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Theoretical investigation of the enzymatic phosphoryl transfer of ?-phosphoglucomutase: revisiting both steps of the catalytic cycle.  


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 Mg(2+). 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 Mg(2+) 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)). PMID:22238068

Elsässer, Brigitta; Dohmeier-Fischer, Silvia; Fels, Gregor



Vimentin Binding to Phosphorylated Erk Sterically Hinders Enzymatic Dephosphorylation of the Kinase  

E-print Network

Vimentin Binding to Phosphorylated Erk Sterically Hinders Enzymatic Dephosphorylation of the Kinase with phosphorylated Erk1 and Erk2 MAP kinases (pErk) in injured sciatic nerve, thus linking pErk to a signaling for this interaction, which explains how pErk is protected from dephos- phorylation while bound to vimentin. Pull

Fainzilber, Michael


Common Hydrogen Bond Interactions in Diverse Phosphoryl Transfer Active Sites  

PubMed Central

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

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



Vibrationally enhanced tunneling as a mechanism for enzymatic hydrogen transfer.  

PubMed Central

We present a theory of enzymatic hydrogen transfer in which hydrogen tunneling is mediated by thermal fluctuations of the enzyme's active site. These fluctuations greatly increase the tunneling rate by shortening the distance the hydrogen must tunnel. The average tunneling distance is shown to decrease when heavier isotopes are substituted for the hydrogen or when the temperature is increased, leading to kinetic isotope effects (KIEs)--defined as the factor by which the reaction slows down when isotopically substituted substrates are used--that need be no larger than KIEs for nontunneling mechanisms. Within this theory we derive a simple KIE expression for vibrationally enhanced ground state tunneling that is able to fit the data for the bovine serum amine oxidase (BSAO) system, correctly predicting the large temperature dependence of the KIEs. Because the KIEs in this theory can resemble those for nontunneling dynamics, distinguishing the two possibilities requires careful measurements over a range of temperatures, as has been done for BSAO. PMID:1420907

Bruno, W J; Bialek, W



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


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 Cys(189) and Cys(195). Enzymatic phosphoryl transfer from ?Glc 1-P to water in H2 (18)O solvent proceeded with complete (18)O 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

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



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

Microsoft Academic Search

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



Phosphoryl transfer by a concerted reaction mechanism in UMP/CMP-kinase.  

PubMed Central

The reaction mechanism of phosphoryl transfer catalyzed by UMP/CMP-kinase from Dictyostelium discoideum was investigated by semiempirical AM1 molecular orbital computations of an active site model system derived from crystal structures that contain a transition state analog or a bisubstrate inhibitor. The computational results suggest that the nucleoside monophosphate must be protonated for the forward reaction while it is unprotonated in the presence of aluminium fluoride, a popular transition state analog for phosphoryl transfer reactions. Furthermore, a compactification of the active site model system during the reaction and for the corresponding complex containing AlF3 was observed. For the active site residues that are part of the LID domain, conformational flexibility during the reaction proved to be crucial. On the basis of the calculations, a concerted phosphoryl transfer mechanism is suggested that involves the synchronous shift of a proton from the monophosphate to the transferred PO3-group. The proposed mechanism is thus analogous to the phosphoryl transfer mechanism in cAMP-dependent protein kinase that phosphorylates the hydroxyl groups of serine residues. PMID:11152133

Hutter, M. C.; Helms, V.



Specific Reaction Parametrization of the AM1/d Hamiltonian for Phosphoryl Transfer Reactions: H, O,  

E-print Network

to model phosphoryl transfer reactions catalyzed by enzymes and ribozymes for use in linear, and ribozymes and to ultimately facilitate the design of improved next- generation multiscale quantum models. 1 or ribozymes.5 The understanding of the molecular mecha- nisms of ribozyme catalysis has been greatly

Minnesota, University of


Evidence for an associative mechanism in the phosphoryl transfer step catalyzed by rabbit muscle creatine kinase.  


Creatine kinase does not catalyze the scrambling of 18O in adenosine 5'-[alpha beta-18O, beta-18O2]triphosphate in the absence of creatine, in the presence of L-arginine or taurocyamine (competitive inhibitors of creatine), or in the presence of poor substrates where single turnover experiments were performed. In order to support this prima facie evidence for an associative mechanism of phosphoryl transfer, an investigation was undertaken of 1-carboxymethyl-2-aminoimidazole, a new substrate analogue of creatine. This analogue has a binding constant for rabbit muscle creatine kinase similar to creatine and 1-carboxymethyl-2-iminoimidazolidine, but the initial rate of phoshorylation by MgATP in the presence of creatine kinase is almost 5 orders of magnitude slower. The phosphorylation product, assigned the structure 1-carboxymethyl-2-imino-3-phospho-4-imidazoline is also a poor substrate for the phosphorylation of MgADP by creatine kinase. These observations can be accounted for by an associative SN2(P) mechanism of phosphoryl transfer and by a microenvironment of the enzyme-bound creatine (or creatine analogue) which lowers the pKa of the guanidino group by several pH units compared with that in aqueous solution. PMID:7372661

Lowe, G; Sproat, B S



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

NASA Astrophysics Data System (ADS)

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.

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



Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry  

Microsoft Academic Search

We present a strategy for the analysis of the yeast phosphoproteome that uses endo-Lys C as the proteolytic enzyme, immobilized metal affinity chromatography for phosphopeptide enrichment, a 90-min nanoflow-HPLC\\/electrospray-ionization MS\\/MS experiment for phosphopeptide fractionation and detection, gas phase ion\\/ion chemistry, electron transfer dissociation for peptide fragmentation, and the Open Mass Spectrometry Search Algorithm for phosphoprotein identification and assignment of phosphorylation

A. Chi; Curtis Huttenhower; L. Y. Geer; Joshua J. Coon; J. E. P. Syka; D. L. Bai; Jeffrey Shabanowitz; D. J. Burke; O. G. Troyanskaya; D. F. Hunt



Enzymatic Activity and Substrate Specificity of Mitogen-activated Protein Kinase p38? in Different Phosphorylation States*S?  

