysics Data System (ADS)

    Semenov, Sergey N.; Wong, Albert S. Y.; van der Made, R. Martijn; Postma, Sjoerd G. J.; Groen, Joost; van Roekel, Hendrik W. H.; de Greef, Tom F. A.; Huck, Wilhelm T. S.

    2015-02-01

    Life is sustained by complex systems operating far from equilibrium and consisting of a multitude of enzymatic reaction networks. The operating principles of biology's regulatory networks are known, but the in vitro assembly of out-of-equilibrium enzymatic reaction networks has proved challenging, limiting the development of synthetic systems showing autonomous behaviour. Here, we present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behaviour. We demonstrate that a network built around autoactivation and delayed negative feedback of the enzyme trypsin is capable of producing sustained oscillating concentrations of active trypsin for over 65?h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equilibrium systems, are obtained by linking multiple network modules in microfluidic flow reactors. The methodology developed here provides a general framework to construct dissipative, tunable and robust (bio)chemical reaction networks.

  6. Enzymatic synthesis of biodiesel via alcoholysis of palm oil.

    PubMed

    Matassoli, André L F; Corrêa, Igor N S; Portilho, Márcio F; Veloso, Cláudia O; Langone, Marta A P

    2009-05-01

    The enzymatic alcoholysis of crude palm oil with methanol and ethanol was investigated using commercial immobilized lipases (Lipozyme RM IM, Lipozyme TL IM). The effect of alcohol (methanol or ethanol), molar ratio of alcohol to crude palm oil, and temperature on biodiesel production was determined. The best ethyl ester yield was about 25 wt.% and was obtained with ethanol/oil molar ratio of 3.0, temperature of 50 degrees C, enzyme concentration of 3.0 wt.%, and stepwise addition of the alcohol after 4 h of reaction. Experiments with 1 and 3 wt.% of KOH and 3 wt.% of MgO were carried out to compare their catalytic behavior with the enzymatic transesterification results. The commercial immobilized lipase, Lipozyme TL IM, showed the best catalytic performance. PMID:19023524

  7. Enzymatic hydrolysis and fermentation of agricultural residues to ethanol

    SciTech Connect

    Mes-Hartree, M.; Hogan, C.M.; Saddler, J.N.

    1984-01-01

    A combined enzymatic hydrolysis and fermentation process was used to convert steam-treated wheat and barley straw to ethanol. Maximum conversion efficiencies were obtained when the substrates were steamed for 90 s. These substrates could yield over 0.4 g ethanol/g cellulose following a combined enzymatic hydrolysis and fermentation process procedure using culture filtrates derived from Trichoderma harzianum E58. When culture filtrates from Trichoderma reesei C30 and T. reesei QM9414 were used, the ethanol yields obtained were 0.32 and 0.12 g ethanol/g cellulose utilized, respectively. The lower ethanol yields obtained with these strains were attributed to the lower amounts of ..beta..-glucosidase detected in the T. reesei culture filtrates.

  8. Analytical applications of enzymatic growth of quantum dots.

    PubMed

    Saa, Laura; Virel, Ana; Sanchez-Lopez, Jose; Pavlov, Valery

    2010-06-01

    We have developed an analytical assay to detect the enzymatic activity of acetylcholine esterase and alkaline phosphatase based on the generation of quantum dots by enzymatic products. Acetylcholine esterase converts acetylthiocholine into thiocholine. The latter enhances the rate of decomposition of sodium thiosulfate into H(2)S, which in the presence of cadmium sulfate yields CdS quantum dots showing a time dependent exponential growth, typical of autocatalytic processes. This assay was also applied to detect acetylcholine esterase inhibitors. Alkaline phosphatase hydrolyzes thiophosphate and yields H(2)S, which instantly reacts with Cd(2+) to give CdS quantum dots. The formation of CdS quantum dots in both reactions was followed by fluorescence spectroscopy and showed dependence on the concentration of enzyme and substrate. PMID:20432413

  9. 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

  10. 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.

  11. Utilizing enzymatic digestion procedures in the bioanalytical laboratory.

    PubMed

    Szapacs, Matthew; Mencken, Thomas; Williams, Jon; Li, Yi

    2016-01-01

    In recent years, the use of LC-MS technologies in the bioanlytical laboratory for quantitation of peptide/protein biomarkers and biotherapeutics has increased dramatically. The increased interest is due to the improvement in sensitivity of MS instruments over the last 5-10 years, as well as its proven ability to overcome some common issues associated with immunoassay, namely selectivity and reagent availability. However, large proteins (>10 kDa) chromatograph and ionize poorly. To overcome this challenge, LC-MS/MS workflows for proteins larger than 10 kDa utilize enzymatic digestion procedures with subsequent quantitation of one or more of these enzymatically derived peptides to act as a surrogate for the intact protein. Here, recommendations of digestion technique and potential internal standards are summarized. PMID:26648043

  12. Enzymatic hydrolysis of steryl glycosides for their analysis in foods.

    PubMed

    Münger, Linda H; Nyström, Laura

    2014-11-15

    Steryl glycosides (SG) contribute significantly to the total intake of phytosterols. The standard analytical procedure involving acid hydrolysis fails to reflect the correct sterol profile of SG due to isomerization of some of the labile sterols. Therefore, various glycosylases were evaluated for their ability to hydrolyse SG under milder conditions. Using a pure SG mixture in aqueous solution, the highest glycolytic activity, as demonstrated by the decrease in SG and increase in free sterols was achieved using inulinase preparations (decrease of >95%). High glycolytic activity was also demonstrated using hemicellulase (63%). The applicability of enzymatic hydrolysis using inulinase preparations was further verified on SG extracted from foods. For example in potato peel ?(5)-avenasteryl glucoside, a labile SG, was well preserved and contributed 26.9% of the total SG. Therefore, enzymatic hydrolysis is suitable for replacing acid hydrolysis of SG in food lipid extracts to accurately determine the sterol profile of SG. PMID:24912717

  13. 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.

  14. Improving biogas production from microalgae by enzymatic pretreatment.

    PubMed

    Passos, Fabiana; Hom-Diaz, Andrea; Blanquez, Paqui; Vicent, Teresa; Ferrer, Ivet

    2016-01-01

    In this study, enzymatic pretreatment of microalgal biomass was investigated under different conditions and evaluated using biochemical methane potential (BMP) tests. Cellulase, glucohydrolase and an enzyme mix composed of cellulase, glucohydrolase and xylanase were selected based on the microalgae cell wall composition (cellulose, hemicellulose, pectin and glycoprotein). All of them increased organic matter solubilisation, obtaining high values already after 6h of pretreatment with an enzyme dose of 1% for cellulase and the enzyme mix. BMP tests with pretreated microalgae showed a methane yield increase of 8 and 15% for cellulase and the enzyme mix, respectively. Prospective research should evaluate enzymatic pretreatments in continuous anaerobic reactors so as to estimate the energy balance and economic cost of the process. PMID:26343574

  15. Mitochondrial cytochrome c oxidase: mechanism of action and role in regulating oxidative phosphorylation.

    PubMed

    Wilson, David F; Vinogradov, Sergei A

    2014-12-15

    Mitochondrial oxidative phosphorylation has a central role in eukaryotic metabolism, providing the energy (ATP) required for survival. Regulation of this important pathway is, however, still not understood, largely due to limitations in the ability to measure the essential metabolites, including oxygen (pO2, oxygen pressure), ADP, and AMP. In addition, neither the mechanism of oxygen reduction by mitochondrial cytochrome c oxidase nor how its rate is controlled is understood, although this enzyme determines the rate of oxygen consumption and thereby the rate of ATP synthesis. Cytochrome c oxidase is responsible for reduction of molecular oxygen to water using reducing equivalents donated by cytochrome c and for site 3 energy coupling in oxidative phosphorylation. A mechanism-based model of the cytochrome c oxidase reaction is presented in which transfer of reducing equivalents from the lower- to the higher-potential region of the coupling site occurs against an opposing energy barrier, Q. The steady-state rate equation is fitted to data for the dependence of mitochondrial respiratory rate on cytochrome c reduction, oxygen pressure (pO2), and [ATP]/[ADP][Pi] at pH 6.5 to 8.35 (where Pi is inorganic phosphate). The fit of the rate expression to the experimental data is very good for all experimental conditions. Levels of the intermediates in oxygen reduction in the oxidase reaction site have been calculated. An intermediate in the reaction, tentatively identified as peroxide, bridged between the iron and copper atoms of the reaction site has a central role in coupling mitochondrial respiration to the [ATP]/[ADP][Pi]. PMID:25324518

  16. Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding

    SciTech Connect

    Baek, Kyuwon; Liu, Xiong; Ferron, Francois; Shu, Shi; Korn, Edward D.; Dominguez, Roberto

    2008-08-27

    On starvation, Dictyostelium cells aggregate to form multicellular fruiting bodies containing spores that germinate when transferred to nutrient-rich medium. This developmental cycle correlates with the extent of actin phosphorylation at Tyr-53 (pY53-actin), which is low in vegetative cells but high in viable mature spores. Here we describe high-resolution crystal structures of pY53-actin and unphosphorylated actin in complexes with gelsolin segment 1 and profilin. In the structure of pY53-actin, the phosphate group on Tyr-53 makes hydrogen-bonding interactions with residues of the DNase I-binding loop (D-loop) of actin, resulting in a more stable conformation of the D-loop than in the unphosphorylated structures. A more rigidly folded D-loop may explain some of the previously described properties of pY53-actin, including its increased critical concentration for polymerization, reduced rates of nucleation and pointed end elongation, and weak affinity for DNase I. We show here that phosphorylation of Tyr-53 inhibits subtilisin cleavage of the D-loop and reduces the rate of nucleotide exchange on actin. The structure of profilin-Dictyostelium-actin is strikingly similar to previously determined structures of profilin-{beta}-actin and profilin-{alpha}-actin. By comparing this representative set of profilin-actin structures with other structures of actin, we highlight the effects of profilin on the actin conformation. In the profilin-actin complexes, subdomains 1 and 3 of actin close around profilin, producing a 4.7 deg. rotation of the two major domains of actin relative to each other. As a result, the nucleotide cleft becomes moderately more open in the profilin-actin complex, probably explaining the stimulation of nucleotide exchange on actin by profilin.

  17. Linker histone partial phosphorylation: effects on secondary structure and chromatin condensation.

    PubMed

    Lopez, Rita; Sarg, Bettina; Lindner, Herbert; Bartolomé, Salvador; Ponte, Inma; Suau, Pedro; Roque, Alicia

    2015-05-19

    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

  18. Isoform-specific phosphorylation-dependent regulation of connexin hemichannels.

    PubMed

    Alstrøm, Jette Skov; Hansen, Daniel Bloch; Nielsen, Morten Schak; MacAulay, Nanna

    2015-11-15

    Connexins form gap junction channels made up of two connexons (hemichannels) from adjacent cells. Unopposed hemichannels may open toward the extracellular space upon stimulation by, e.g., removal of divalent cations from the extracellular solution and allow isoform-specific transmembrane flux of fluorescent dyes and physiologically relevant molecules, such as ATP and ions. Connexin (Cx)43 and Cx30 are the major astrocytic connexins. Protein kinase C (PKC) regulates Cx43 in its cell-cell gap junction configuration and may also act to keep Cx43 hemichannels closed. In contrast, the regulation of Cx30 hemichannels by PKC is unexplored. To determine phosphorylation-dependent regulation of Cx30 and Cx43 hemichannels, these were heterologously expressed in Xenopus laevis oocytes and opened with divalent cation-free solution. Inhibition of PKC activity did not affect hemichannel opening of either connexin. PKC activation had no effect on Cx43-mediated hemichannel activity, whereas both dye uptake and current through Cx30 hemichannels were reduced. We detected no PKC-induced connexin internalization from the plasma membrane, indicating that PKC reduced Cx30 hemichannel activity by channel closure. In an attempt to resolve the PKC phosphorylation site(s) on Cx30, alanine mutations of putative cytoplasmic PKC consensus sites were created to prevent phosphorylation (T5A, T8A, T102A, S222A, S225A, S239A, and S258A). These Cx30 mutants responded to PKC activation, suggesting that Cx30 hemichannels are not regulated by phosphorylation of a single site. In conclusion, Cx30, but not Cx43, hemichannels close upon PKC activation, illustrating that connexin hemichannels display not only isoform-specific permeability profiles but also isoform-specific regulation by PKC. PMID:26400258

  19. Phosphorylation of Phosducin Accelerates Rod Recovery from Transducin Translocation

    PubMed Central

    Belcastro, Marycharmain; Song, Hongman; Sinha, Satyabrata; Song, Chunyan; Mathers, Peter H; Sokolov, Maxim

    2012-01-01

    Purpose. In rods saturated by light, the G protein transducin undergoes translocation from the outer segment compartment, which results in the uncoupling of transducin from its innate receptor, rhodopsin. We measured the kinetics of recovery from this adaptive cellular response, while also investigating the role of phosducin, a phosphoprotein binding transducin ?? subunits in its de-phosphorylated state, in regulating this process. Methods. Mice were exposed to a moderate rod-saturating light triggering transducin translocation, and then allowed to recover in the dark while free running. The kinetics of the return of the transducin subunits to the outer segments were compared in transgenic mouse models expressing full-length phosducin, and phosducin lacking phosphorylation sites serine 54 and 71, using Western blot analysis of serial tangential sections of the retina. Results. In mice expressing normal phosducin, transducin ? and ?? subunits returned to the outer segments with a half-time (t1/2) of ?24 and 29 minutes, respectively. In the phosducin phosphorylation mutants, the transducin ? subunit moved four times slower, with t1/2 ?95 minutes, while the movement of transducin ?? was less affected. Conclusions. We demonstrate that the recovery of rod photoreceptors from the ambient saturating levels of illumination, in terms of the return of the light-dispersed transducin subunits to the rod outer segments, occurs six times faster than reported previously. Our data also support the notion that the accumulation of transducin ? subunit in the outer segment is driven by its re-binding to the transducin ?? dimer, because this process is accelerated significantly by phosducin phosphorylation. PMID:22491418

  20. Phosphorylation of Glyceric Acid in Aqueous Solution Using Trimetaphosphate

    NASA Technical Reports Server (NTRS)

    Kolb, Vera; Orgel, Leslie E.

    1996-01-01

    The phosphorylation of glyceric acid is an interesting prebiotic reaction because it converts a simple, potentially prebiotic organic molecule into phosphate derivatives that are central to carbohydrate metabolism. We find that 0.05 M glyceric acid in the presence of 0.5 M trimetaphosphate in alkaline solution gives a mixture of 2- and 3-phosphoglyceric acids in combined yields of up to 40%.

  1. Serine-71 phosphorylation of Rac1 modulates downstream signaling.

    PubMed

    Schwarz, Janett; Proff, Julia; Hävemeier, Anika; Ladwein, Markus; Rottner, Klemens; Barlag, Britta; Pich, Andreas; Tatge, Helma; Just, Ingo; Gerhard, Ralf

    2012-01-01

    The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways. PMID:22970203

  2. Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

    Cancer.gov

    In cooperation with the RAS Initiative, the NCI's Clinical Proteomic Tumor Analysis Consortium (CPTAC) has launched a project to develop quantitative assays for proteins and phosphopeptides involved in RAS signaling. Within the next 1-2 years these assays should allow the amounts and phosphorylation states of tens of RAS and RAS-related proteins to be determined in tumor samples, cell lines, or cancer models in a single run.