PubMed Central

The mitogen-activated protein (MAP) kinases are essential signaling molecules that mediate many cellular effects of growth factors, cytokines, and stress stimuli. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Down-regulation of MAP kinase activity can be initiated by multiple serine/threonine phosphatases, tyrosine-specific phosphatases, and dual specificity phosphatases (MAP kinase phosphatases). This would inevitably lead to the formation of monophosphorylated MAP kinases. However, the biological functions of these monophosphorylated MAP kinases are currently not clear. In this study, we have prepared MAP kinase p38?, a member of the MAP kinase family, in all phosphorylated forms and characterized their biochemical properties. Our results indicated the following: (i) p38? phosphorylated at both Thr-180 and Tyr-182 was 10–20-fold more active than p38? phosphorylated at Thr-180 only, whereas p38? phosphorylated at Tyr-182 alone was inactive; (ii) the dual-specific MKP5, the tyrosine-specific hematopoietic protein-tyrosine phosphatase, and the serine/threonine-specific PP2C? are all highly specific for the dephosphorylation of p38?, and the dephosphorylation rates were significantly affected by different phosphorylated states of p38?; (iii) the N-terminal domain of MPK5 has no effect on enzyme catalysis, whereas deletion of the MAP kinase-binding domain in MKP5 leads to a 370-fold decrease in kcat/Km for the dephosphorylation of p38?. This study has thus revealed the quantitative contributions of phosphorylation of Thr, Tyr, or both to the activation of p38? and to the substrate specificity for various phosphatases. PMID:18669639

Zhang, Yuan-Yuan; Mei, Zi-Qing; Wu, Jia-Wei; Wang, Zhi-Xin



Application of Electron Transfer Dissociation Mass Spectrometry in Analyses of Non-enzymatically Glycated Peptides  

SciTech Connect

Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in diabetes mellitus research, particularly in the context of development of diabetic complications. The fragmentation behavior of glycated peptides produced from reaction of D-glucose with lysine residues was investigated by electron transfer dissociation (ETD) and collision induced dissociation (CID) tandem mass spectrometry. It was found that high abundance ions corresponding to various degrees of neutral water losses, as well as furylium ion production, dominate the CID spectra, and that the sequence informative b and y ions were rarely observed when Amadori-modified peptides were fragmented. Contrary to what was observed under CID conditions, ions corresponding to neutral losses of water or furylium ion production were not observed in the ETD spectra. Instead, abundant and almost complete series of c and z type ions were observed regardless of whether the modification site was located in the middle of the sequence or close to the N-terminus, greatly facilitating the peptide sequencing. This study strongly suggests that ETD is a better technique for proteomics studies of non-enzymatically glycated peptides and proteins.

Zhang, Qibin; Frolov, Andrej; Tang, Ning; Hoffman, Ralf; van der Goor, Tom; Metz, Thomas O.; Smith, Richard D.



The power of vanadate in crystallographic investigations of phosphoryl transfer enzymes.  


The formation of transition state mimics of phosphoryl transfer reactions with the metal oxoanion vanadate is a powerful technique in macromolecular crystallography. The tendency of vanadate to form pentacovalent complexes exhibiting trigonal bipyramidal geometry makes this compound a close approximation of the transition state for such reactions. In many cases, vanadate complexes provide the most accurate visualization of the transition state that can be reasonably achieved. A survey of the Protein Data Bank reveals that a relatively small number of structures (39, representing 23 unique proteins) include vanadate, yet these structures represent four of the six E.C. categories of enzymes, and were obtained in crystals with pH values ranging from 5.0 to 7.8. Vanadate has additional advantages over other compounds such as aluminum fluoride, beryllium fluoride and nitrate used for visualization of transition state mimics in that vanadate readily forms covalent bonds with a variety of ligands and has produced a wider variety of transition state mimics. Given the hundreds of crystal structures that have been solved for phosphoryl transfer enzymes, it is surprising that vanadate has not been used more frequently for visualization of transition state analogs. We propose that an opportunity exists for vanadate to become a more commonly utilized component of the macromolecular crystallographer's toolbox. PMID:15556602

Davies, Douglas R; Hol, Wim G J



Formycin triphosphate-terbium complex: a novel spectroscopic probe for phosphoryl transfer enzymes.  


The conditions under which the fluorescent pyrazolopyrimidine nucleotide formycin A triphosphate (7-amino-3-(beta-D-(5'- tripolyphosphate)ribofuranosyl)pyrazolo[4,3-d]pyrimidine, FTP) forms a 1:1 complex in solution with Tb3+ have been characterized. The complex has a dissociation constant of approx. 10(-7) M. Within the complex, the luminescence of Tb3+ is dramatically sensitized by energy transfer from formycin. The value for 50% transfer efficiency, Förster's R0 (Förster, T. (1964) in Modern Quantum Chemistry (Sinanoglu, O., ed.), pp. 93-137, Academic Press, New York) was determined to be 3.34 +/- 0.4 A, and the effective distance between the donor and acceptor transition dipoles, R, in the complex was estimated to be 6.6 +/- 1.0 A. The quantum yield of Tb3+ in the complex is sensitive to the number of O-H oscillators bound to the Tb3+, which allows determination of the number of waters bound to it (approx. 4). Preliminary results show that the complex binds to the phosphoryl transfer enzyme hexokinase in the presence of the glucose analogs N-acetylglucosamine, frucose and xylose, which are not phosphorylated by the enzyme. The binding occurs with a loss of energy efficiency consistent with a new distance from the effective transition dipole of formycin to that of terbium of approx. 9.6 A. The FTP-terbium complex can be used as both a spectroscopic and an X-ray diffraction probe. Studies with this compound should be most valuable for correlating solution and crystallographic data. PMID:3689792

Kirk, W R; Amzel, L M



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

NASA Astrophysics Data System (ADS)

The synthesis of uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P) has been accomplished under simulated prebiotic conditions using urea and cyanamide, two condensing agents considered to have been present on the primitive Earth. The synthesis of UDPG was carried out by reacting G1P and UTP at 70 °C for 24 hours in the presence of the condensing agents in an aqueous medium. CDP-choline was obtained under the same conditions by reacting choline phosphate and CTP. G1P and G6P were synthesized from glucose and inorganic phosphate at 70 °C for 16 hours. Separation and identification of the reaction products have been performed by paper chromatography, thin layer chromatography, enzymatic analysis and ion pair reverse phase high performance liquid chromatography. These results suggest that metabolic intermediates could have been synthesized on the primitive Earth from simple precursors by means of prebiotic condensing agents.

Mar, A.; Dworkin, J.; Oró, J.



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


The synthesis of uridine diphosphate glucose (UDPG), cytidine diphosphate choline (CDP-choline), glucose-1-phosphate (G1P) and glucose-6-phosphate (G6P) has been accomplished under simulated prebiotic conditions using urea and cyanamide, two condensing agents considered to have been present on the primitive Earth. The synthesis of UDPG was carried out by reacting G1P and UTP at 70 degrees C for 24 hours in the presence of the condensing agents in an aqueous medium. CDP-choline was obtained under the same conditions by reacting choline phosphate and CTP X G1P and G6P were synthesized from glucose and inorganic phosphate at 70 degrees C for 16 hours. Separation and identification of the reaction products have been performed by paper chromatography, thin layer chromatography, enzymatic analysis and ion pair reverse phase high performance liquid chromatography. These results suggest that metabolic intermediates could have been synthesized on the primitive Earth from simple precursors by means of prebiotic condensing agents. PMID:2819807

Mar, A; Dworkin, J; Oró, J



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

PubMed Central

Enzymes stabilize transition states of reactions while limiting binding to ground states, as is generally required for any catalyst. Alkaline Phosphatase (AP) and other nonspecific phosphatases are some of Nature's most impressive catalysts, achieving preferential transition state over ground state stabilization of more than 1022-fold while utilizing interactions with only the five atoms attached to the transferred phosphorus. We tested a model that AP achieves a portion of this preference by destabilizing ground state binding via charge repulsion between the anionic active site nucleophile, Ser102, and the negatively charged phosphate monoester substrate. Removal of the Ser102 alkoxide by mutation to glycine or alanine increases the observed Pi affinity by orders of magnitude at pH 8.0. To allow precise and quantitative comparisons, the ionic form of bound Pi was determined from pH dependencies of the binding of Pi and tungstate, a Pi analog lacking titratable protons over the pH range of 5–11, and from the 31P chemical shift of bound Pi. The results show that the Pi trianion binds with an exceptionally strong femtomolar affinity in the absence of Ser102, show that its binding is destabilized by ?108-fold by the Ser102 alkoxide, and provide direct evidence for ground state destabilization. Comparisons of X-ray crystal structures of AP with and without Ser102 reveal the same active site and Pi binding geometry upon removal of Ser102, suggesting that the destabilization does not result from a major structural rearrangement upon mutation of Ser102. Analogous Pi binding measurements with a protein tyrosine phosphatase suggest the generality of this ground state destabilization mechanism. Our results have uncovered an important contribution of anionic nucleophiles to phosphoryl transfer catalysis via ground state electrostatic destabilization and an enormous capacity of the AP active site for specific and strong recognition of the phosphoryl group in the transition state. PMID:23843744

Andrews, Logan D.; Fenn, Tim D.; Herschlag, Daniel



Triple isotopic labeling and kinetic isotope effects: Exposing H-transfer steps in enzymatic systems†  

PubMed Central

Kinetic isotope effect (KIE) studies can provide insight into the mechanism and kinetics of specific chemical steps in complex catalytic cascades. Recent results from hydrogen KIE measurements have examined correlations between enzyme dynamics and catalytic function, leading to a surge of studies in this area. Unfortunately, most enzymatic H-transfer reactions are not rate-limiting and the observed KIEs do not reliably reflect the intrinsic KIEs on the chemical step of interest. Given their importance to understanding the chemical step under study, accurate determination of the intrinsic KIE from the observed data is essential. In 1975, Northrop developed an elegant method to assess intrinsic KIEs from their observed values [Northrop, D. B. (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions, Biochemistry, 14, 2644–2651]. The Northrop method involves KIE measurements using all three hydrogen isotopes, where one of them serves as the reference isotope. This method has been successfully used with different combinations of observed KIEs over the years but criteria for a rational choice of reference isotope have never before been experimentally determined. Here we compare different reference isotopes (and hence distinct experimental designs) using the reduction of dihydrofolate and dihydrobiopterin by two dissimilar enzymes as model reactions. A number of isotopic labeling patterns have been applied to facilitate the comparative study of reference isotopes. The results demonstrate the versatility of the Northrop method, and that such experiments are limited only by synthetic techniques, availability of starting materials, and the experimental error associated with the use of distinct combinations of isotopologues. PMID:21688781

Sen, Arundhuti; Yahashiri, Atsushi; Kohen, Amnon



Molecular Basis of 1,6-Anhydro Bond Cleavage and Phosphoryl Transfer by Pseudomonas aeruginosa 1,6-Anhydro-N-acetylmuramic Acid Kinase*  

PubMed Central

Anhydro-N-acetylmuramic acid kinase (AnmK) catalyzes the ATP-dependent conversion of the Gram-negative peptidoglycan (PG) recycling intermediate 1,6-anhydro-N-acetylmuramic acid (anhMurNAc) to N-acetylmuramic acid-6-phosphate (MurNAc-6-P). Here we present crystal structures of Pseudomonas aeruginosa AnmK in complex with its natural substrate, anhMurNAc, and a product of the reaction, ADP. AnmK is homodimeric, with each subunit comprised of two subdomains that are separated by a deep active site cleft, which bears similarity to the ATPase core of proteins belonging to the hexokinase-hsp70-actin superfamily of proteins. The conversion of anhMurNAc to MurNAc-6-P involves both cleavage of the 1,6-anhydro ring of anhMurNAc along with addition of a phosphoryl group to O6 of the sugar, and thus represents an unusual enzymatic mechanism involving the formal addition of H3PO4 to anhMurNAc. The structural complexes and NMR analysis of the reaction suggest that a water molecule, activated by Asp-182, attacks the anomeric carbon of anhMurNAc, aiding cleavage of the 1,6-anhydro bond and facilitating the capture of the ? phosphate of ATP by O6 via an in-line phosphoryl transfer. AnmK is active only against anhMurNAc and not the metabolically related 1,6-anhydro-N-acetylmuramyl peptides, suggesting that the cytosolic N-acetyl-anhydromuramyl-l-alanine amidase AmpD must first remove the stem peptide from these PG muropeptide catabolites before anhMurNAc can be acted upon by AnmK. Our studies provide the foundation for a mechanistic model for the dual activities of AnmK as a hydrolase and a kinase of an unusual heterocyclic monosaccharide. PMID:21288904

Bacik, John-Paul; Whitworth, Garrett E.; Stubbs, Keith A.; Yadav, Anuj K.; Martin, Dylan R.; Bailey-Elkin, Ben A.; Vocadlo, David J.; Mark, Brian L.