  3. 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

  4. Protein tyrosine phosphorylation during meiotic divisions of starfish oocytes

    SciTech Connect

    Peaucellier, G.; Andersen, A.C.; Kinsey, W.H. )

    1990-04-01

    We have used an antibody specific for phosphotyrosine to investigate protein phosphorylation on tyrosine during hormone-induced maturation of starfish oocytes. Analysis of immunoprecipitates from cortices of in vivo labeled Marthasterias glacialis oocytes revealed the presence of labeled phosphotyrosine-containing proteins only after hormone addition. Six major phosphoproteins of 195, 155, 100, 85, 45, and 35 kDa were detected. Total activity in immunoprecipitates increased until first polar body emission and was greatly reduced upon completion of meiosis but some proteins exhibited different kinetics. The labeling of the 155-kDa protein reached a maximum at germinal vesicle breakdown, while the 35-kDa appeared later and disappeared after polar body emission. Similar results were obtained with Asterias rubens oocytes. In vitro phosphorylation of cortices showed that tyrosine kinase activity is a major protein kinase activity in this fraction, the main endogenous substrate being a 68-kDa protein. The proteins phosphorylated on tyrosine in vitro were almost similar in extracts from oocytes treated or not with the hormone.

  5. 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

  6. Catalytic constants enable the emergence of bistability in dual phosphorylation

    PubMed Central

    Conradi, Carsten; Mincheva, Maya

    2014-01-01

    Dual phosphorylation of proteins is a principal component of intracellular signalling. Bistability is considered an important property of such systems and its origin is not yet completely understood. Theoretical studies have established parameter values for multistationarity and bistability for many types of proteins. However, up to now no formal criterion linking multistationarity and bistability to the parameter values characterizing dual phosphorylation has been established. Deciding whether an unclassified protein has the capacity for bistability, therefore requires careful numerical studies. Here, we present two general algebraic conditions in the form of inequalities. The first employs the catalytic constants, and if satisfied guarantees multistationarity (and hence the potential for bistability). The second involves the catalytic and Michaelis constants, and if satisfied guarantees uniqueness of steady states (and hence absence of bistability). Our method also allows for the direct computation of the total concentration values such that multistationarity occurs. Applying our results yields insights into the emergence of bistability in the ERK–MEK–MKP system that previously required a delicate numerical effort. Our algebraic conditions present a practical way to determine the capacity for bistability and hence will be a useful tool for examining the origin of bistability in many models containing dual phosphorylation. PMID:24647909

  7. On the localized coupling of respiration and phosphorylation in mitochondria.

    PubMed

    Yaguzhinsky, Lev S; Yurkov, Vladimir I; Krasinskaya, Inna P

    2006-01-01

    This paper is an overview of the theoretical and experimental studies performed in our laboratory to answer the question whether there exist conditions where the hypothetical mechanism of the localized coupling of respiration and phosphorylation postulated by R. Williams in 1961 operates. These studies were undertaken to verify the earlier suggestion that mitochondria may exist in two structural and functional states. Correspondingly, there are two operation modes of oxidative phosphorylation, one of which corresponds to the Williams' mechanism of localized coupling and the other, to the Mitchell's mechanism of delocalized coupling. The paper considers the principle of the energy conservation of oxidative reactions in mitochondrial membranes in the form of the thermodynamic potential of hydrogen ions (Deltamusol) lacking, in part, the solvation shell. We present experimental evidence for the existence of the mechanism of localized coupling and describes the conditions favorable for its implementation. The experiments described in this paper show that the aforementioned models for proton coupling are not necessarily alternative. A conclusion is made that, depending on the particular conditions, either localized or delocalized coupling mechanisms of oxidative phosphorylation may come into operation. PMID:16730641

  8. Expression, purification, phosphorylation and characterization of recombinant human statherin.

    PubMed

    Manconi, Barbara; Cabras, Tiziana; Vitali, Alberto; Fanali, Chiara; Fiorita, Antonella; Inzitari, Rosanna; Castagnola, Massimo; Messana, Irene; Sanna, Maria Teresa

    2010-02-01

    This work reports the successful recombinant expression of human statherin in Escherichia coli, its purification and in vitro phosphorylation. Human statherin is a 43-residue peptide, secreted by parotid and submandibular glands and phosphorylated on serine 2 and 3. The codon-optimized statherin gene was synthesized and cloned into commercial pTYB11 plasmid to allow expression of statherin as a fusion protein with intein containing a chitin-binding domain. The plasmid was transformed into E. coli strains and cultured in Luria-Bertani medium, which gave productivity of soluble statherin fusion protein of up to 47mg per liter of cell culture, while 112mg of fusion protein were in the form of inclusion bodies. No significant refolded target protein was obtained from inclusion bodies. The amount of r-h-statherin purified by RP-HPLC corresponded to 0.6mg per liter of cell culture. Attenuated total reflection-Fourier transform infrared spectroscopy experiments performed on human statherin isolated from saliva and r-h-statherin assessed the correct folding of the recombinant peptide. Recombinant statherin was transformed into the diphosphorylated biologically active form by in vitro phosphorylation using the Golgi-enriched fraction of pig parotid gland containing the Golgi-casein kinase. PMID:19651217

  9. Pom1 gradient buffering through intermolecular auto-phosphorylation

    PubMed Central

    Hersch, Micha; Hachet, Olivier; Dalessi, Sascha; Ullal, Pranav; Bhatia, Payal; Bergmann, Sven; Martin, Sophie G

    2015-01-01

    Concentration gradients provide spatial information for tissue patterning and cell organization, and their robustness under natural fluctuations is an evolutionary advantage. In rod-shaped Schizosaccharomyces pombe cells, the DYRK-family kinase Pom1 gradients control cell division timing and placement. Upon dephosphorylation by a Tea4-phosphatase complex, Pom1 associates with the plasma membrane at cell poles, where it diffuses and detaches upon auto-phosphorylation. Here, we demonstrate that Pom1 auto-phosphorylates intermolecularly, both in vitro and in vivo, which confers robustness to the gradient. Quantitative imaging reveals this robustness through two system’s properties: The Pom1 gradient amplitude is inversely correlated with its decay length and is buffered against fluctuations in Tea4 levels. A theoretical model of Pom1 gradient formation through intermolecular auto-phosphorylation predicts both properties qualitatively and quantitatively. This provides a telling example where gradient robustness through super-linear decay, a principle hypothesized a decade ago, is achieved through autocatalysis. Concentration-dependent autocatalysis may be a widely used simple feedback to buffer biological activities. PMID:26150232

  10. Phosphorylation Site Dynamics of Early T-cell Receptor Signaling

    PubMed Central

    Rigbolt, Kristoffer T. G.; Hu, Bin; Hlavacek, William S.; Blagoev, Blagoy

    2014-01-01

    In adaptive immune responses, T-cell receptor (TCR) signaling impacts multiple cellular processes and results in T-cell differentiation, proliferation, and cytokine production. Although individual protein–protein interactions and phosphorylation events have been studied extensively, we lack a systems-level understanding of how these components cooperate to control signaling dynamics, especially during the crucial first seconds of stimulation. Here, we used quantitative proteomics to characterize reshaping of the T-cell phosphoproteome in response to TCR/CD28 co-stimulation, and found that diverse dynamic patterns emerge within seconds. We detected phosphorylation dynamics as early as 5 s and observed widespread regulation of key TCR signaling proteins by 30 s. Development of a computational model pointed to the presence of novel regulatory mechanisms controlling phosphorylation of sites with central roles in TCR signaling. The model was used to generate predictions suggesting unexpected roles for the phosphatase PTPN6 (SHP-1) and shortcut recruitment of the actin regulator WAS. Predictions were validated experimentally. This integration of proteomics and modeling illustrates a novel, generalizable framework for solidifying quantitative understanding of a signaling network and for elucidating missing links. PMID:25147952

  11. Angling for Uniqueness in Enzymatic Preparation of Glycosides

    PubMed Central

    Trincone, Antonio

    2013-01-01

    In the early days of biocatalysis, limitations of an enzyme modeled the enzymatic applications; nowadays the enzyme can be engineered to be suitable for the process requirements. This is a general bird’s-eye view and as such cannot be specific for articulated situations found in different classes of enzymes or for selected enzymatic processes. As far as the enzymatic preparation of glycosides is concerned, recent scientific literature is awash with examples of uniqueness related to the features of the biocatalyst (yield, substrate specificity, regioselectivity, and resistance to a particular reaction condition). The invention of glycosynthases is just one of the aspects that has thrust forward the research in this field. Protein engineering, metagenomics and reaction engineering have led to the discovery of an expanding number of novel enzymes and to the setting up of new bio-based processes for the preparation of glycosides. In this review, new examples from the last decade are compiled with attention both to cases in which naturally present, as well as genetically inserted, characteristics of the catalysts make them attractive for biocatalysis. PMID:24970171

  12. Recent Research Trends on the Enzymatic Synthesis of Structured Lipids.

    PubMed

    Kim, Byung Hee; Akoh, Casimir C

    2015-08-01

    Structured lipids (SLs) are lipids that have been chemically or enzymatically modified from their natural biosynthetic form. Because SLs are made to possess desired nutritional, physicochemical, or textural properties for various applications in the food industry, many research activities have been aimed at their commercialization. The production of SLs by enzymatic procedures has a great potential in the future market because of the specificity of lipases and phospholipases used as the biocatalysts. The aim of this review is to provide concise information on the recent research trends on the enzymatic synthesis of SLs of commercial interest, such as medium- and long-chain triacylglycerols, human milk fat substitutes, cocoa butter equivalents, trans-free or low-trans plastic fats (such as margarines and shortenings), low-calorie fats/oils, health-beneficial fatty acid-rich fats/oils, mono- or diacylglycerols, and structurally modified phospholipids. This limited review covers 108 research articles published between 2010 and 2014 which were searched in Web of Science. PMID:26189491

  13. Enzymatic technologies for remediation of hydrophobic organic pollutants in soil.

    PubMed

    Eibes, G; Arca-Ramos, A; Feijoo, G; Lema, J M; Moreira, M T

    2015-11-01

    Worldwide there are numerous contaminated sites as a result of the widespread production and use of chemicals in industrial and military activities as well as poor schemes of waste disposal and accidental spillages. The implementation of strategies for decontamination and restoration of polluted sites has become a priority, being bioremediation with biological agents a promising alternative. Enzyme-based technologies offer several advantages over the use of microbial cells, provided that the biocatalyst meets specific requirements: efficiency to remove the target pollutant/s, non-dependency on expensive coenzymes or cofactors, enzyme stability, and an affordable production system. In this mini-review, the direct application of enzymes for in situ soil bioremediation is explored, and also novel ex situ enzymatic technologies are presented. This new perspective provides a valuable insight into the different enzymatic alternatives for decontamination of soils. Examples of recent applications are reported, including pilot-scale treatments and patented technologies, and the principles of operation and the main requirements associated are described. Furthermore, the main challenges regarding the applicability of enzymatic technologies for remediation of hydrophobic organic pollutants from soil are discussed. PMID:26293336

  14. Immobilization and enzymatic properties of Bacillus megaterium glucose dehydrogenase

    SciTech Connect

    Baron, M.; Fontana, J.D.; Guimaraes, M.F.; Woodward, J.

    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.

  15. Enzymatic hydrolysis of cellulose and various pretreated wood fractions

    SciTech Connect

    Saddler, J.N.; Brownell, H.H.; Clermont, L.P.; Levitin, N.

    1982-06-01

    Three strains of Trichoderma-Trichoderma reesei C30, Trichoderma reesei QM9414, and Trichoderma species E58-were used to study the enzymatic hydrolysis of pretreated wood substrates. Each of the culture filtrates was incubated with a variety of commercially prepared cellulose substrates and pretreated wood substrates. Solka floc was the most easily degraded commercial cellulose. The enzyme accessibility of steam-exploded samples which has been alkali extracted and then stored wet decreased with the duration of the steam treatment. Air drying reduced the extent of hydrolysis of all the samples but had a greater effect on the samples which had previously shown the greatest hydrolysis. Mild pulping using 2% chlorite increased the enzymatic hydrolysis of all the samples. Steam explosion was shown to be an excellent pretreatment method for aspen wood and was much superior to dilute nitric acid pretreatment. The results indicate that the distribution of the lignin as well as the surface area of the cellulosic substrate are important features in enzymatic hydrolysis. (Refs 17).

  16. Self-evolving microstructured systems upon enzymatic catalysis.

    PubMed

    Chopineau, J; Lesieur, S; Carion-Taravella, B; Ollivon, M

    1998-01-01

    The consequences of cell microstructuration on enzyme functions is discussed in the framework of self-evolving microstructured systems. Molecular assemblies of amphiphiles or lipids are spontaneously formed by self-organisation. Among these different structures, reversed micelles, liquid crystalline mesophases and vesicles are hosts for enzymatic reaction studies. Inside a living cell, phospholipid metabolism is responsible for membrane structural modifications; the catalytic behaviour of lipolytic enzymes, mainly phospholipase (PL) A2, is described in relation with structural aspects of biological membranes. The implication in cellular regulation events of PLC and PLD is discussed in relation with the role of their reaction products as second messengers in membrane fusion processes. The in vitro synthesis of dialkyl phosphatidylcholines, via the enzymatic 'salvage pathway' which leads to the formation of vesicles upon phospholipid formation, is considered in relation with autopoiesis. More recent studies on self-evolving systems based on enzyme-surfactants reactions are detailed. The interactions between amphiphilic aggregates and enzymes allow to explore the OG/octanol/water phase diagram. Enzymatic formation of dipalmitoylphosphatidylcholine (DPPC) liposomes and non-ionic surfactant vesicles (NSV), starting from mixed micelles or open structures, finally sets an example of a biomimetic self-evolving system. PMID:9782383

  17. Enzymatic hydrolysis of fractionated products from oil thermally oxidated

    SciTech Connect

    Yashida, H.; Alexander, J.C.

    1983-01-01

    Enzymatic hydrolysis of the acylglycerol products obtained from thermally oxidized vegetable oils was studied. Corn, sunflower and soybean oils were heated in the laboratory at 180/sup 0/C for 50, 70 and 100 hr with aeration and directly fractionated by silicic acid column chromatography. By successive elution with 20%, then 60% isopropyl ether in n-hexane, and diethyl ether, the thermally oxidized oils were separated into three fractions: the nonpolar fraction (monomeric compounds), slightly polar fraction (dimeric compounds), and polar fraction comprising oligomeric compounds. Enzymatic hydrolysis with pancreatic lipase showed that the monomers were hydrolyzed as rapidly as the corresponding unheated oils, the dimers much more slowly, and the oligomeric compounds barely at all. Overall, the hydrolysis of the dimers was less than 23% of that for the monomers, with small differences among the oils. Longer heating periods resulted in greater reductions in hydrolysis of the dimeric compounds. These results suggest that the degree of enzymatic hydrolysis of the fractionated acylglycerol compounds is related to differences in the thermal oxidative deterioration, and amounts of polar compounds in the products. (33 Refs.)