Physical Properties and Enzymatic Digestibility of Phosphorylated ae, wx , and Normal Maize Starch Prepared at Different pH Levels 1  

Microsoft Academic Search

Cereal Chem. 76(6):938-943 Phosphorylated starches were prepared with sodium tripolyphosphate (STPP) at pH 6, 8, and 10 from waxy (wx, 3.3% amylose), normal (22.4% amylose), and two high-amylose (ae, 47 and 66% amylose) maize starches. After phosphorylation, the gelatinization peak temperature ( Tp) decreased and pasting peak viscosity (PV) increased for all the starches except wx, which showed a slight

Huijun Liu; Lawrence Ramsden; Harold Corke



Cardiac mitochondrial matrix and respiratory complex protein phosphorylation  

PubMed Central

It has become appreciated over the last several years that protein phosphorylation within the cardiac mitochondrial matrix and respiratory complexes is extensive. Given the importance of oxidative phosphorylation and the balance of energy metabolism in the heart, the potential regulatory effect of these classical signaling events on mitochondrial function is of interest. However, the functional impact of protein phosphorylation and the kinase/phosphatase system responsible for it are relatively unknown. Exceptions include the well-characterized pyruvate dehydrogenase and branched chain ?-ketoacid dehydrogenase regulatory system. The first task of this review is to update the current status of protein phosphorylation detection primarily in the matrix and evaluate evidence linking these events with enzymatic function or protein processing. To manage the scope of this effort, we have focused on the pathways involved in energy metabolism. The high sensitivity of modern methods of detecting protein phosphorylation and the low specificity of many kinases suggests that detection of protein phosphorylation sites without information on the mole fraction of phosphorylation is difficult to interpret, especially in metabolic enzymes, and is likely irrelevant to function. However, several systems including protein translocation, adenine nucleotide translocase, cytochrome c, and complex IV protein phosphorylation have been well correlated with enzymatic function along with the classical dehydrogenase systems. The second task is to review the current understanding of the kinase/phosphatase system within the matrix. Though it is clear that protein phosphorylation occurs within the matrix, based on 32P incorporation and quantitative mass spectrometry measures, the kinase/phosphatase system responsible for this process is ill-defined. An argument is presented that remnants of the much more labile bacterial protein phosphoryl transfer system may be present in the matrix and that the evaluation of this possibility will require the application of approaches developed for bacterial cell signaling to the mitochondria. PMID:22886415

Covian, Raul



From 10,000 to 1: Selective synthesis and enzymatic evaluation of fluorescence resonance energy transfer peptides as specific substrates for chymopapain  

Microsoft Academic Search

The synthesis and detailed enzymatic analysis of fluorescence resonance energy transfer (FRET)-based peptides as substrates for chymopapain are reported. The design of these substrates arose from a massively parallel high-throughput microarray screening process using peptide nucleic acid (PNA) encoding technology, allowing the identification of detailed substrate specificities of any protease. Two peptides so identified with chymopapain were observed to be

Juan J. Diaz-Mochon; Songsak Planonth; Mark Bradley



Using positive-ion electrospray ionization mass spectrometry and H/D exchange study phosphoryl group transfer reactions involved in amino acid ester isopropyl phosphoramidates of Brefeldin A.  


As mini-chemical models, amino acid ester isopropyl phosphoramidates of Brefeldin A (compounds 2a-2d) were synthesized and investigated by electrospray ionization tandem mass spectrometry in combination with H/D exchange. To further confirm the fragments's structures, off-line Fourier transform resonance tandem mass spectrometry (FT-ICR-MS/MS) was also performed. The fragmentation rules of compounds 2a-2d have been summarized and the plausible schemes for the fragmentation pathways were proposed. In this study, one dephosphorylated ion and two phosphorylated ions were observed in ESI-MS(2) spectra of [M+Na](+) ions for compounds 2a-2d. The possible mechanisms about phosphorylation and dephosphorylation were proposed and confirmed by H/D exchange. For the "dephosphorylation" rearrangement, a nitrogen atom was migrated from the phosphoryl group to the carbon atom of Brefeldin A's backbone with losing a molecule of C3H7PO3 (122Da). For the "phosphorylation" rearrangement, an oxygen atom of one phosphoryl group attacked the sideward phosphorus atom to form a nine-member ring intermediate, then two steps of CH covalent bond cleavage with consecutive migration of hydrogen atom to lose a molecule of C16H20O2 (244Da). The two proposed rearrangement mechanisms about phosphoryl group transfer might be valuable for the structure analysis of other analogs and provide insights into elucidating the dynamic process of the phosphorylation-dephosphorylation of proteins. PMID:25467484

Fang, Mei-Juan; Zhang, He; Liao, Chao; Qiu, Ying-Kun; Fang, Hua; Zheng, Zhen-Yu; Gao, Xiang; Zhao, Yu-Fen; Wu, Zhen



Lewis acid catalysis of phosphoryl transfer from a copper(II)-NTP complex in a kinase ribozyme  

PubMed Central

The chemical strategies used by ribozymes to enhance reaction rates are revealed in part from their metal ion and pH requirements. We find that kinase ribozyme K28(1-77)C, in contrast with previously characterized kinase ribozymes, requires Cu2+ for optimal catalysis of thiophosphoryl transfer from GTP?S. Phosphoryl transfer from GTP is greatly reduced in the absence of Cu2+, indicating a specific catalytic role independent of any potential interactions with the GTP?S thiophosphoryl group. In-line probing and ATP?S competition both argue against direct Cu2+ binding by RNA; rather, these data establish that Cu2+ enters the active site within a Cu2+•GTP?S or Cu2+•GTP chelation complex, and that Cu2+•nucleobase interactions further enforce Cu2+ selectivity and position the metal ion for Lewis acid catalysis. Replacing Mg2+ with [Co(NH3)6]3+ significantly reduced product yield, but not kobs, indicating that the role of inner-sphere Mg2+ coordination is structural rather than catalytic. Replacing Mg2+ with alkaline earths of increasing ionic radii (Ca2+, Sr2+ and Ba2+) gave lower yields and approximately linear rates of product accumulation. Finally, we observe that reaction rates increased with pH in log-linear fashion with an apparent pKa = 8.0 ± 0.1, indicating deprotonation in the rate-limiting step. PMID:23358821

Biondi, Elisa; Poudyal, Raghav R.; Forgy, Joshua C.; Sawyer, Andrew W.; Maxwell, Adam W. R.; Burke, Donald H.