  18. Enzymatic transition states and dynamic motion in barrier crossing

    PubMed Central

    Schwartz, Steven D.; Schramm, Vern L.

    2010-01-01

    What are the atomic motions at enzymatic catalytic sites on the timescale of chemical change? Combined experimental and computational chemistry approaches take advantage of transition-state analogs to reveal dynamic motions linked to transition-state formation. QM/MM transition path sampling from reactive complexes provides both temporal and dynamic information for barrier crossing. Fast (femtosecond to picosecond) dynamic motions provide essential links to enzymatic barrier crossing by local or promoting-mode dynamic searches through bond-vibrational space. Transition-state lifetimes are within the femtosecond timescales of bond vibrations and show no manifestations of stabilized, equilibrated complexes. The slow binding and protein conformational changes (microsecond to millisecond) also required for catalysis are temporally decoupled from the fast dynamic motions forming the transition state. According to this view of enzymatic catalysis, transition states are formed by fast, coincident dynamic excursions of catalytic site elements, while the binding of transition-state analogs is the conversion of the dynamic excursions to equilibrated states. PMID:19620996

  19. 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

  20. Phosphorylation/dephosphorylation of reconstituted shark Na+,K(+)-ATPase: one phosphorylation site per alpha beta protomer.

    PubMed

    Cornelius, F

    1995-05-01

    In the present investigation reconstitution of Na+,K(+)-ATPase increases the number of phosphorylation sites (EP) of solubilized enzyme from 4.2 +/- 0.3 nmol/mg to 6.9 +/- 0.6 nmol/mg. The latter figure corresponds to one phosphorylation site per alpha beta-promoter. A cholesterol content > 10 mol% in the liposome bilayer and a high extracellular [Na+] are necessary to obtain this high value. Spontaneous dephosphorylation after maximum phosphorylation in Na+ is biphasic both in solubilized enzyme and after reconstitution. The rate of dephosphorylation compares with the specific hydrolytic Na(+)-ATPase activity measured at exactly identical conditions for all three preparations assuming parallel dephosphorylation of at least two phosphointermediates. The distribution of EP-species is found to vary among the three enzyme preparation used, i.e., membrane bound, solubilized, and reconstituted Na+,K(+)-ATPase, however in all the equilibrium is strongly poised away from the E1P-form. PMID:7756326

  1. Local modulation of steroid action: rapid control of enzymatic activity

    PubMed Central

    Charlier, Thierry D.; Cornil, Charlotte A.; Patte-Mensah, Christine; Meyer, Laurence; Mensah-Nyagan, A. Guy; Balthazart, Jacques

    2015-01-01

    Estrogens can induce rapid, short-lived physiological and behavioral responses, in addition to their slow, but long-term, effects at the transcriptional level. To be functionally relevant, these effects should be associated with rapid modulations of estrogens concentrations. 17?-estradiol is synthesized by the enzyme aromatase, using testosterone as a substrate, but can also be degraded into catechol-estrogens via hydroxylation by the same enzyme, leading to an increase or decrease in estrogens concentration, respectively. The first evidence that aromatase activity (AA) can be rapidly modulated came from experiments performed in Japanese quail hypothalamus homogenates. This rapid modulation is triggered by calcium-dependent phosphorylations and was confirmed in other tissues and species. The mechanisms controlling the phosphorylation status, the targeted amino acid residues and the reversibility seem to vary depending of the tissues and is discussed in this review. We currently do not know whether the phosphorylation of the same amino acid affects both aromatase and/or hydroxylase activities or whether these residues are different. These processes provide a new general mechanism by which local estrogen concentration can be rapidly altered in the brain and other tissues. PMID:25852459

  2. Local modulation of steroid action: rapid control of enzymatic activity.

    PubMed

    Charlier, Thierry D; Cornil, Charlotte A; Patte-Mensah, Christine; Meyer, Laurence; Mensah-Nyagan, A Guy; Balthazart, Jacques

    2015-01-01

    Estrogens can induce rapid, short-lived physiological and behavioral responses, in addition to their slow, but long-term, effects at the transcriptional level. To be functionally relevant, these effects should be associated with rapid modulations of estrogens concentrations. 17?-estradiol is synthesized by the enzyme aromatase, using testosterone as a substrate, but can also be degraded into catechol-estrogens via hydroxylation by the same enzyme, leading to an increase or decrease in estrogens concentration, respectively. The first evidence that aromatase activity (AA) can be rapidly modulated came from experiments performed in Japanese quail hypothalamus homogenates. This rapid modulation is triggered by calcium-dependent phosphorylations and was confirmed in other tissues and species. The mechanisms controlling the phosphorylation status, the targeted amino acid residues and the reversibility seem to vary depending of the tissues and is discussed in this review. We currently do not know whether the phosphorylation of the same amino acid affects both aromatase and/or hydroxylase activities or whether these residues are different. These processes provide a new general mechanism by which local estrogen concentration can be rapidly altered in the brain and other tissues. PMID:25852459

  3. 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

  4. Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3 proteins converts a dimer into a hexamer

    PubMed Central

    Kanczewska, Justyna; Marco, Sergio; Vandermeeren, Caroline; Maudoux, Olivier; Rigaud, Jean-Louis; Boutry, Marc

    2005-01-01

    Plant plasma membrane H+-ATPases (PMAs) can be activated by phosphorylation of their penultimate residue (a Thr) and the subsequent binding of regulatory 14-3-3 proteins. Although 14-3-3 proteins usually exist as dimers and can bind two targets, the in vivo effects of their binding on the quaternary structure of H+-ATPases have never been examined. To address this question, we used a Nicotiana tabacum cell line expressing the Nicotiana plumbaginifolia PMA2 isoform with a 6-His tag. The purified PMA2 was mainly nonphosphorylated and 14-3-3-free, and it was shown by blue native gel electrophoresis and chemical cross-linking to exist as a dimer. Fusicoccin treatment of the cells resulted in a dramatic increase in Thr phosphorylation, 14-3-3 binding, and in vivo and in vitro ATPase activity, as well as in the conversion of the dimer into a larger, possibly hexameric, complex. PMA2 phosphorylation and 14-3-3 binding were observed also when cells in stationary growth phase were metabolically activated by transfer to fresh medium. When expressed in yeast, PMA2 was also phosphorylated and formed a complex with 14-3-3 proteins without requiring fusicoccin; no complex was observed when phosphorylation was prevented by mutagenesis. Single-particle analysis by cryoelectron microscopy showed that the PMA2–14-3-3 complex is a wheel-like structure with a 6-fold symmetry, suggesting that the activated complex consists of six H+-ATPase molecules and six 14-3-3 molecules. PMID:16081536

  5. 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

  6. Phosphorylation reactions in bovine rod outer segments studied by 32P-labelling of intact retina.

    PubMed

    Kamps, K M; Dratz, E A; Daemen, F J; de Grip, W J

    1986-04-11

    The protein phosphorylation pattern in the intact bovine retina has been investigated by labelling with 32P-phosphate under incubation conditions that preserve the electrical photoresponse of the photoreceptor cells. The phosphorylation of rod outer segment proteins was analysed after isolation of outer segments from the labelled retina. The global influence of light, Ca2+ and the phosphodiesterase inhibitor, isobutylmethylxanthine, on protein phosphorylation in rod outer segments was analysed. A 12 kDa protein is the most prominent phosphorylated species in the intact bovine retina. Its phosphorylation is increased by light and/or Ca2+. Evidence is presented that this strongly phosphorylated protein is not located in the outer segment, and we suggest that it may be a synaptic protein. Retinal rod outer segment membrane proteins with apparent molecular weights of 245, 226, 125, 110, 50, 46, 38 and 20 all show light-stimulated phosphorylation. Lowering the extracellular Ca2+ levels results in a decrease of the phosphorylation level of some of these proteins, viz. at 125, 50, 38 and probably at 20 kDa. Such proteins, whose phosphorylation level is influenced both by light and by elevated Ca2+, are candidates for mediators of phototransduction. The phosphorylated species at 245, 226, 110, 50 and 20 kDa are enriched in rod outer segment plasma membrane preparations. These protein species could participate in the light-regulated modulation of the Na+-conductance of the plasma membrane. PMID:2420366

  7. Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

    PubMed Central

    Safran, A; Neumann, D; Fuchs, S

    1986-01-01

    Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms. Images Fig. 2. Fig. 4. Fig. 5. PMID:3816758

  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. Changes in the phosphorylation of claudins during the course of experimental colitis

    PubMed Central

    Li, Jing; Li, Yong-Xiang; Chen, Mei-Hua; Li, Jie; Du, Juan; Shen, Bing; Xia, Xian-Ming

    2015-01-01

    The phosphorylation of the tight-junction protein claudin causes allosterism, endocytosis and changes in the polarity of the epithelium, thus affecting the barrier function. The phosphorylation status of claudin during the course of colitis has not been demonstrated. In the present study, we found that the phosphorylated claudin-4 and claudin-7 contents were increased in experimental colitis at days 6 and 8, and colonic phosphorylated claudin-6 was found to be increased at day 4 and day 8. Colonic phosphorylated claudin-5 was found to be decreased at day 4 but increased at day 6. These changes were accompanied by increases in intestinal permeability. In T84 cells, phosphorylated claudin-3 was increased at 48 h but decreased at 72 h after lipopolysaccharide (LPS) treatment. Phosphorylated claudin-5 and claudin-7 were decreased 72 h after LPS treatment, while phosphorylated claudin-6 was increased at 72 h after LPS treatment. We conclude that the phosphorylation of colonic claudins was changed during the course of colitis, which may be related to the change in the intestinal barrier function. Cytokine such as LPS was found to affect the phosphorylation of colonic claudins. PMID:26722407

  10. dbPSP: a curated database for protein phosphorylation sites in prokaryotes.

    PubMed

    Pan, Zhicheng; Wang, Bangshan; Zhang, Ying; Wang, Yongbo; Ullah, Shahid; Jian, Ren; Liu, Zexian; Xue, Yu

    2015-01-01

    As one of the most important post-translational modifications, phosphorylation is highly involved in almost all of biological processes through temporally and spatially modifying substrate proteins. Recently, phosphorylation in prokaryotes attracted much attention for its critical roles in various cellular processes such as signal transduction. Thus, an integrative data resource of the prokaryotic phosphorylation will be useful for further analysis. In this study, we presented a curated database of phosphorylation sites in prokaryotes (dbPSP, Database URL: http://dbpsp.biocuckoo.org) for 96 prokaryotic organisms, which belong to 11 phyla in two domains including bacteria and archaea. From the scientific literature, we manually collected experimentally identified phosphorylation sites on seven types of residues, including serine, threonine, tyrosine, aspartic acid, histidine, cysteine and arginine. In total, the dbPSP database contains 7391 phosphorylation sites in 3750 prokaryotic proteins. With the dataset, the sequence preferences of the phosphorylation sites and functional annotations of the phosphoproteins were analyzed, while the results shows that there were obvious differences among the phosphorylation in bacteria, archaea and eukaryotes. All the phosphorylation sites were annotated with original references and other descriptions in the database, which could be easily accessed through user-friendly website interface including various search and browse options. Taken together, the dbPSP database provides a comprehensive data resource for further studies of protein phosphorylation in prokaryotes. Database URL: http://dbpsp.biocuckoo.org PMID:25841437

  11. Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1.

    PubMed

    Alessi, D R; Saito, Y; Campbell, D G; Cohen, P; Sithanandam, G; Rapp, U; Ashworth, A; Marshall, C J; Cowley, S

    1994-04-01

    Many growth factors whose receptors are protein tyrosine kinases stimulate the MAP kinase pathway by activating first the GTP-binding protein Ras and then the protein kinase p74raf-1. p74raf-1 phosphorylates and activates MAP kinase kinase (MAPKK). To understand the mechanism of activation of MAPKK, we have identified Ser217 and Ser221 of MAPKK1 as the sites phosphorylated by p74raf-1. This represents the first characterization of sites phosphorylated by this proto-oncogene product. Ser217 and Ser221 lie in a region of the catalytic domain where the activating phosphorylation sites of several other protein kinases are located. Among MAPKK family members, this region is the most conserved, suggesting that all members of the family are activated by the phosphorylation of these sites. A 'kinase-dead' MAPKK1 mutant was phosphorylated at the same residues as the wild-type enzyme, establishing that both sites are phosphorylated directly by p74raf-1, and not by autophosphorylation. Only the diphosphorylated form of MAPKK1 (phosphorylated at both Ser217 and Ser221) was detected, even when the stoichiometry of phosphorylation by p74raf-1 was low, indicating that phosphorylation of one of these sites is rate limiting, phosphorylation of the second then occurring extremely rapidly. Ser217 and Ser221 were both phosphorylated in vivo within minutes when PC12 cells were stimulated with nerve growth factor. Analysis of MAPKK1 mutants in which either Ser217 or Ser221 were changed to glutamic acid, and the finding that inactivation of maximally activated MAPKK1 required the dephosphorylation of both serines, shows that phosphorylation of either residue is sufficient for maximal activation. PMID:8157000

  12. Technology Transfer

    NASA Technical Reports Server (NTRS)

    Smith, Nanette R.

    1995-01-01

    The objective of this summer's work was to attempt to enhance Technology Application Group (TAG) ability to measure the outcomes of its efforts to transfer NASA technology. By reviewing existing literature, by explaining the economic principles involved in evaluating the economic impact of technology transfer, and by investigating the LaRC processes our William & Mary team has been able to lead this important discussion. In reviewing the existing literature, we identified many of the metrics that are currently being used in the area of technology transfer. Learning about the LaRC technology transfer processes and the metrics currently used to track the transfer process enabled us to compare other R&D facilities to LaRC. We discuss and diagram impacts of technology transfer in the short run and the long run. Significantly, it serves as the basis for analysis and provides guidance in thinking about what the measurement objectives ought to be. By focusing on the SBIR Program, valuable information regarding the strengths and weaknesses of this LaRC program are to be gained. A survey was developed to ask probing questions regarding SBIR contractors' experience with the program. Specifically we are interested in finding out whether the SBIR Program is accomplishing its mission, if the SBIR companies are providing the needed innovations specified by NASA and to what extent those innovations have led to commercial success. We also developed a survey to ask COTR's, who are NASA employees acting as technical advisors to the SBIR contractors, the same type of questions, evaluating the successes and problems with the SBIR Program as they see it. This survey was developed to be implemented interactively on computer. It is our hope that the statistical and econometric studies that can be done on the data collected from all of these sources will provide insight regarding the direction to take in developing systematic evaluations of programs like the SBIR Program so that they can reach their maximum effectiveness.

  13. Transfer Investigational Agent Form

    Cancer.gov

    10/02 Transfer Investigational Agent Form This form is to be used for an intra-institutional transfer, one transfer/form. Division of Cancer Prevention National Cancer Institute National Institutes of Health TRANSFER FROM: Investigator transferring agent:

  14. Investigation of the enzymatic digestion of plant cell walls using reflectance Fourier Transform Infrared spectroscopy.

    PubMed

    Hopkinson, J; Moustou, C; Charlwood, K A; Newbery, J E; Charlwood, B V

    1985-12-01

    A method has been developed to determine the reflectance Fourier Transform Infrared spectra of plant cells grown in vitro and of the protoplasts released from such cells by enzymatic digestion. It is demonstrated that there is a smooth and reproducible transition in spectral detail as enzymatic digestion procedes. Reflectance Fourier Transform Infrared spectroscopy has been used to monitor the progress of protoplast release during enzymatic digestion of cell wall material. PMID:24254073

  15. Injectable hydrogels derived from phosphorylated alginic acid calcium complexes.

    PubMed

    Kim, Han-Sem; Song, Minsoo; Lee, Eun-Jung; Shin, Ueon Sang

    2015-06-01

    Phosphorylation of sodium alginate salt (NaAlg) was carried out using H3PO4/P2O5/Et3PO4 followed by acid-base reaction with Ca(OAc)2 to give phosphorylated alginic acid calcium complexes (CaPAlg), as a water dispersible alginic acid derivative. The modified alginate derivatives including phosphorylated alginic acid (PAlg) and CaPAlg were characterized by nuclear magnetic resonance spectroscopy for (1)H, and (31)P nuclei, high resolution inductively coupled plasma optical emission spectroscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. CaPAlg hydrogels were prepared simply by mixing CaPAlg solution (2w/v%) with NaAlg solution (2w/v%) in various ratios (2:8, 4:6, 6:4, 8:2) of volume. No additional calcium salts such as CaSO4 or CaCl2 were added externally. The gelation was completed within about 3-40min indicating a high potential of hydrogel delivery by injection in vivo. Their mechanical properties were tested to be ?6.7kPa for compressive strength at break and about 8.4kPa/mm for elastic modulus. SEM analysis of the CaPAlg hydrogels showed highly porous morphology with interconnected pores of width in the range of 100-800?m. Cell culture results showed that the injectable hydrogels exhibited comparable properties to the pure alginate hydrogel in terms of cytotoxicity and 3D encapsulation of cells for a short time period. The developed injectable hydrogels showed suitable physicochemical and mechanical properties for injection in vivo, and could therefore be beneficial for the field of soft tissue engineering. PMID:25842118

  16. Further studies on phosphorylated pituitary somatotropin (growth hormone)

    SciTech Connect

    Kornberg, L.J.; Liberti, J.P.

    1987-05-01

    This laboratory made the original observation that naturally-occurring ovine growth hormone (GH) is phosphorylated and that slices of pituitary glands from male rats synthesize and secrete /sup 32/P-GH. This observation has been extended to explore the generality of this process. After incubation in PO/sub 4/-free Ham's F-10 medium (PFH) or in saline/Hepes (SH) containing 300..mu..Ci /sup 32/Pi/mL, tissue and medium were separated and a cell extract was prepared. GH in the medium and extract was recovered by immunoprecipitation using rat GH antiserum. The samples were electrophoresed under denaturating conditions and processed for autoradiography. /sup 32/P-GH was characterized by the presence of a protein-staining band and radioactive area which migrated the same as authentic GH and /sup 125/I-GH. Slices of glands from male rats incubated for 2h in PFH secreted /sup 32/P-GH. Similar results were found upon incubation of slices from female rats in the presence of SH. Short-term incubations of acutely dispersed pituitary cells obtained from young and old male rats also synthesized and secreted /sup 32/P-GH. Thus, the production of /sup 32/P-GH occurs (a) in simple and complex incubaton media, (b) in slices and cells from glands from older and younger rats and (c) in female as well as male rats. Therefore, phosphorylation of GH appears to be a general phenomenon. The physiological action(s) of phosphorylated GH in growth and development is under study.

  17. Protein phosphorylation profiling identifies potential mechanisms for direct immunotoxicity.

    PubMed

    Shao, Jia; Stout, Inge; Hendriksen, Peter J M; van Loveren, Henk; Peijnenburg, Ad A C M; Volger, Oscar L

    2016-01-01

    Signaling networks are essential elements that are involved in diverse cellular processes. One group of fundamental components in various signaling pathways concerns protein tyrosine kinases (PTK). Various toxicants have been demonstrated to exert their toxicity via modulation of tyrosine kinase activity. The present study aimed to identify common cellular signaling pathways that are involved in chemical-induced direct immunotoxicity. To this end, an antibody array-based profiling approach was applied to assess effects of five immunotoxicants, two immunosuppressive drugs and two non-immunotoxic control chemicals on the phosphorylation of 28 receptor tyrosine kinases and 11 crucial signaling nodes in Jurkat T-cells. The phosphorylation of ribosomal protein S6 (RPS6) and of kinases Akt, Src and p44/42 were found to be commonly regulated by immunotoxicants and/or immunosuppressive drugs (at least three compounds), with the largest effect observed upon RPS6. Flow cytometry and Western blotting were used to further examine the effect of the model immunotoxicant TBTO on the components of the mTOR-p70S6K-RPS6 pathway. These analyses revealed that both TBTO and the mTOR inhibitor rapamycin inactivate RPS6, but via different mechanisms. Finally, a comparison of the protein phosphorylation data to previously obtained transcriptome data of TBTO-treated Jurkat cells resulted in a good correlation at the pathway level and indicated that TBTO affects ribosome biogenesis and leukocyte migration. The effect of TBTO on the latter process was confirmed using a CXCL12 chemotaxis assay. PMID:25715851

  18. Functionalization of monolithic and porous three-dimensional graphene by one-step chitosan electrodeposition for enzymatic biosensor.

    PubMed

    Liu, Jiyang; Wang, Xiaohui; Wang, Tianshu; Li, Dan; Xi, Fengna; Wang, Jin; Wang, Erkang

    2014-11-26

    Biological modification of monolithic and porous 3D graphene is of great significance for extending its application in fabricating highly sensitive biosensors. The present work reports on the first biofunctionalization of monolithic and freestanding 3D graphene foam for one-step preparation of reagentless enzymatic biosensors by controllable chitosan (CS) electrodeposition technology. Using a homogeneous three-component electrodeposition solution containing a ferrocene (Fc) grafted CS hybrid (Fc-CS), glucose oxidase (GOD), and single-walled carbon nanotubes (SWNTs), a homogeneous biocomposite film of Fc-CS/SWNTs/GOD was immobilized on the surface of 3D graphene foam by one-step electrodeposition. The Fc groups grafted on chitosan can be stably immobilized on the 3D graphene surface and keep their original electrochemical activity. The SWNTs doped into the Fc-CS matrix act as a nanowire to facilitate electron transfer and improve the conductivity of the biocomposite film. Combined with the extraordinary properties of 3D graphene foam including large active surface area, high conductivity, and fast mass transport dynamics, the 3D graphene based enzymatic biosensor achieved a large linear range (5.0 ?M to 19.8 mM), a low detection limit (1.2 ?M), and rapid response (reaching the 95% steady-state response within 8 s) for reagentless detection of glucose in the phosphate buffer solution. PMID:25384251

  19. Functional conservation of phosphorylation-specific prolyl isomerases in plants.

    PubMed

    Yao, J L; Kops, O; Lu, P J; Lu, K P

    2001-04-27

    The phosphorylation-specific peptidyl prolyl cis/trans isomerase (PPIase) Pin1 in humans and its homologues in yeast and animal species play an important role in cell cycle regulation. These PPIases consist of an NH(2)-terminal WW domain that binds to specific phosphoserine- or phosphothreonine-proline motifs present in a subset of phosphoproteins and a COOH-terminal PPIase domain that specifically isomerizes the phosphorylated serine/threonine-proline peptide bonds. Here, we describe the isolation of MdPin1, a Pin1 homologue from the plant species apple (Malus domestica) and show that it has the same phosphorylation-specific substrate specificity and can be inhibited by juglone in vitro, as is the case for Pin1. A search in the plant expressed sequence tag data bases reveals that the Pin1-type PPIases are present in various plants, and there are multiple genes in one organism, such as soybean (Glycine max) and tomato (Lycopersicon esculentum). Furthermore, all these plant Pin1-type PPIases, including AtPin1 in Arabidopsis thaliana, do not have a WW domain, but all contain a four-amino acid insertion next to the phospho-specific recognition site of the active site. Interestingly, like Pin1, both MdPin1 and AtPin1 are able to rescue the lethal mitotic phenotype of a temperature-sensitive mutation in the Pin1 homologue ESS1/PTF1 gene in Saccharomyces cerevisiae. However, deleting the extra four amino acid residues abolished the ability of AtPin1 to rescue the yeast mutation under non-overexpression conditions, indicating that these extra amino acids may be important for mediating the substrate interaction of plant enzymes. Finally, expression of MdPin1 is tightly associated with cell division both during apple fruit development in vivo and during cell cultures in vitro. These results have demonstrated that phosphorylation-specific PPIases are highly conserved functionally in yeast, animal, and plant species. Furthermore, the experiments suggest that although plant Pin1-type enzymes do not have a WW domain, they may fulfill the same functions as Pin1 and its homologues do in other organisms. PMID:11118438

  20. Protein Ser/Thr/Tyr Phosphorylation in the Archaea*

    PubMed Central

    Kennelly, Peter J.

    2014-01-01

    The third domain of life, the Archaea (formerly Archaebacteria), is populated by a physiologically diverse set of microorganisms, many of which reside at the ecological extremes of our global environment. Although ostensibly prokaryotic in morphology, the Archaea share much closer evolutionary ties with the Eukarya than with the superficially more similar Bacteria. Initial genomic, proteomic, and biochemical analyses have revealed the presence of “eukaryotic” protein kinases and phosphatases and an intriguing set of serine-, threonine-, and tyrosine-phosphorylated proteins in the Archaea that may offer new insights into this important regulatory mechanism. PMID:24554702

  1. Protein phosphorylation cascades associated with methamphetamine-induced glial activation.

    PubMed

    Hebert, M A; O'Callaghan, J P

    2000-09-01

    Reactive gliosis is the most prominent response to diverse forms of central nervous system (CNS) injury. The signaling events that mediate this characteristic response to neural injury are under intense investigation. Several studies have demonstrated the activation of phosphoproteins within the mitogen-activated protein kinase (MAPK) and Janus kinase (JAK) pathways following neural insult. These signaling pathways may be involved or responsible for the glial response following injury, by virtue of their ability to phosphorylate and dynamically regulate the activity of various transcription factors. This study sought to delineate, in vivo, the relative contribution of MAPK- and JAK-signaling components to reactive gliosis as measured by induction of glial-fibrillary acidic protein (GFAP), following chemical-induced neural damage. At time points (6, 24, and 48 h) following methamphetamine (METH, 10 mg/kg x 4, s.c.) administration, female C57BL/6J mice were sacrificed by focused microwave irradiation, a technique that preserves steady-state phosphorylation. Striatal (target) and nontarget (hippocampus) homogenates were assayed for METH-induced changes in markers of dopamine (DA) neuron integrity as well as differences in the levels of activated phosphoproteins. GFAP upregulation occurred as early as 6 h, reaching a threefold induction 48 h following METH exposure. Neurotoxicant-induced reductions in striatal levels of DA and tyrosine hydroxylase (TH) paralleled the temporal profile of GFAP induction. Blots of striatal homogenates, probed with phosphorylation-state specific antibodies, demonstrated significant changes in activated forms of extracellular-regulated kinase 1/2 (ERK 1/2), c-jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), MAPK/ERK kinase (MEK1/2), 70-kDa ribosomal S6 kinase (p70 S6), cAMP responsive element binding protein (CREB), and signal transducer and activator of transcription 3 (STAT3). MAPK-related phosphoproteins exhibited an activation profile that peaked at 6 h, remained significantly increased at 24, and fell to baseline levels 48 h following neurotoxicant treatment. The ribosomal S6 kinase was enhanced over 60% for all time points examined. Immunoreactivity profiles for the transcription factors CREB and STAT3 indicated maximal increases in phosphorylation occurring at 24 h, and measuring greater than 2- or 17-fold, respectively. Specific signaling events were found to occur with a time course suggestive of their involvement in the gliotic response. The toxicant-induced activation of these growth-associated signaling cascades suggests that these pathways could be obligatory for the triggering and/or persistence of reactive gliosis and may therefore serve as potential targets for modulation of glial response to neural damage. PMID:11085325

  2. Phosphorylation energy hypothesis: open chemical systems and their biological functions.

    PubMed

    Qian, Hong

    2007-01-01

    Biochemical systems and processes in living cells generally operate far from equilibrium. This review presents an overview of a statistical thermodynamic treatment for such systems, with examples from several key components in cellular signal transduction. Open-system nonequilibrium steady-state (NESS) models are introduced. The models account quantitatively for the energetics and thermodynamics in phosphorylation-dephosphorylation switches, GTPase timers, and specificity amplification through kinetic proofreading. The chemical energy derived from ATP and GTP hydrolysis establishes the NESS of a cell and makes the cell--a mesoscopic-biochemical reaction system that consists of a collection of thermally driven fluctuating macromolecules--a genetically programmed chemical machine. PMID:17059360

  3. Phosphorylation Energy Hypothesis: Open Chemical Systems and Their Biological Functions

    NASA Astrophysics Data System (ADS)

    Qian, Hong

    2007-05-01

    Biochemical systems and processes in living cells generally operate far from equilibrium. This review presents an overview of a statistical thermodynamic treatment for such systems, with examples from several key components in cellular signal transduction. Open-system nonequilibrium steady-state (NESS) models are introduced. The models account quantitatively for the energetics and thermodynamics in phosphorylation-dephosphorylation switches, GTPase timers, and specificity amplification through kinetic proofreading. The chemical energy derived from ATP and GTP hydrolysis establishes the NESS of a cell and makes the cell—a mesoscopic-biochemical reaction system that consists of a collection of thermally driven fluctuating macromolecules—a genetically programmed chemical machine.

  4. Simple rapid method for gene transfer

    SciTech Connect

    Cockburn, A.F.; Meier, H.

    1990-01-30

    The object of the present invention is to provide methods for gene transfer that reduce or eliminate cellular pretreatment steps, e.g., the removal of cell wall by chemical or enzymatic methods, is rapid and can be practiced without the need of additional expensive equipment. Cells, embryos or tissues selected for genetic manipulation are suspended in an Eppendorf tube in an aliquot of the desired genetic material to be transferred to which the resulting mixture is added and is agitated by vortexing from about 30 to about 90 seconds. The cells, embryos or tissue are sedimented and the DNA supernatant removed. After sedimentation, the injected material is resuspended in or on a growth medium to assay for expression.

  5. Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices

    SciTech Connect

    Stratton, K.R.; Worley, P.F.; Huganir, R.L.; Baraban, J.M. )

    1989-04-01

    The authors have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment of intact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine- and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipid system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation.

  6. Auxin-regulated changes in protein phosphorylation in pea epicotyl segments

    SciTech Connect

    Reddy, A.S.N.; Chengappa, S.; Raghothama, K.G.; Poovaiah, B.W.