Miniature direct electron transfer based sulphite/oxygen enzymatic fuel cells.  


A direct electron transfer (DET) based sulphite/oxygen biofuel cell is reported that utilises human sulphite oxidase (hSOx) and Myrothecium verrucaria bilirubin oxidase (MvBOx) and nanostructured gold electrodes. For bioanode construction, the nanostructured gold microelectrodes were further modified with 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester) to which polyethylene imine was covalently attached. hSOx was adsorbed onto this chemically modified nanostructured electrode with high surface loading of electroactive enzyme and in presence of sulphite high anodic bioelectrocatalytic currents were generated with an onset potential of 0.05V vs. NHE. The biocathode contained MvBOx directly adsorbed to the deposited gold nanoparticles for cathodic oxygen reduction starting at 0.71V vs. NHE. Both enzyme electrodes were integrated to a DET-type biofuel cell. Power densities of 8 and 1?Wcm(-2) were achieved at 0.15V and 0.45V of cell voltages, respectively, with the membrane based biodevices under aerobic conditions. PMID:25460879

Zeng, T; Pankratov, D; Falk, M; Leimkühler, S; Shleev, S; Wollenberger, U



An enzymatic bridge between carbohydrate and amino acid metabolism: regulation of glutamate dehydrogenase by reversible phosphorylation in a severe hypoxia-tolerant crayfish.  


Glutamate dehydrogenase (GDH) (EC is a crucial enzyme involved in bridging two metabolic pathways, gating the use of glutamate for either amino acid metabolism, or carbohydrate metabolism. The present study investigated GDH from tail muscle of the freshwater crayfish Orconectes virilis exploring changes to kinetic properties, phosphorylation levels and structural stability between two forms of the enzyme (aerobic control and 20-h severe hypoxic). Evidence indicated that GDH was converted to a high phosphate form under oxygen limitation. ProQ Diamond phosphoprotein staining showed a 42% higher bound phosphate content on GDH from muscle of severely hypoxic crayfish compared with the aerobic form, and treatment of this GDH with commercial phosphatase (alkaline phosphatase), and treatments that stimulated the activities of different endogenous protein phosphatases (stimulating PP1 + PP2A, PP2B, and PP2C) yielded significant increases in the fold activation by ADP of GDH from both control and severe hypoxic conditions. By contrast, stimulation of the activities of endogenous protein kinases (AMPK, PKA or CaMK) significantly reduced the ADP fold activation from control animals. The physiological consequence of severe hypoxia-induced GDH phosphorylation may be to suppress GDH activity under low oxygen, shutting off this critical bridge point between two metabolic pathways. PMID:22076534

Dawson, Neal J; Storey, Kenneth B



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

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.

Barry, R.J.



Enzymatic regeneration of adenosine triphosphate cofactor  

NASA Technical Reports Server (NTRS)

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.

Marshall, D. L.



Spin chemistry of enzymatic processes  

NASA Astrophysics Data System (ADS)

The results of spin chemistry studies of the single electron transfer step and the role of spin states of paramagnetic intermediates in the enzymatic oxidation of organic substrates induced by horseradish peroxidase are generalised. Detailed theoretical description of the approach used to analyse the magnetic field effects in enzymatic multispin systems is presented. Prospects for the use of spin chemistry techniques for elucidation of the mechanisms of radical steps of enzymatic processes are considered.

Afanasyeva, M. S.; Purtov, P. A.; Taraban, Mark B.; Leshina, Tatyana V.



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

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.

Kajikawa, Takao; Kataoka, Kunishige [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan)] [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan); Sakurai, Takeshi, E-mail: [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan)] [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan)



Summarizing lecture: factors influencing enzymatic H-transfers, analysis of nuclear tunnelling isotope effects and thermodynamic versus specific effects  

PubMed Central

In the articles in this Discussion, a wide variety of topics are treated, including reorganization energy, initially introduced for electron transfers (‘environmentally assisted tunnelling’), nuclear tunnelling, H/D and C12/C13 kinetic isotope effects (KIEs), the effect of changes of distal and nearby amino acid residues using site-directed mutagenesis, and dynamics versus statistical effects. A coordinate-free form of semi-classical theory is used to examine topics on data such as tunnelling versus ‘over-the-barrier’ paths and temperature and pressure effects on KIEs. The multidimensional semi-classical theory includes classically allowed and classically forbidden transitions. More generally, we address the question of relating kinetic to thermodynamic factors, as in the electron transfer field, so learning about specific versus thermodynamic effects in enzyme catalysis and KIEs. PMID:16873131

Marcus, R.A



Catalytic metal ions and enzymatic processing of DNA and RNA.  


Conspectus Two-metal-ion-dependent nucleases cleave the phosphodiester bonds of nucleic acids via the two-metal-ion (2M) mechanism. Several high-resolution X-ray structures portraying the two-metal-aided catalytic site, together with mutagenesis and kinetics studies, have demonstrated a functional role of the ions for catalysis in numerous metallonucleases. Overall, the experimental data confirm the general mechanistic hypothesis for 2M-aided phosphoryl transfer originally reported by Steitz and Steitz ( Proc. Natl. Acad. Sci. U.S.A. 1993 , 90 ( 14 ), 6498 - 6502 ). This seminal paper proposed that one metal ion favors the formation of the nucleophile, while the nearby second metal ion facilitates leaving group departure during RNA hydrolysis. Both metals were suggested to stabilize the enzymatic transition state. Nevertheless, static X-ray structures alone cannot exhaustively unravel how the two ions execute their functional role along the enzymatic reaction during processing of DNA or RNA strands when moving from reactants to products, passing through metastable intermediates and high-energy transition states. In this Account, we discuss the role of multiscale molecular simulations in further disclosing mechanistic insights of 2M-aided catalysis for two prototypical enzymatic targets for drug discovery, namely, ribonuclease H (RNase H) and type II topoisomerase (topoII). In both examples, first-principles molecular simulations, integrated with structural data, emphasize a cooperative motion of the bimetal motif during catalysis. The coordinated motion of both ions is crucial for maintaining a flexible metal-centered structural architecture exquisitely tailored to accommodate the DNA or RNA sugar-phosphate backbone during phosphodiester bond cleavage. Furthermore, our analysis of RNase H and the N-terminal domain (PAN) of influenza polymerase shows that classical molecular dynamics simulations coupled with enhanced sampling techniques have contributed to describe the modulatory effect of metal ion concentration and metal uptake on the 2M mechanism and efficiency. These aspects all point to the emerging and intriguing role of additional adjacent ions potentially involved in the modulation of phosphoryl transfer reactions and enzymatic turnover in 2M-catalysis, as recently observed experimentally in polymerase ? and homing endonuclease I-DmoI. These computational results, integrated with experimental findings, describe and reinforce the nascent concept of a functional and cooperative dynamics of the catalytic metal ions during the 2M-dependent enzymatic processing of DNA and RNA. Encouraged by the insights provided by computational approaches, we foresee further experiments that will feature the functional and joint dynamics of the catalytic metal ions for nucleic acid processing. This could impact the de novo design of artificial metallonucleases and the rational design of potent metal-chelating inhibitors of pharmaceutically relevant enzymes. PMID:25590654