    1987-04-01

    Auxin-regulated changes in protein phosphorylation were studied by labeling pea epicotyl segments with (/sup 32/P) PO/sub 4//sup 3 -/ and analyzing the phosphoproteins by two dimensional (2-D) gel electrophoresis. Analysis of phosphoproteins revealed auxin-regulated changes in the phosphorylation of specific polypeptides. In the presence of auxin, phosphorylation of 23,000, 82,000, 105,000 and 110,000 molecular weight polypeptides was markedly decreased whereas phosphorylation of 19,000, 24,000, 28,000 molecular weight polypeptides was increased. Some of these changes are very rapid and could be observed within minutes. Furthermore, their studies with calmodulin antagonists indicate the possible involvement of calmodulin-dependent protein kinases and/or phosphatases in auxin-regulated changes in protein phosphorylation. In view of these results, they suggest that auxin-regulated protein phosphorylation could be the one of the earliest events in regulating diverse physiological processes by this hormone.

  7. Systematic profiling of the bacterial phosphoproteome reveals bacterium-specific features of phosphorylation.

    PubMed

    Lin, Miao-Hsia; Sugiyama, Naoyuki; Ishihama, Yasushi

    2015-09-15

    Protein phosphorylation is a crucial posttranslational modification for regulating cellular processes in bacteria; however, it has not been extensively studied because of technical difficulties in the enrichment of phosphopeptides. We devised an enrichment protocol that enabled the identification of >1000 phosphopeptides from a single bacterial sample. We discovered three high-confidence serine and threonine phosphorylation motifs, as well as 29 other motifs at various levels of confidence, from three distinct bacterial phosphoproteomes. We found that the proline-directed and basophilic phosphorylation motifs that are commonly enriched in eukaryotes were not observed in bacteria. Unlike eukaryotes, bacteria had a low occurrence of both phosphorylation and acetylation in N-terminal phosphopeptides. Because infection of host cells by bacterial pathogens is often accompanied by kinase-mediated phosphorylation events, the differences in phosphorylation preferences between bacteria and eukaryotes revealed by this study could be useful in identifying bacterial-specific targets for future therapies. PMID:26373674

  8. Rapid Identification of Protein Kinase Phosphorylation Site Motifs Using Combinatorial Peptide Libraries.

    PubMed

    Miller, Chad J; Turk, Benjamin E

    2016-01-01

    Eukaryotic protein kinases phosphorylate substrates at serine, threonine, and tyrosine residues that fall within the context of short sequence motifs. Knowing the phosphorylation site motif for a protein kinase facilitates designing substrates for kinase assays and mapping phosphorylation sites in protein substrates. Here, we describe an arrayed peptide library protocol for rapidly determining kinase phosphorylation consensus sequences. This method uses a set of peptide mixtures in which each of the 20 amino acid residues is systematically substituted at nine positions surrounding a central site of phosphorylation. Peptide mixtures are arrayed in multiwell plates and analyzed by radiolabel assay with the kinase of interest. The preferred sequence is determined from the relative rate of phosphorylation of each peptide in the array. Consensus peptides based on these sequences typically serve as efficient and specific kinase substrates for high-throughput screening or incorporation into biosensors. PMID:26501912

  9. Crystal Structure of a Phosphorylated Light Chain Domain of Scallop Smooth-Muscle Myosin

    SciTech Connect

    Kumar, V.S.; Robinson, H.; O-Neall-Hennessey, E.; Reshetnikova, L.; Brown, J. H.; Szent-Gyorgyi, A. G.; Cohen, C.

    2011-11-02

    We have determined the crystal structure of a phosphorylated smooth-muscle myosin light chain domain (LCD). This reconstituted LCD is of a sea scallop catch muscle myosin with its phosphorylatable regulatory light chain (RLC SmoA). In the crystal structure, Arg{sup 16}, an arginine residue that is present in this isoform but not in vertebrate smooth-muscle RLC, stabilizes the phosphorylation site. This arginine interacts with the carbonyl group of the phosphorylation-site serine in the unphosphorylated LCD (determined previously), and with the phosphate group when the serine is phosphorylated. However, the overall conformation of the LCD is essentially unchanged upon phosphorylation. This result provides additional evidence that phosphorylation of the RLC is unlikely to act as an on-switch in regulation of scallop catch muscle myosin.

  10. The Effects of Surfactant Pretreatment and Xylooligomers on Enzymatic Hydrolysis of Cellulose and Pretreated Biomass

    E-print Network

    Qing, Qing

    2010-01-01

    Maize Cell Walls Following Thermochemical Pretreatment. Biotechnology andMaize Cell Walls Following Thermochemical Pretreatment. Biotechnology andmaize stems retards enzymatic hydrolysis of cellulose. Biotechnology

  11. Enzymatic production of lactulose and epilactose in milk.

    PubMed

    Rentschler, Eva; Schuh, Katharina; Krewinkel, Manuel; Baur, Claudia; Claaßen, Wolfgang; Meyer, Susanne; Kuschel, Beatrice; Stressler, Timo; Fischer, Lutz

    2015-10-01

    The enzymatic production of lactulose was described recently through conversion of lactose by a thermophilic cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE). In the current study, we examined the application of CsCE for lactulose and epilactose production in milk (1.5% fat). The bioconversions were carried out in stirred reaction vessels at 2 different temperatures (50 and 8°C) at a scale of 25 mL volume. At 50°C, 2 highly different CsCE amounts were investigated for the time course of formation of lactulose and epilactose. The conversion of milk lactose (initial lactose content of 48.5±2.1 g/L) resulted in a final yield of 57.7% (28.0 g/L) lactulose and 15.5% (7.49 g/L) epilactose in the case of the approximately 9.5-fold higher CsCE amount (39.5 µkatepilactose, 50°C) after 24 h. Another enzymatic lactose conversion was carried out at low 8°C, an industrially relevant temperature for milk processing. Although the CsCE originated from a thermophilic microorganism, it was still applicable at 8°C. This enzymatic lactose conversion resulted in 56.7% (27.5 g/L) lactulose and 13.6% (6.57 g/L) epilactose from initial milk lactose after 72 h. The time courses of lactose conversion by CsCE suggested that first epilactose formed and afterward lactulose via epilactose. To the best of our knowledge, this is the first time that an enzyme has produced lactulose directly in milk in situ at industrially relevant temperatures. PMID:26254523

  12. 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

  13. 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].

  14. Study on sterilization and storage of enzymatic preparations by irradiation

    NASA Astrophysics Data System (ADS)

    Dengyi, Xu; Guangzheng, Liu; Yunsen, Long; Xiaoping, Liu; Fengxin, Yi; Xiaqdong, Zhao

    1993-10-01

    Effects of irradiation with cobalt-60 ?-rays on sterilization, storage and enzymes activity of four kinds of biological enzymatic preparations, namely trypsin, pepsin, amylase and liquid carbohydrase, were investigated. The results showed that these enzymes have different sensitivity to irradiation, amylase being the most sensitive. The enzyme activity and enzyme effect of amylase were lowered with irradiation dose higher than 7 KGY. With irradiation doses between 7-10 KGY, enzyme activity of trypsin and pepsin were stable. Liquid carbohydrase can be stored more than half a year under a natural temperature with a stable enzyme activity without molding.

  15. Non-enzymatic glucose detection using magnetic nanoemulsions

    NASA Astrophysics Data System (ADS)

    Mahendran, V.; Philip, John

    2014-09-01

    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.

  16. Enzymatic transformation of stevioside using a ?-galactosidase from Sulfolobus sp.

    PubMed

    Wan, Hui-da; Xia, Yong-Mei

    2015-10-01

    Enzymatic hydrolysis and transgalactosylation of stevioside (St) were investigated using a ?-galactosidase from Sulfolobus sp. The hydrolysis yielded steviol as the main final product. Under the optimal transgalactosylation conditions, the highest conversion of stevioside was 87.3% with lactose as a donor, several galactosylated products (St-Gals) were obtained. Metal ions such as Na(+), K(2+), Ca(2+), Ba(2+), Mn(2+) and Mg(2+) (2 mM) did not affect the transgalactosylation activity, while Fe(2+), Fe(3+) and Cu(2+) reduced the transgalactosylation activity of ?-galactosidase to 64%, 33% and 18%, respectively. PMID:26242384

  17. Evaluation of pretreatments for enzymatic conversion of agricultural residues

    SciTech Connect

    Fan, L.T.; Gharpuray, M.M.; Lee, Y.H.

    1981-01-01

    The effectiveness of various physical and chemical pretreatments on the enzymatic digestibility of wheat straw was evaluated. Structural features, including crystallinity and lignin content, were measured after pretreatments. Several promising pretreatments were identified. In general, chemical pretreatments were more effective than physical pretreatments. Although the hydrolysis rate was dependent appreciably on the crystallinity index, lignin content of the substrate appeared predominant. The hydrolysis rate increased with an increase in the degree of delignification only up to 50%; it remained unchanged thereafter. Scanning electron microscopic observations were used to facilitate further insight into structural modification of the substrate. A preliminary cost analysis was conducted.

  18. Non-enzymatic glucose detection using magnetic nanoemulsions

    SciTech Connect

    Mahendran, V.; Philip, John

    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.

  19. p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts.

    PubMed

    Furukawa, Fukiko; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yoshida, Katsunori; Sugano, Yasushi; Yamagata, Hideo; Matsushita, Masanori; Seki, Toshihito; Inagaki, Yutaka; Nishizawa, Mikio; Fujisawa, Junichi; Inoue, Kyoichi

    2003-10-01

    Hepatic stellate cells (HSCs) spontaneously transdifferentiate into myofibroblast (MFB)-phenotype on plastic dishes. This response recapitulates the features of activation in vivo. Transforming growth factor beta (TGF-beta) plays a prominent role in stimulating liver fibrogenesis by MFBs. In quiescent HSCs, TGF-beta signaling involves TGF-beta type I receptor (TbetaRI)-mediated phosphorylation of serine residues within the conserved SSXS motif at the C-terminus of Smad2 and Smad3. The middle linker regions of Smad2 and Smad3 also are phosphorylated by mitogen-activated protein kinase (MAPK). This study elucidates the change of Smad3-mediated signals during the transdifferentiation process. By using antibodies highly specific to the phosphorylated C-terminal region and the phosphorylated linker region of Smad3, we found that TGF-beta-dependent Smad3 phosphorylation at the C-terminal region decreased, but that the phosphorylation at the linker region increased in the process of transdifferentiation. TGF-beta activated the p38 MAPK pathway, further leading to Smad3 phosphorylation at the linker region in the cultured MFBs, irrespective of Smad2. The phosphorylation promoted hetero-complex formation and nuclear translocation of Smad3 and Smad4. Once combined with TbetaRI-phosphorylated Smad2, the Smad3 and Smad4 complex bound to plasminogen activator inhibitor-type I promoter could enhance the transcription. In addition, Smad3 phosphorylation mediated by the activated TbetaRI was impaired severely in MFBs during chronic liver injury, whereas Smad3 phosphorylation at the linker region was remarkably induced by p38 MAPK pathway. In conclusion, p38 MAPK-dependent Smad3 phosphorylation promoted extracellular matrix production in MFBs both in vitro and in vivo. PMID:14512875

  20. Estrogen receptor alpha phosphorylation and its functional impact in human breast cancer.

    PubMed

    Anbalagan, Muralidharan; Rowan, Brian G

    2015-12-15

    Estrogen receptor ? (ER?) is a member of the nuclear receptor superfamily of transcription factors that regulates cell proliferation, differentiation and homeostasis in various tissues. Sustained exposure to estrogen/estradiol (E2) increases the risk of breast, endometrial and ovarian cancers. ER? function is also regulated by phosphorylation through various kinase signaling pathways that will impact various ER? functions including chromatin interaction, coregulator recruitment and gene expression, as well impact breast tumor growth/morphology and breast cancer patient response to endocrine therapy. However, many of the previously characterized ER? phosphorylation sites do not fully explain the impact of receptor phosphorylation on ER? function. This review discusses work from our laboratory toward understanding a role of ER? site-specific phosphorylation in ER? function and breast cancer. The key findings discussed in this review are: (1) the effect of site specific ER? phosphorylation on temporal recruitment of ER? and unique coactivator complexes to specific genes; (2) the impact of stable disruption of ER? S118 and S167 phosphorylation in breast cancer cells on eliciting unique gene expression profiles that culminate in significant effects on breast cancer growth/morphology/migration/invasion; (3) the Src kinase signaling pathway that impacts ER? phosphorylation to alter ER? function; and (4) circadian disruption by light exposure at night leading to elevated ERK1/2 and Src kinase and phosphorylation of ER?, concomitant with tamoxifen resistance in breast tumor models. Results from these studies demonstrate that even changes to single ER? phosphorylation sites can have a profound impact on ER? function in breast cancer. Future work will extend beyond single site phosphorylation analysis toward identification of specific patterns/profiles of ER? phosphorylation under different physiological/pharmacological conditions to understand how common phosphorylation profiles in breast cancer program specific physiological endpoints such as growth, apoptosis, migration/invasion, and endocrine therapy response. PMID:25597633

  1. Musite, a tool for global prediction of general and kinase-specific phosphorylation sites.

    PubMed

    Gao, Jianjiong; Thelen, Jay J; Dunker, A Keith; Xu, Dong

    2010-12-01

    Reversible protein phosphorylation is one of the most pervasive post-translational modifications, regulating diverse cellular processes in various organisms. High throughput experimental studies using mass spectrometry have identified many phosphorylation sites, primarily from eukaryotes. However, the vast majority of phosphorylation sites remain undiscovered, even in well studied systems. Because mass spectrometry-based experimental approaches for identifying phosphorylation events are costly, time-consuming, and biased toward abundant proteins and proteotypic peptides, in silico prediction of phosphorylation sites is potentially a useful alternative strategy for whole proteome annotation. Because of various limitations, current phosphorylation site prediction tools were not well designed for comprehensive assessment of proteomes. Here, we present a novel software tool, Musite, specifically designed for large scale predictions of both general and kinase-specific phosphorylation sites. We collected phosphoproteomics data in multiple organisms from several reliable sources and used them to train prediction models by a comprehensive machine-learning approach that integrates local sequence similarities to known phosphorylation sites, protein disorder scores, and amino acid frequencies. Application of Musite on several proteomes yielded tens of thousands of phosphorylation site predictions at a high stringency level. Cross-validation tests show that Musite achieves some improvement over existing tools in predicting general phosphorylation sites, and it is at least comparable with those for predicting kinase-specific phosphorylation sites. In Musite V1.0, we have trained general prediction models for six organisms and kinase-specific prediction models for 13 kinases or kinase families. Although the current pretrained models were not correlated with any particular cellular conditions, Musite provides a unique functionality for training customized prediction models (including condition-specific models) from users' own data. In addition, with its easily extensible open source application programming interface, Musite is aimed at being an open platform for community-based development of machine learning-based phosphorylation site prediction applications. Musite is available at http://musite.sourceforge.net/. PMID:20702892

  2. Exosome-associated Tau Is Secreted in Tauopathy Models and Is Selectively Phosphorylated in Cerebrospinal Fluid in Early Alzheimer Disease*

    PubMed Central

    Saman, Sudad; Kim, WonHee; Raya, Mario; Visnick, Yvonne; Miro, Suhad; Saman, Sarmad; Jackson, Bruce; McKee, Ann C.; Alvarez, Victor E.; Lee, Norman C. Y.; Hall, Garth F.