Palermo, Giulia; Cavalli, Andrea; Klein, Michael L; Alfonso-Prieto, Mercedes; Dal Peraro, Matteo; De Vivo, Marco



Insights into the Phosphoryl Transfer Catalyzed by cAMP-Dependent Protein Kinase: An X-ray Crystallographic Study of Complexes with Various Metals and Peptide Substrate SP20  

PubMed Central

X-ray structures of several ternary substrate and product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with different bound metal ions. In the PKAc complexes, Mg2+, Ca2+, Sr2+, and Ba2+ metal ions could bind to the active site and facilitate the phosphoryl transfer reaction. ATP and a substrate peptide (SP20) were modified, and the reaction products ADP and the phosphorylated peptide were found trapped in the enzyme active site. Finally, we determined the structure of a pseudo-Michaelis complex containing Mg2+, nonhydrolyzable AMP-PCP (?,?-methyleneadenosine 5?-triphosphate) and SP20. The product structures together with the pseudo-Michaelis complex provide snapshots of different stages of the phosphorylation reaction. Comparison of these structures reveals conformational, coordination, and hydrogen bonding changes that might occur during the reaction and shed new light on its mechanism, roles of metals, and active site residues. PMID:23672593



Identification of a potential general acid/base in the reversible phosphoryl transfer reactions catalyzed by tyrosine recombinases: Flp H305.  


Flp provides a unique opportunity to apply the tools of chemical biology to phosphoryl transfer reactions. Flp and other tyrosine recombinases catalyze site-specific DNA rearrangements via a phosphotyrosine intermediate. Unlike most related enzymes, Flp's nucleophilic tyrosine derives from a different protomer than the remainder of its active site. Because the tyrosine can be supplied exogenously, nonnatural synthetic analogs can be used. Here we examine the catalytic role of Flp's conserved H305. DNA cleavage was studied using a peptide containing either tyrosine (pKa congruent with 10) or 3-fluoro-tyrosine (pKa congruent with 8.4). Religation was studied using DNA substrates with 3'-phospho-cresol (pKa congruent with 10) or 3'-para-nitro-phenol (pKa congruent with 7.1). In both cases, the tyrosine analog with the lower pKa specifically restored the activity of an H305 mutant. These results provide experimental evidence that this conserved histidine functions as a general acid/base catalyst in tyrosine recombinases. PMID:17317566

Whiteson, Katrine L; Chen, Yu; Chopra, Neeraj; Raymond, Amy C; Rice, Phoebe A



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


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

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



Phosphorylation of Raf1 Serine 338Serine 339 Is an Essential Regulatory Event for Ras-Dependent Activation and Biological Signaling  

Microsoft Academic Search

Activation of the Raf serine\\/threonine protein kinases is tightly regulated by multiple phosphorylation events. Phosphorylation of either tyrosine 340 or 341 in the catalytic domain of Raf-1 has been previously shown to induce the ability of the protein kinase to phosphorylate MEK. By using a combination of mitogenic and enzymatic assays, we found that phosphorylation of the adjacent residue, serine




The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VII. Chemical synthesis by phosphorylation of 6-thioguanosine 5'-monophosphate, 5'-diphosphate and 5'-triphosphate, and their purification and identification by reversed-phase/ion-pair high-performance liquid chromatography and by various enzymatic assays.  


A fast and reliable two-step method has been established for the chemical synthesis of 6-thioguanosine 5'-monophosphate, 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate starting from the ribonucleoside. In the first step, 6-thioguanosine dissolved in triethyl phosphate, at high yield reacts with phosphorus oxide trichloride to 6-thioguanosine 5'-monophosphate which is purified by anion-exchange chromatography on DEAE-Sephadex using a step gradient of hydrochloric acid. In the second step, 6-thioguanosine 5'-monophosphate dissolved in water, reacts with phosphoric acid in the presence of pyridine/dicyclohexyl carbodiimide and is converted to 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate which are separated from each other and from the 6-thioguanosine 5'-monophosphate by anion-exchange chromatography on DEAE-Sephadex using a gradient of ammonium bicarbonate. Material from each step of the preparation procedure is separated by reversed-phase HPLC chromatography and analyzed for its free ribonucleoside content, 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and small amounts of unidentified phosphorylated compounds. The purity of the final preparations and the identity of each 6-thioguanosine 5'-phosphate are proven by highly specific enzymatic peak-shifting/HPLC analyses using alkaline phosphatase, 5'-nucleotidase, pyruvate kinase, nucleoside diphosphate kinase and combined hexokinase/glucose 6-phosphate dehydrogenase. PMID:2306458

Breter, H J; Mertes, H



Enzymatic hydrolysis of cellulose  

SciTech Connect

This book reviews the theory and application of enzymatic hydrolysis of cellulosic biomass; with implications for genetic engineering techniques. State of the art and potential industrial processes are detailed, including high productivity fermentation systems for the production of ethanol. Contents: Theory of Enzymatic Hydrolysis; Production of Cellulase and Xylanase; Hydrolysis of Agricultural Residues; Enzymatic Hydrolysis Processes; High Productivity Ethanol Fermentation; Ethanol Economics.

Wilke, C.R.



Requirements for Phosphorylation of MAP Kinase During Meiosis in Xenopus Oocytes  

Microsoft Academic Search

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

James Posada; Jonathan A. Cooper



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

PubMed Central

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

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



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

SciTech Connect

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.

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



Toward a systems-level view of dynamic phosphorylation networks  

PubMed Central

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

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



Microwave-mediated enzymatic modifications of DNA.  


Here we report microwave-induced specific cleavage, ligation, dephosphorylation, and phosphorylation of nucleic acids catalyzed by restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase, and calf intestinal alkaline phosphatase. The microwave-mediated method has dramatically reduced the reaction time to 20 to 50s. In control experiments, the same reactions failed to give the desired reaction products when carried out in the same time periods but without microwave irradiation. Because the microwave method is rapid, it could be a useful alternative to the time-consuming conventional procedure for enzymatic modification of DNA. PMID:25447491

Das, Rakha Hari; Ahirwar, Rajesh; Kumar, Saroj; Nahar, Pradip



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

PubMed Central

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 Ĺ 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 Ĺ 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

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



Enzymatic desulfurization of coal  

SciTech Connect

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.

Marquis, J.K. (Boston Univ., MA (United States). School of Medicine); Kitchell, J.P. (Holometrix, Inc., Cambridge, MA (United States))



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


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

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



Reversible phosphorylation in mitochondria.  

E-print Network

??Mitochondria represent an underappreciated site of regulation by reversible phosphorylation. Our work has focused on the identification of proteins involved in regulating mitochondria by reversible… (more)

Rardin, Matthew James



Protein Scaffolds Can Enhance the Bistability of Multisite Phosphorylation Systems  

PubMed Central

The phosphorylation of a substrate at multiple sites is a common protein modification that can give rise to important structural and electrostatic changes. Scaffold proteins can enhance protein phosphorylation by facilitating an interaction between a protein kinase enzyme and its target substrate. In this work we consider a simple mathematical model of a scaffold protein and show that under specific conditions, the presence of the scaffold can substantially raise the likelihood that the resulting system will exhibit bistable behavior. This phenomenon is especially pronounced when the enzymatic reactions have sufficiently large KM, compared to the concentration of the target substrate. We also find for a closely related model that bistable systems tend to have a specific kinetic conformation. Using deficiency theory and other methods, we provide a number of necessary conditions for bistability, such as the presence of multiple phosphorylation sites and the dependence of the scaffold binding/unbinding rates on the number of phosphorylated sites. PMID:22737061

Chan, Carlo; Liu, Xinfeng; Wang, Liming; Bardwell, Lee



Reduction of Cysteine Sulfinic Acid in Peroxiredoxin by Sulfiredoxin Proceeds Directly Through a Sulfinic Phosphoryl Ester Intermediate  

SciTech Connect

Sulfiredoxin (Srx) catalyzes a novel enzymatic reaction, the reduction of protein cysteine sulfinic acid, Prx-SO{sub 2}{sup -}. This reaction is unique to the typical 2-Cys peroxiredoxins (Prx) and plays a role in peroxide-mediated signaling by regulating the activity of Prxs. Two mechanistic schemes have been proposed that differ regarding the first step of the reaction. This step involves either the direct transfer of the {gamma}-phosphate of ATP to the Prx molecule or through Srx acting as a phosphorylated intermediary. In an effort to clarify this step of the Srx reaction, we have determined the 1.8 Angstroms resolution crystal structure of Srx in complex with ATP and Mg2+. This structure reveals the role of the Mg2+ ion to position the {gamma}-phosphate toward solvent, thus preventing an in-line attack by the catalytic residue Cys-99 of Srx. A model of the quaternary complex is consistent with this proposal. Furthermore, phosphorylation studies on several site-directed mutants of Srx and Prx, including the Prx-Asp mimic of the Prx-SO{sub 2}{sup -} species, support a mechanism where phosphorylation of Prx-SO{sub 2}{sup -} is the first chemical step.

Jonsson,T.; Murray, M.; Johnson, L.; Lowther, W.



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

PubMed Central

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

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



Calcium Activation of the Ca-ATPase Enhances Conformational Heterogeneity Between Nucleotide Binding and Phosphorylation Domains  

SciTech Connect

High-resolution crystal structures obtained in two conformations of the Ca-ATPase suggest that a large-scale rigid-body domain reorientation of approximately 50 involving the nucleotide-binding (N) domain is required to permit the transfer of the -phosphoryl group of ATP to Asp351 in the phosphorylation (P) domain during coupled calcium transport. However, variability observed in the orientation of the N-domain relative to the P-domain in both different crystal structures of the Ca-ATPase following calcium activation, and structures of other P-type ATPases, suggests the presence of conformational heterogeneity in solution which may be modulated by contact interactions within the crystal. Therefore, to address the extent of conformational heterogeneity between these domains in solution, we have used fluorescence resonance energy transfer (FRET) to measure the spatial separation and conformational heterogeneity between donor (i.e., 5-[[2-[(iodoacetyl)amino]ethyl]amino] naphthalene-1-sulfonic acid) and acceptor (i.e., fluorescein 5-isothiocyanate) chromophores covalently bound to the P- and N-domains, respectively, within the Ca-ATPase stabilized in different enzymatic states associated with the transport cycle. In comparison to the unliganded enzyme, the spatial separation and conformational heterogeneity between these domains is unaffected by enzyme phosphorylation. However, calcium-activation results in a 3.4 increase in the average spatial separation, which increases from 29.4 to 32.8 , in good agreement with the high-resolution structures where these sites are respectively separated by 31.6 (1 IWO.pdb) and 35.9 (1EUL.pdb). Thus, the crystal structures accurately reflect the average solution structures of the Ca-ATPase. However, there is substantial conformational heterogeneity for all enzyme states measured, indicating that formation of catalytically important transition states involves a subpopulation of enzyme intermediates. These results suggest that the approximation of cytoplasmic domains accompanying calcium transport, as observed from crystal structures, occurs in solution within the context of large amplitude domain motions important for catalysis. These domain motions permit substrate (ATP) access and product (ADP) egress, and enhance the probability of a productive juxtaposition of the -phosphoryl moiety of ATP with Asp351 on the phosphosphorylation domain to facilitate enzyme phosphorylation and calcium transport.

Chen, Baowei; Squier, Thomas C.; Bigelow, Diana J.



Enzymatic DNA molecules  

NASA Technical Reports Server (NTRS)

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.

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



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

E-print Network

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 ...

Gonzalez-Vera, Juan A.


Enzymatic desulfurization of coal  

SciTech Connect

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)

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



Enzymatic production of cyclodextrins.  