    2012-01-01

    Recent demonstrations that the secretion, uptake, and interneuronal transfer of tau can be modulated by disease-associated tau modifications suggest that secretion may be an important element in tau-induced neurodegeneration. Here, we show that much of the tau secreted by M1C cells occurs via exosomal release, a widely characterized mechanism that mediates unconventional secretion of other aggregation-prone proteins (?-synuclein, prion protein, and ?-amyloid) in neurodegenerative disease. Exosome-associated tau is also present in human CSF samples and is phosphorylated at Thr-181 (AT270), an established phosphotau biomarker for Alzheimer disease (AD), in both M1C cells and in CSF samples from patients with mild (Braak stage 3) AD. A preliminary analysis of proteins co-purified with tau in secreted exosomes identified several that are known to be involved in disease-associated tau misprocessing. Our results suggest that exosome-mediated secretion of phosphorylated tau may play a significant role in the abnormal processing of tau and in the genesis of elevated CSF tau in early AD. PMID:22057275

  3. Efficient transfer of information from hexitol nucleic acids to RNA during nonenzymatic oligomerization

    NASA Technical Reports Server (NTRS)

    Kozlov, I. A.; De Bouvere, B.; Van Aerschot, A.; Herdewijn, P.; Orgel, L. E.

    1999-01-01

    Hexitol nucleic acids (HNAs) are DNA analogues that contain the standard nucleoside bases attached to a phosphorylated 1,5-anhydrohexitol backbone. We find that HNAs support efficient information transfer in nonensymatic template-directed reactions. HNA heterosequences appeared to be superior to the corresponding DNA heterosequences in facilitating synthesis of complementary oligonucleotides from nucleoside-5'-phosphoro-2-methyl imidazolides.

  4. Phosphorylation of Human CTP Synthetase 1 by Protein Kinase C IDENTIFICATION OF Ser462 AND Thr455 AS MAJOR SITES OF PHOSPHORYLATION*

    PubMed Central

    Chang, Yu-Fang; Martin, Shelley S.; Baldwin, Enoch P.; Carman, George M.

    2007-01-01

    Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined. Using purified Escherichia coli-expressed CTP synthetase 1 as a substrate, protein kinase C activity was time-and dose-dependent, and dependent on the concentrations of ATP and CTP synthetase 1. The protein kinase C phosphorylation of the recombinant enzyme was accompanied by a 95-fold increase in CTP synthetase 1 activity. Phosphopeptide mapping and phosphoamino acid analyses showed that CTP synthetase 1 was phosphorylated on multiple serine and threonine residues. The induction of PKC1R398A-encoded protein kinase C resulted in a 50% increase for human CTP synthetase 1 phosphorylation in the Saccharomyces cerevisiae ura7? ura8? mutant lacking yeast CTP synthetase activity. Synthetic peptides that contain the protein kinase C motif for Ser462 and Thr455 were substrates for mammalian protein kinase C, and S462A and T455A mutations resulted in decreases in the extent of CTP synthetase 1 phosphorylation that occurred in vivo. Phosphopeptide mapping analysis of S. cerevisiae-expressed CTP synthetase 1 mutant enzymes phosphorylated with mammalian protein kinase C confirmed that Ser462 and Thr455 were phosphorylation sites. The S. cerevisiae-expressed and purified S462A mutant enzyme exhibited a 2-fold reduction in CTP synthetase 1 activity, whereas the purified T455A mutant enzyme exhibits a 2-fold elevation in CTP synthetase 1 activity (Choi, M.-G., and Carman, G.M. (2006) J. Biol. Chem. 282, 5367–5377). These data indicated that protein kinase C phosphorylation at Ser462 stimulates human CTP synthetase 1 activity, whereas phosphorylation at Thr455 inhibits activity. PMID:17463002

  5. Identification of four novel phosphorylation sites in estrogen receptor ?: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2

    PubMed Central

    2009-01-01

    Background Estrogen receptor ? (ER?) phosphorylation is important for estrogen-dependent transcription of ER-dependent genes, ligand-independent receptor activation and endocrine therapy response in breast cancer. However ER? phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ER? phosphorylation sites exist, COS-1 cells expressing human ER? were labeled with [32P]H3PO4 in vivo and ER? tryptic phosphopeptides were isolated to identify phosphorylation sites. Results Previously uncharacterized phosphorylation sites at serines 46/47, 282, 294, and 559 were identified by manual Edman degradation and phosphoamino acid analysis and confirmed by mutagenesis and phospho-specific antibodies. Antibodies detected phosphorylation of endogenous ER? in MCF-7, MCF-7-LCC2, and Ishikawa cancer cell lines by immunoblot. Mutation of Ser-282 and Ser-559 to alanine (S282A, S559A) resulted in ligand independent activation of ER? as determined by both ERE-driven reporter gene assays and endogenous pS2 gene expression in transiently transfected HeLa cells. Mutation of Ser-46/47 or Ser-294 to alanine markedly reduced estradiol dependent reporter activation. Additionally protein kinase CK2 was identified as a kinase that phosphorylated ER? at S282 and S559 using motif analysis, in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor. Conclusion These novel ER? phosphorylation sites represent new means for modulation of ER? activity. S559 represents the first phosphorylation site identified in the extreme C-terminus (F domain) of a steroid receptor. PMID:20043841

  6. Compensatory phosphorylation and protein-protein interactions revealed by loss of function and gain of function mutants of multiple serine phosphorylation sites in endothelial nitric-oxide synthase.

    PubMed

    Bauer, Philip M; Fulton, David; Boo, Yong Chool; Sorescu, George P; Kemp, Bruce E; Jo, Hanjoong; Sessa, William C

    2003-04-25

    We examined the influence of individual serine phosphorylation sites in endothelial nitric-oxide synthase (eNOS) on basal and stimulated NO release, cooperative phosphorylation, and co-association with hsp90 and Akt. Mutation of the serine phosphorylation sites 116, 617, and 1179 to alanines affected the phospho-state of at least one other site, demonstrating cooperation between multiple phosphorylation events, whereas mutation of serine 635 to alanine did not cause compensation. Mutation of serines 116 and 617 to alanine promoted a greater protein-protein interaction with hsp90 and Akt and greater phosphorylation on serine 1179, the major site for Akt phosphorylation. More importantly, using alanine substitutions, Ser-116 is important for agonist, but not basal NO release, Ser-635 is important for basal, but not stimulated, Ser-617 negatively regulates basal and stimulated NO release, and Ser-1179 phosphorylation is stimulatory for both basal and agonist-mediated NO release. Using putative "gain of function" mutants (serine to aspartate) serines 635 and 1179 are important positive regulators of basal and stimulated NO release. S635D eNOS is the most efficacious, yielding 5-fold increases in basal and 2-fold increases in stimulated NO release from cells. However, S617A and S617D eNOS both increased NO release with opposite actions in NOS activity assays. Thus, multiple serine phosphorylation events regulate basal and stimulate NO release with Ser-635 and Ser-1179 being important positive regulatory sites and Ser-116 as a negative regulatory. Ser-617 may not be important for directly regulating NO release but is important as a modulator of phosphorylation at other sites and protein-protein interactions. PMID:12591925

  7. Mitosis-specific phosphorylation of nucleolin by p34cdc2 protein kinase.

    PubMed Central

    Belenguer, P; Caizergues-Ferrer, M; Labbé, J C; Dorée, M; Amalric, F

    1990-01-01

    Nucleolin is a ubiquitous multifunctional protein involved in preribosome assembly and associated with both nucleolar chromatin in interphase and nucleolar organizer regions on metaphasic chromosomes in mitosis. Extensive nucleolin phosphorylation by a casein kinase (CKII) occurs on serine in growing cells. Here we report that while CKII phosphorylation is achieved in interphase, threonine phosphorylation occurs during mitosis. We provide evidence that this type of in vivo phosphorylation involves a mammalian homolog of the cell cycle control Cdc2 kinase. In vitro M-phase H1 kinase from starfish oocytes phosphorylated threonines in a TPXK motif present nine times in the amino-terminal part of the protein. The same sites which matched the p34cdc2 consensus phosphorylation sequence were used in vivo during mitosis. We propose that successive Cdc2 and CKII phosphorylation could modulate nucleolin function in controlling cell cycle-dependent nucleolar function and organization. Our results, along with previous studies, suggest that while serine phosphorylation is related to nucleolin function in the control of rDNA transcription, threonine phosphorylation is linked to mitotic reorganization of nucleolar chromatin. Images PMID:2192260

  8. TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

    SciTech Connect

    Yang, Lifen; Section of Nephrology, Department of Medicine, The University of Chicago, Chicago, IL 60637; The Committees on Immunology, The University of Chicago, Chicago, IL 60637 ; Qiao, Guilin; Ying, Haiyan; The Committees on Immunology, The University of Chicago, Chicago, IL 60637 ; Zhang, Jian; Section of Nephrology, Department of Medicine, The University of Chicago, Chicago, IL 60637; The Committees on Immunology, The University of Chicago, Chicago, IL 60637; The Committees on Molecular Medicine, The University of Chicago, Chicago, IL 60637 ; Yin, Fei

    2010-09-10

    Research highlights: {yields} Conventional PKC positively regulates TCR-induced phosphorylation of Akt. {yields} PKC-alpha is the PDK-2 responsible for phosphorylating Akt at Ser{sup 473} upon TCR stimulation. {yields} Knockdown of PKC-alpha decreases TCR-induced Akt phosphorylation. -- Abstract: Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser{sup 473} in the hydrophobic motif, along with Thr{sup 308} in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr{sup 308}, but the kinase(s) responsible for phosphorylating Akt at Ser{sup 473} (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser{sup 473} phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser{sup 473} in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

  9. Identification of a phosphorylation site in cyclobutane pyrimidine dimer photolyase of rice.

    PubMed

    Teranishi, Mika; Nakamura, Kentaro; Furukawa, Haruya; Hidema, Jun

    2013-02-01

    Cyclobutane pyrimidine dimer (CPD) photolyase monomerises ultraviolet (UV) radiation-induced CPDs present in DNA, using energy from UVA and visible light. In plants, CPD photolyase activity is a crucial factor for determining UVB sensitivity. We previously demonstrated that native rice CPD photolyase is phosphorylated. To determine the phosphorylation site(s), the phosphorylation status of CPD photolyase was analyzed in rice varieties that have amino acid alterations at the potential phosphorylation sites. In wild-rice species, CPD photolyase was phosphorylated. In Poaceae species, CPD photolyase was phosphorylated in wheat but not in maize. Mutant CPD photolyase proteins, in which these putative phosphorylated residues were replaced with alanine residues, were synthesized using an insect cell-free translation system. A slow-migrating band disappeared when the serine residue at position 7 was mutated. A phospho-specific antibody was generated to determine whether this residue is phosphorylated in CPD photolyase. Only the slow-migrating band of native rice CPD photolyase was detected using this antibody, indicating that the serine residue at position 7 is a phosphorylation site in native rice CPD photolyase. PMID:23220084

  10. A flexible codon in genomically recoded Escherichia coli permits programmable protein phosphorylation

    PubMed Central

    Pirman, Natasha L.; Barber, Karl W.; Aerni, Hans R.; Ma, Natalie J.; Haimovich, Adrian D.; Rogulina, Svetlana; Isaacs, Farren J.; Rinehart, Jesse

    2015-01-01

    Biochemical investigation of protein phosphorylation events is limited by inefficient production of the phosphorylated and non-phosphorylated forms of full-length proteins. Here using a genomically recoded strain of E. coli with a flexible UAG codon we produce site-specific serine- or phosphoserine-containing proteins, with purities approaching 90%, from a single recombinant DNA. Specifically, we synthesize human MEK1 kinase with two serines or two phosphoserines, from one DNA template, and demonstrate programmable kinase activity. Programmable protein phosphorylation is poised to help reveal the structural and functional information encoded in the phosphoproteome. PMID:26350500

  11. Detection of the Phosphorylation of the Estrogen Receptor ? as an Outcome of GPR30 Activation.

    PubMed

    Clark, Sara; Pollard, Kevin; Rainville, Jennifer; Vasudevan, Nandini

    2016-01-01

    Phosphorylation of the serine residues in estrogen receptor (ER) ? is important in transcriptional activation. Hence, methods to detect such posttranslational modification events are valuable. We describe, in detail, the analysis of the phosphorylated ER? by electrophoretic separation of proteins and subsequent immuno-blotting techniques. In particular, phosphorylation of the ER? is one possible outcome of activation of the putative membrane estrogen receptor (mER), GPR30. Hence, phosphorylation represents a cross talk event between GPR30 and ER? and may be important in estrogen-regulated physiology. PMID:26585157

  12. Histone H3 Ser57 and Thr58 phosphorylation in the brain of 5XFAD mice

    PubMed Central

    Anderson, Kyle W.; Mast, Natalia; Pikuleva, Irina A.; Turko, Illarion V.

    2015-01-01

    Alzheimer’s disease has been shown to have a global reduction in gene expression, called an epigenetic blockade, which may be regulated by histone post-translational modifications. Histone H3 has been shown to be highly regulated by phosphorylation. We, therefore, chose H3 for investigation of phosphorylation of the core sites serine-57 (S57) and threonine-58 (T58). Hemispheres of brains from a mouse model of rapid amyloid deposition (5XFAD) were used for measurement of S57 and T58 phosphorylation. Multiple reaction monitoring (MRM) was used to measure the level of phosphorylation, which was normalized to a non-modified “housekeeping” peptide of H3. S57 phosphorylation was decreased by 40%, T58 phosphorylation was decreased by 45%, and doubly phosphorylated S57pT58p was decreased by 30% in 5XFAD brain in comparison to C57BL/6J age- and sex-matched wild type controls. Amyloid-? (A?) and amyloid precursor protein were also measured to confirm that 5XFAD mice produced high levels of A?. Decreased phosphorylation of these sites in close proximity to DNA may lead to stabilization of DNA–histone interactions and a condensed chromatin state, consistent with the epigenetic blockade associated with AD. Our findings of H3 sites S57 and T58 exhibiting lower levels of phosphorylation in 5XFAD model compared to wild type control implicate these sites in the epigenetic blockade in neurodegeneration pathology. PMID:26199864

  13. Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

    SciTech Connect

    Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao

    2013-04-05

    Highlights: •VCIP135 is mitotically phosphorylated on Threonine-760 and Serine-767 by Cdc2. •Phosphorylated VCIP135 does not bind to p97ATPase. •The phosphorylation of VCIP135 inhibits p97ATPase-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

  14. Phosphorylation of p37 is important for Golgi disassembly at mitosis

    SciTech Connect

    Kaneko, Yayoi; Mitsubishi Kagaku Institute of Life Sciences, Tokyo 194-8511 ; Tamura, Kaori; Totsukawa, Go; Mitsubishi Kagaku Institute of Life Sciences, Tokyo 194-8511 ; Kondo, Hisao

    2010-11-05

    Research highlights: {yields} p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis. {yields} Phosphorylated p37 does not bind to Golgi membranes. {yields} p37 phosphorylation inhibits p97/p37-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled at early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 by Cdc2 results in mitotic inhibition of the p97/p47 pathway . In this study, we demonstrate that p37 is phosphorylated on Serine-56 and Threonine-59 by Cdc2 at mitosis, and this phosphorylated p37 does not bind to Golgi membranes. Using an in vitro Golgi reassembly assay, we show that mutated p37(S56D, T59D), which mimics mitotic phosphorylation, does not cause any cisternal regrowth, indicating that p37 phosphorylation inhibits the p97/p37 pathway. Our results demonstrate that p37 phosphorylation on Serine-56 and Threonine-59 is important for Golgi disassembly at mitosis.

  15. Study of the docking-dependent PLK1 phosphorylation of the CDC25B phosphatase

    SciTech Connect

    Lobjois, Valerie; CNRS, ITAV-UMS3039, F-31106 Toulouse ; Froment, Carine; Universite de Toulouse, UPS, IPBS, F-31077 Toulouse ; Braud, Emmanuelle; Universite Paris Descartes, F-75270 Paris Cedex 06 ; Grimal, Fanny; Burlet-Schiltz, Odile; Universite de Toulouse, UPS, IPBS, F-31077 Toulouse ; Ducommun, Bernard; CNRS, ITAV-UMS3039, F-31106 Toulouse; CHU de Toulouse, F-31059 Toulouse ; Bouche, Jean-Pierre

    2011-06-24

    Highlights: {yields} Phosphorylation of CDC25B by CDK1 enhances its substrate properties for PLK1 in vitro. {yields} Sequential phosphorylation of CDC25B is analyzed using {sup 16}O and {sup 18}O ATP. {yields} Thirteen sites phosphorylated by PLK1 have been identified. -- Abstract: CDC25 (A, B and C) phosphatases control cell cycle progression through the timely dephosphorylation and activation of cyclin-dependent kinases (CDK). At mitosis the CDC25B phosphatase activity is dependent on its phosphorylation by multiple kinases impinging on its localisation, stability and catalytic activity. Here we report that prior phosphorylation of CDC25B by CDK1 enhances its substrate properties for PLK1 in vitro, and we also show that phosphorylated S50 serves as a docking site for PLK1. Using a sophisticated strategy based on the sequential phosphorylation of CDC25B with {sup 16}O and {sup 18}O ATP prior to nanoLC-MS/MS analysis we identified 13 sites phosphorylated by PLK1. This study illustrates the complexity of the phosphorylation pattern and of the subsequent regulation of CDC25B activity.

  16. Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP.

    PubMed Central

    Miles, K; Anthony, D T; Rubin, L L; Greengard, P; Huganir, R L

    1987-01-01

    The nicotinic acetylcholine receptor (AcChoR) from rat myotubes prelabeled in culture with [32P]orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the beta and delta subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the delta subunit 20-fold over basal phosphorylation and induced phosphorylation of the alpha subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 microM in the presence of 35 microM Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of delta subunit phosphorylation was almost maximal within 5 min, whereas stimulation of alpha subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the delta subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the delta subunit increases the rate of AcChoR desensitization in rat myotubes. Images PMID:2819883

  17. Actin Polymerization: An Event Regulated by Tyrosine Phosphorylation During Buffalo Sperm Capacitation.

    PubMed

    Naresh, S; Atreja, S K

    2015-12-01

    In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time-dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)-induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H-89, PD9809 and GF-109) and enhancer (dbcAMP, H2 O2 and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F-actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa. PMID:26514336

  18. Case study: using sequence homology to identify putative phosphorylation sites in an evolutionarily distant species (honeybee).

    PubMed

    Trost, Brett; Napper, Scott; Kusalik, Anthony

    2015-09-01

    The majority of scientific resources are devoted to studying a relatively small number of model species, meaning that the ability to translate knowledge across species is of considerable importance. Obtaining species-specific knowledge enables targeted investigations of the biology and pathobiology of a particular species, and facilitates comparative analyses. Phosphorylation is the most widespread posttranslational modification in eukaryotes, and although many phosphorylation sites have been experimentally identified for some species, little or no data are available for others. Using the honeybee as a test organism, this case study illustrates the process of using protein sequence homology to identify putative phosphorylation sites in a species of interest using experimentally determined sites from other species. A number of issues associated with this process are examined and discussed. Several databases of experimentally determined phosphorylation sites exist; however, it can be difficult for the nonspecialist to ascertain how their contents compare. Thus, this case study assesses the content and comparability of several phosphorylation site databases. Additional issues examined include the efficacy of homology-based phosphorylation site prediction, the impact of the level of evolutionary relatedness between species in making these predictions, the ability to translate knowledge of phosphorylation sites across large evolutionary distances and the criteria that should be used in selecting probable phosphorylation sites in the species of interest. Although focusing on phosphorylation, the issues discussed here also apply to the homology-based cross-species prediction of other posttranslational modifications, as well as to sequence motifs in general. PMID:25380664

  19. Crystal-induced neutrophil activation. IV. Specific inhibition of tyrosine phosphorylation by colchicine.

    PubMed Central

    Roberge, C J; Gaudry, M; de Médicis, R; Lussier, A; Poubelle, P E; Naccache, P H

    1993-01-01

    We recently demonstrated that pathologically relevant inflammatory microcrystals, namely triclinic monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals, potently stimulate a characteristic protein tyrosine phosphorylation pattern in human neutrophils that differed from that observed in response to other soluble or particulate agonists. In this study, the effects of colchicine on protein tyrosine phosphorylation induced by MSU and CPPD crystals in human blood neutrophils were investigated. Immunoblot analysis with antiphosphotyrosine antibodies demonstrated that colchicine dose-dependently inhibited the tyrosine phosphorylation of all the proteins phosphorylated in response to MSU and CPPD crystals. Other microtubule-disruptive agents such as vinblastine, nocodazole, and colcemid also inhibited crystal-induced protein tyrosine phosphorylation while lumicolchicine and trimethylcolchicinic acid were without effect. Indomethacin and phenylbutazone were similarly without effect on microcrystal-induced tyrosine phosphorylation. Colchicine, as well as the other active alkaloids, failed to inhibit the protein tyrosine phosphorylation elicited by FMLP, C5a, leukotriene B4, and unopsonized zymosan. Overall, these results demonstrate that colchicine specifically and significantly inhibits the protein tyrosine phosphorylation induced by MSU and CPPD crystals and suggest that its effects are associated, at least in part, with its interaction with microtubules. Furthermore, the use of microtubule-disrupting drugs demonstrate that the mechanisms implicated in the induction of protein tyrosine phosphorylation by microcrystals differed from those involved in response to other soluble or particulate agonists. Images PMID:7691884

  20. 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. PMID:26150991

  1. Quantum-dynamical picture of a multistep enzymatic process: reaction catalyzed by phospholipase A(2).

    PubMed Central

    Ba?a, P; Grochowski, P; Nowi?ski, K; Lesyng, B; McCammon, J A

    2000-01-01

    A quantum-classical molecular dynamics model (QCMD), applying explicit integration of the time-dependent Schrödinger equation (QD) and Newtonian equations of motion (MD), is presented. The model is capable of describing quantum dynamical processes in complex biomolecular systems. It has been applied in simulations of a multistep catalytic process carried out by phospholipase A(2) in its active site. The process includes quantum-dynamical proton transfer from a water molecule to histidine localized in the active site, followed by a nucleophilic attack of the resulting OH(-) group on a carbonyl carbon atom of a phospholipid substrate, leading to cleavage of an adjacent ester bond. The process has been simulated using a parallel version of the QCMD code. The potential energy function for the active site is computed using an approximate valence bond (AVB) method. The dynamics of the key proton is described either by QD or classical MD. The coupling between the quantum proton and the classical atoms is accomplished via Hellmann-Feynman forces, as well as the time dependence of the potential energy function in the Schrödinger equation (QCMD/AVB model). Analysis of the simulation results with an Advanced Visualization System revealed a correlated rather than a stepwise picture of the enzymatic process. It is shown that an sp(2)--> sp(3) configurational change at the substrate carbonyl carbon is mostly responsible for triggering the activation process. PMID:10968989

  2. 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

  3. Rapid colorimetric determination of reduced and oxidized glutathione using an end point coupled enzymatic assay.

    PubMed

    Cappiello, Mario; Peroni, Eleonora; Lepore, Ambra; Moschini, Roberta; Del Corso, Antonella; Balestri, Francesco; Mura, Umberto

    2013-02-01

    A simple and rapid colorimetric coupled enzymatic assay for the determination of glutathione is described. The proposed method is based on the specific reaction catalyzed by ?-glutamyltransferase, which transfers the ?-glutamyl moiety from glutahione to an acceptor, with the formation of the ?-glutamyl derivative of the acceptor and cysteinylglycine. The latter dipeptide is a substrate of leucyl aminopeptidase, which hydrolyzes cysteinylglycine to glycine and cysteine that can be easily measured spectrophotometrically. The proposed method was used to measure the content of glutathione in acid extracts of bovine lens, to follow the NADPH-dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) catalyzed by the enzyme glutathione reductase and to determine the glutathione content in human astrocytoma ADF cells subjected to oxidative stress. The results obtained showed that the method can be suitably used for the determination of GSH and GSSG in different biological samples and to monitor tissue or cell redox status under different conditions. It is also applicable for following reactions involving GSH and/or GSSG. PMID:23203508

  4. Quantum dots based electrochemiluminescent immunosensor by coupling enzymatic amplification for ultrasensitive detection of clenbuterol.

    PubMed

    Yao, Xun; Yan, Panpan; Tang, Qinghui; Deng, Anping; Li, Jianguo

    2013-10-10

    An ultrasensitive electrochemiluminescence (ECL) immunosensor based on CdSe quantum dots (QDs) has been designed for the detection of clenbuterol. The immunosensor was fabricated by layer by layer and characterized with atomic force microscopic images (AFM) and electrochemical impedance spectra (EIS). In oxygen-saturated pH=9.0 Tris-HCl buffer, a strong ECL emission of QDs could be observed during the cathodic process due to the H2O2 product from electrochemical reduction of dissolved oxygen. Upon the formation of immunocomplex, the second antibody labeled with horseradish peroxidase was simply immobilized on the electrode surface. The ECL emission decreased since steric hindrance of the immunocomplex slowed down the electron-transfer speed of dissolved oxygen, and also could be greatly amplified by an enzymatic cycle to consume the self-produced coreactant. Using clenbuterol as model analyte, the ECL intensity was determined by the concentration of competitive immunoassay of clenbuterol with a wide calibration in the range of 0.05 ng mL(-1) to 1000 ng mL(-1), and a low detection limit was 0.02 ng mL(-1). The immunosensor shows good stability and fabrication reproducibility. It was applied to detecting practical samples with the satisfactory results. This immunosensing strategy opens a new avenue for detection of residue and application of QDs in ECL biosensing. PMID:24070487

  5. A hybrid DNA-templated gold nanocluster for enhanced enzymatic reduction of oxygen

    SciTech Connect

    Chakraborty, Saumen; Babanova, Sofia; Rocha, Reginaldo C.; Desireddy, Anil; Artyushkova, Kateryna; Boncella, Amy E.; Atanassov, Plamen; Martinez, Jennifer S.

    2015-08-19

    We report the synthesis and characterization of a new DNA-templated gold nanocluster (AuNC) of ~1 nm in diameter and possessing ~7 Au atoms. When integrated with bilirubin oxidase (BOD) and single walled carbon nanotubes (SWNTs), the AuNC acts as an enhancer of electron transfer (ET) and lowers the overpotential of electrocatalytic oxygen reduction reaction (ORR) by ~15 mV as compared to the enzyme alone. In addition, the presence of AuNC causes significant enhancements in the electrocatalytic current densities at the electrode. Control experiments show that such enhancement of ORR by the AuNC is specific to nanoclusters and not to plasmonic gold particles. Rotating ring disk electrode (RRDE) measurements confirm 4e– reduction of O2 to H2O with minimal production of H2O2, suggesting that the presence of AuNC does not perturb the mechanism of ORR catalyzed by the enzyme. This unique role of the AuNC as enhancer of ET at the enzyme-electrode interface makes it a potential candidate for the development of cathodes in enzymatic fuel cells, which often suffer from poor electronic communication between the electrode surface and the enzyme active site. In conclusion, the AuNC displays phosphorescence with large Stokes shift and microsecond lifetime.

  6. A hybrid DNA-templated gold nanocluster for enhanced enzymatic reduction of oxygen

    DOE PAGESBeta

    Chakraborty, Saumen; Babanova, Sofia; Rocha, Reginaldo C.; Desireddy, Anil; Artyushkova, Kateryna; Boncella, Amy E.; Atanassov, Plamen; Martinez, Jennifer S.

    2015-08-19

    We report the synthesis and characterization of a new DNA-templated gold nanocluster (AuNC) of ~1 nm in diameter and possessing ~7 Au atoms. When integrated with bilirubin oxidase (BOD) and single walled carbon nanotubes (SWNTs), the AuNC acts as an enhancer of electron transfer (ET) and lowers the overpotential of electrocatalytic oxygen reduction reaction (ORR) by ~15 mV as compared to the enzyme alone. In addition, the presence of AuNC causes significant enhancements in the electrocatalytic current densities at the electrode. Control experiments show that such enhancement of ORR by the AuNC is specific to nanoclusters and not to plasmonicmore »gold particles. Rotating ring disk electrode (RRDE) measurements confirm 4e– reduction of O2 to H2O with minimal production of H2O2, suggesting that the presence of AuNC does not perturb the mechanism of ORR catalyzed by the enzyme. This unique role of the AuNC as enhancer of ET at the enzyme-electrode interface makes it a potential candidate for the development of cathodes in enzymatic fuel cells, which often suffer from poor electronic communication between the electrode surface and the enzyme active site. In conclusion, the AuNC displays phosphorescence with large Stokes shift and microsecond lifetime.« less

  7. Determination of phosphate in soil extracts in the field: A green chemistry enzymatic method.

    PubMed

    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. PMID:26150991

  8. Directed evolution of transketolase activity on non-phosphorylated substrates.

    PubMed

    Hibbert, Edward G; Senussi, Tarik; Costelloe, Sean J; Lei, Wenling; Smith, Mark E B; Ward, John M; Hailes, Helen C; Dalby, Paul A

    2007-09-30

    We have used active-site targeted directed evolution by saturation mutagenesis to improve the activity of E. coli transketolase towards non-phosphorylated substrates. Residues were selected for each set based on either structural proximity to substrate, or on phylogenetic variation. Each library was screened towards the reaction between hydroxypyruvate (HPA) and glycolaldehyde (GA) to form L-erythrulose, and the location of improved mutants related to the natural sequence entropy at each residue. A number of mutants from the phylogenetically defined library were found to outperform the wild-type with up to 3-fold specific activity under biocatalytically relevant conditions, though interestingly with substituted residues that differed from those found in nature. Conserved residues which interact with the phosphate group in natural substrates also yielded mutants with almost 5-fold improved specific activity on the non-phosphorylated substrates. These results suggest that phylogenetically variant active-site residues are useful for modulating activity on natural or structurally-homologous substrates, and that conserved residues which no longer interact with modified target substrates are useful sites to apply saturation mutagenesis for improvement of activity. PMID:17825449

  9. Regulation of Endothelial Adherens Junctions by Tyrosine Phosphorylation

    PubMed Central

    Adam, Alejandro Pablo

    2015-01-01

    Endothelial cells form a semipermeable, regulated barrier that limits the passage of fluid, small molecules, and leukocytes between the bloodstream and the surrounding tissues. The adherens junction, a major mechanism of intercellular adhesion, is comprised of transmembrane cadherins forming homotypic interactions between adjacent cells and associated cytoplasmic catenins linking the cadherins to the cytoskeleton. Inflammatory conditions promote the disassembly of the adherens junction and a loss of intercellular adhesion, creating openings or gaps in the endothelium through which small molecules diffuse and leukocytes transmigrate. Tyrosine kinase signaling has emerged as a central regulator of the inflammatory response, partly through direct phosphorylation and dephosphorylation of the adherens junction components. This review discusses the findings that support and those that argue against a direct effect of cadherin and catenin phosphorylation in the disassembly of the adherens junction. Recent findings indicate a complex interaction between kinases, phosphatases, and the adherens junction components that allow a fine regulation of the endothelial permeability to small molecules, leukocyte migration, and barrier resealing. PMID:26556953

  10. Cholinesterase inhibitors may increase phosphorylated tau in Alzheimer's disease.

    PubMed

    Chalmers, Katy A; Wilcock, Gordon K; Vinters, Harry V; Perry, Elaine K; Perry, Robert; Ballard, Clive G; Love, Seth

    2009-05-01

    Cholinesterase inhibitors (ChEIs) are widely used for the symptomatic treatment of Alzheimer's disease (AD). In vitro and in animal studies, ChEIs have been shown to influence the processing of Abeta and the phosphorylation of tau, proteins that are the principal constituents of the plaques and neurofibrillary tangles, respectively, in AD brain. However, little is known about the effects of these drugs on Abeta and tau pathology in AD. Using avidin-biotin immunohistochemistry and computer-assisted image analysis, we compared Abeta and tau loads in the frontal and temporal cortices of 72 brains from matched cohorts of AD patients who had or had not received ChEIs. Patients treated with ChEIs had accumulated significantly more phospho-tau in their cerebral cortex than had untreated patients (P = 0.004). Abeta accumulation was reduced but not significantly. These data raise the possibility that increased tau phosphorylation may influence long-term clinical responsiveness to ChEIs. PMID:19240967

  11. ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation

    PubMed Central

    Teixeira, Felipe K.; Sanchez, Carlos G.; Hurd, Thomas R.; Seifert, Jessica R. K.; Czech, Benjamin; Preall, Jonathan B.; Hannon, Gregory J.; Lehmann, Ruth

    2015-01-01

    The differentiation of stem cells is a tightly regulated process essential for animal development and tissue homeostasis. Through this process, attainment of new identity and function is achieved by marked changes in cellular properties. Intrinsic cellular mechanisms governing stem cell differentiation remain largely unknown, in part because systematic forward genetic approaches to the problem have not been widely used1,2. Analysing genes required for germline stem cell differentiation in the Drosophila ovary, we find that the mitochondrial ATP synthase plays a critical role in this process. Unexpectedly, the ATP synthesizing function of this complex was not necessary for differentiation, as knockdown of other members of the oxidative phosphorylation system did not disrupt the process. Instead, the ATP synthase acted to promote the maturation of mitochondrial cristae during differentiation through dimerization and specific upregulation of the ATP synthase complex. Taken together, our results suggest that ATP synthase-dependent crista maturation is a key developmental process required for differentiation independent of oxidative phosphorylation. PMID:25915123

  12. (Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation.

    PubMed

    Fiesel, Fabienne C; Ando, Maya; Hudec, Roman; Hill, Anneliese R; Castanedes-Casey, Monica; Caulfield, Thomas R; Moussaud-Lamodière, Elisabeth L; Stankowski, Jeannette N; Bauer, Peter O; Lorenzo-Betancor, Oswaldo; Ferrer, Isidre; Arbelo, José M; Siuda, Joanna; Chen, Li; Dawson, Valina L; Dawson, Ted M; Wszolek, Zbigniew K; Ross, Owen A; Dickson, Dennis W; Springer, Wolfdieter

    2015-09-01

    Mutations in PINK1 and PARKIN cause recessive, early-onset Parkinson's disease (PD). Together, these two proteins orchestrate a protective mitophagic response that ensures the safe disposal of damaged mitochondria. The kinase PINK1 phosphorylates ubiquitin (Ub) at the conserved residue S65, in addition to modifying the E3 ubiquitin ligase Parkin. The structural and functional consequences of Ub phosphorylation (pS65-Ub) have already been suggested from in vitro experiments, but its (patho-)physiological significance remains unknown. We have generated novel antibodies and assessed pS65-Ub signals in vitro and in cells, including primary neurons, under endogenous conditions. pS65-Ub is dependent on PINK1 kinase activity as confirmed in patient fibroblasts and postmortem brain samples harboring pathogenic mutations. We show that pS65-Ub is reversible and barely detectable under basal conditions, but rapidly induced upon mitochondrial stress in cells and amplified in the presence of functional Parkin. pS65-Ub accumulates in human brain during aging and disease in the form of cytoplasmic granules that partially overlap with mitochondrial, lysosomal, and total Ub markers. Additional studies are now warranted to further elucidate pS65-Ub functions and fully explore its potential for biomarker or therapeutic development. PMID:26162776

  13. Uncovering Phosphorylation-Based Specificities through Functional Interaction Networks.

    PubMed

    Wagih, Omar; Sugiyama, Naoyuki; Ishihama, Yasushi; Beltrao, Pedro

    2016-01-01

    Protein kinases are an important class of enzymes involved in the phosphorylation of their targets, which regulate key cellular processes and are typically mediated by a specificity for certain residues around the target phospho-acceptor residue. While efforts have been made to identify such specificities, only ?30% of human kinases have a significant number of known binding sites. We describe a computational method that utilizes functional interaction data and phosphorylation data to predict specificities of kinases. We applied this method to human kinases to predict substrate preferences for 57% of all known kinases and show that we are able to reconstruct well-known specificities. We used an in vitro mass spectrometry approach to validate four understudied kinases and show that predicted models closely resemble true specificities. We show that this method can be applied to different organisms and can be extended to other phospho-recognition domains. Applying this approach to different types of posttranslational modifications (PTMs) and binding domains could uncover specificities of understudied PTM recognition domains and provide significant insight into the mechanisms of signaling networks. PMID:26572964

  14. Functional analyses of phosphorylation events in human Argonaute 2.

    PubMed

    Lopez-Orozco, Joaquin; Pare, Justin M; Holme, Andrea L; Chaulk, Steven G; Fahlman, Richard P; Hobman, Tom C

    2015-12-01

    Argonaute 2 (Ago2) protein is a central effector of RNA interference (RNAi) pathways and regulates mammalian genes on a global level. The mechanisms of Ago2-mediated silencing are well understood, but less is known about its regulation. Recent reports indicate that phosphorylation significantly affects Ago2 activity. Here, we investigated the effect of mutating all known phospho-residues within Ago2 on its localization and activity. Ago2 associates with two different cytoplasmic RNA granules known as processing bodies (P-bodies) and stress granules, but the nature of this phenomenon is controversial. We report that replacing serine with a phospho-mimetic aspartic acid at position 798 completely abrogates association of Ago2 with P-bodies and stress granules. The effect of this mutation on its activity in gene silencing was modest, which was surprising because association of Ago2 with cytoplasmic RNA granules is thought to be a consequence of its role in RNAi. As such, our data indicate that targeting of Ago2 to P-bodies and stress granules is separable from its role in RNAi and likely requires dynamic phosphorylation of serine 798. PMID:26443379

  15. Mercury Alters B-Cell Protein Phosphorylation Profiles

    PubMed Central

    Carruthers, Nicholas J.; Stemmer, Paul M.; Shin, Namhee; Dombkowski, Alan; Caruso, Joseph A.; Gill, Randal; Rosenspire, Allen

    2014-01-01

    Environmental exposure to mercury is suggested to contribute to human immune dysfunction. To shed light on the mechanism we identified changes in the phosphoproteomic profile of the WEHI-231 B cell line after intoxication with Hg2+. These changes were compared to changes in the phosphoproteome that were induced by pervanadate or okadaic acid exposure. Both 250 ?M HgCl2 and pervanadate, a known phosphotyrosine phosphatase inhibitor, caused an increase in the number of proteins identified after TiO2 affinity selection and LC-MS/MS analysis. Pervanadate treatment had a larger effect than Hg2+ on the number of Scansite motifs which were tyrosine-phosphorylated, 17, and Ingenuity canonical signaling pathways activated, 4 with score > 5.0. However, Hg2+ had a more focused effect, primarily causing tyrosine-phosphorylation in SH2 domains in proteins that are in the B cell receptor signaling pathway. The finding that many of the changes induced by Hg2+ overlap with those of pervanadate, indicates that at high concentrations Hg2+ inhibits protein tyrosine phosphatases. PMID:24224561

  16. The threonine residues in MAP kinase kinase 1 phosphorylated by MAP kinase in vitro are also phosphorylated in nerve growth factor-stimulated rat phaeochromocytoma (PC12) cells.

    PubMed

    Saito, Y; Gomez, N; Campbell, D G; Ashworth, A; Marshall, C J; Cohen, P

    1994-03-14

    The residues on MAP kinase kinase-1 (MAPKK1) phosphorylated by MAP kinase in vitro have been identified as Thr-291 and Thr-385. Both threonines are phosphorylated in PC12 cells and the 32P-labelling of each residue increases after stimulation with nerve growth factor (NGF). The results establish that MAPKK1 is a physiological substrate for MAP kinase. The two active forms of MAPKK that are resolved by Mono Q chromatography of PC12 cell extracts are both phosphorylated at Thr-291 and Thr-385, demonstrating that neither species is the MAPKK2 isoform which lacks Thr-291. PMID:8137910

  17. High LET-induced H2AX phosphorylation around the Bragg curve

    NASA Astrophysics Data System (ADS)

    Desai, N.; Durante, M.; Lin, Z. W.; Cucinotta, F.; Wu, H.

    We investigated the spatial distribution of the induction of the phosphorylated form of the histone protein H2AX (?-H2AX), known to be activated by DSBs. Following irradiation of human fibroblast cells with 600 MeV/nucleon silicon and 600 MeV/nucleon iron ions we observed the formation of ?-H2AX aggregates in the shape of streaks stretching over several micrometers in an x/ y plane. Polyethylene shielding was used to achieve a Bragg curve distribution with beam geometry parallel to the monolayer of cells. We present data that highlights the formation of immunofluorescent ?-H2AX tracks showing the ion trajectories across the Bragg peak of irradiated human fibroblast cells. Qualitative analyses of these distributions indicated potentially increased clustering of DNA damage before the Bragg peak, enhanced ?-H2AX distribution at the peak, and provided visual evidence of high-linear energy transfer particle traversal of cells beyond the Bragg peak in agreement with one-dimensional transport approximations. Spatial assessment of ?-H2AX fluorescence may provide direct insights into DNA damage across the Bragg curve for high charge and energy ions including the biological consequences of shielding and possible contributors to bystander effects.

  18. Mutant SOD1 Increases APP Expression and Phosphorylation in Cellular and Animal Models of ALS

    PubMed Central

    Rabinovich-Toidman, Polina; Rabinovich-Nikitin, Inna; Ezra, Assaf; Barbiro, Beka; Fogel, Hilla; Slutsky, Inna; Solomon, Beka

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease and it is the most common adult onset neurodegenerative disorder affecting motor neurons. There is currently no effective treatment for ALS and our understanding of the pathological mechanism is still far away from prevention and/or treatment of this devastating disease. Amyloid precursor protein (APP) is a transmembrane protein that undergoes processing either by ?-secretase or ?-secretase, followed by ?-secretase. In the present study, we show that APP levels, and aberrant phosphorylation, which is associated with enhanced ?-secretase cleavage, are increased in SOD1G93A ALS mouse model. Fluorescence resonance energy transfer (FRET) analysis suggests a close interaction between SOD1 and APP at hippocampal synapses. Notably, SOD1G93A mutation induces APP-SOD1 conformational changes, indicating a crosstalk between these two signaling proteins. Inhibition of APP processing via monoclonal antibody called BBS that blocks APP ?-secretase cleavage site, resulted in reduction of mutant SOD1G93A levels in animal and cellular models of ALS, significantly prolonged life span of SOD1G93A mice and diminished inflammation. Beyond its effect on toxic mutant SOD1G93A, BBS treatment resulted in a reduction in the levels of APP, its processing product soluble APP? and pro-apoptotic p53. This study demonstrates that APP and its processing products contribute to ALS pathology through several different pathways; thus BBS antibody could be a promising neuroprotective strategy for treatment of this disease. PMID:26600047

  19. Discovery of causal mechanisms: Oxidative phosphorylation and the Calvin-Benson cycle.

    PubMed

    Scholl, Raphael; Nickelsen, Kärin

    2015-06-01

    We investigate the context of discovery of two significant achievements of twentieth century biochemistry: the chemiosmotic mechanism of oxidative phosphorylation (proposed in 1961 by Peter Mitchell) and the dark reaction of photosynthesis (elucidated from 1946 to 1954 by Melvin Calvin and Andrew A. Benson). The pursuit of these problems involved discovery strategies such as the transfer, recombination and reversal of previous causal and mechanistic knowledge in biochemistry. We study the operation and scope of these strategies by careful historical analysis, reaching a number of systematic conclusions: (1) even basic strategies can illuminate "hard cases" of scientific discovery that go far beyond simple extrapolation or analogy; (2) the causal-mechanistic approach to discovery permits a middle course between the extremes of a completely substrate-neutral and a completely domain-specific view of scientific discovery; (3) the existing literature on mechanism discovery underemphasizes the role of combinatorial approaches in defining and exploring search spaces of possible problem solutions; (4) there is a subtle interplay between a fine-grained mechanistic and a more coarse-grained causal level of analysis, and both are needed to make discovery processes intelligible. PMID:26013645

  20. Application of response surface methodology in enzymatic synthesis: a review.

    PubMed

    Ghaffari-Moghaddam, Mansour; Yekke-Ghasemi, Zahra; Khajeh, Mostafa; Rakhshanipour, Mansoureh; Yasin, Yamin

    2014-01-01

    There are very chemical reactions with very slow rates which can be catalyzed by enzymes. These biocatalysts need to moderate conditions for their catalytic activity and are stable in low temperature (between 15-50°C), average pH (5-10) and aqueous media. One of important things in enzymatic synthesis which has been recently noticed is the yield of reactions. Nowadays wide application of response surface methodology (RSM) was observed in organic chemistry. In one-variable-at-a-time technique only one parameter is changed and other parameters are kept at a constant level. It does not study the interactive effects among the variables, and does not illustrate the complete effects of the parameters on the process. Increasing the yield of product without increase in casts is carried out by modeling and optimization of reaction variables through statistical techniques such as RSM. In this paper, we reviewed some articles that used the RSM for optimization in the enzymatic synthesis. PMID:25898733