Cyclodextrins (CD) are enzymatically modified starches with a wide range of applications in food, pharmaceutical and chemical industries, agriculture and environmental engineering. They are produced from starch via enzymatic conversion using cyclodextrin glycosyl transferases (CGTases) and partly alpha-amylases. Due to its low solubility in water, separation and purification of beta-CD is relatively easy compared to alpha- and gamma-CD. In recent years more economic processes for gamma-CD and especially alpha-CD production have been developed using improved CGTases and downstream processing. New purification steps, e.g. affinity adsorption, may reduce the use of complexing agents. The implementation of thermostable CGTases can simplify the production process and increase the selectivity of the reaction. A tabular overview of alpha-CD production processes is presented. PMID:12226716

Biwer, A; Antranikian, G; Heinzle, E



Screening for protein phosphorylation using nanoscale reactions on microdroplet arrays.  


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

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



Enzymatic temperature change indicator  


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.

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



Oxidative phosphorylation revisited.  


The fundamentals of oxidative phosphorylation and photophosphorylation are revisited. New experimental data on the involvement of succinate and malate anions respectively in oxidative phosphorylation and photophosphorylation are presented. These new data offer a novel molecular mechanistic explanation for the energy coupling and ATP synthesis carried out in mitochondria and chloroplast thylakoids. The mechanism does not suffer from the flaws in Mitchell's chemiosmotic theory that have been pointed out in many studies since its first appearance 50 years ago, when it was hailed as a ground-breaking mechanistic explanation of what is perhaps the most important process in cellular energetics. The new findings fit very well with the predictions of Nath's torsional mechanism of energy transduction and ATP synthesis. It is argued that this mechanism, based on at least 15 years of experimental and theoretical work by Sunil Nath, constitutes a fundamentally different theory of the energy conversion process that eliminates all the inconsistencies in Mitchell's chemiosmotic theory pointed out by other authors. It is concluded that the energy-transducing complexes in oxidative phosphorylation and photosynthesis are proton-dicarboxylic acid anion cotransporters and not simply electrogenic proton translocators. These results necessitate revision of previous theories of biological energy transduction, coupling, and ATP synthesis. The novel molecular mechanism is extended to cover ATP synthesis in prokaryotes, in particular to alkaliphilic and haloalkaliphilic bacteria, essentially making it a complete theory addressing mechanistic, kinetic, and thermodynamic details. Finally, based on the new interpretation of oxidative phosphorylation, quantitative values for the P/O ratio, the amount of ATP generated per redox package of the reduced substrates, are calculated and compared with experimental values for fermentation on different substrates. It is our hope that the presentation of oxidative phosphorylation and photophosphorylation from a wholly new perspective will rekindle scientific discussion of a key process in bioenergetics and catalyze new avenues of research in a truly interdisciplinary field. Biotechnol. Bioeng. 2015;112: 429-437. © 2014 Wiley Periodicals, Inc. PMID:25384602

Nath, Sunil; Villadsen, John



Determining in vivo Phosphorylation Sites using Mass Spectrometry  

PubMed Central

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

Breitkopf, Susanne B.; Asara, John M.



Cross-talk between GlcNAcylation and phosphorylation: site-specific phosphorylation dynamics in response to globally elevated O-GlcNAc.  


Protein GlcNAcylation serves as a nutrient/stress sensor to modulate the functions of many nuclear and cytoplasmic proteins. O-GlcNAc cycles on serine or threonine residues like phosphorylation, is nearly as abundant, and functions, at least partially, via its interplay with phosphorylation. Here, we describe changes in site-specific phosphorylation dynamics in response to globally elevated GlcNAcylation. By combining sequential phospho-residue enrichment, iTRAQ labeling, and high throughput mass spectrometric analyses, phosphorylation dynamics on 711 phosphopeptides were quantified. Based upon their insensitivity to phosphatase inhibition, we conclude that approximately 48% of these phosphorylation sites were not actively cycling in the conditions of the study. However, increased GlcNAcylation influenced phosphate stoichiometry at most of the sites that did appear to be actively cycling. Elevated GlcNAcylation resulted in lower phosphorylation at 280 sites and caused increased phosphorylation at 148 sites. Thus, the cross-talk or interplay between these two abundant posttranslational modifications is extensive, and may arises both by steric competition for occupancy at the same or proximal sites and by each modification regulating the other's enzymatic machinery. The phosphoproteome dynamics presented by this large set of quantitative data not only delineates the complex interplay between phosphorylation and GlcNAcyation, but also provides insights for more focused investigations of specific roles of O-GlcNAc in regulating protein functions and signaling pathways. PMID:18779572

Wang, Zihao; Gucek, Marjan; Hart, Gerald W



Enzymatic modification of schizophyllan.  


An enzymatic method was developed for the progressive modification of the polysaccharide schizophyllan. Fungal strains Hypocrea nigricans NRRL 62555, Penicillium crustosum NRRL 62558, and Penicillium simplicissimum NRRL 62550 were previously identified as novel sources of ?-endoglucanase with specificity towards schizophyllan. Concentrated enzyme preparations from these strains showed specific activities of 1.7-4.3 U ?-glucanase/mg protein. Using dilutions of these enzymes in time course digestions, schizophyllan was progressively modified to reduced molecular weight species. Glucose and oligosaccharides were found only in the more complete digestions, and thus modified schizophyllan can be produced quantitatively, without loss, to small molecules. Permethylation analysis confirmed that modified schizophyllan retains the fundamental linkage structure of native schizophyllan. Modified schizophyllan species showed progressively reduced viscosity profiles, and all exhibited pseudoplasticity in response to shear thinning. PMID:25335747

Leathers, Timothy D; Sutivisedsak, Nongnuch; Nunnally, Melinda S; Price, Neil P J; Stanley, April M



Phosphorylation-mediated PTEN conformational closure and deactivation revealed with protein semisynthesis  

PubMed Central

The tumor suppressor PIP3 phosphatase PTEN is phosphorylated on four clustered Ser/Thr on its C-terminal tail (aa 380–385) and these phosphorylations are proposed to induce a reduction in PTEN’s plasma membrane recruitment. How these phosphorylations affect the structure and enzymatic function of PTEN is poorly understood. To gain insight into the mechanistic basis of PTEN regulation by phosphorylation, we generated semisynthetic site-specifically tetra-phosphorylated PTEN using expressed protein ligation. By employing a combination of biophysical and enzymatic approaches, we have found that purified tail-phosphorylated PTEN relative to its unphosphorylated counterpart shows reduced catalytic activity and membrane affinity and undergoes conformational compaction likely involving an intramolecular interaction between its C-tail and the C2 domain. Our results suggest that there is a competition between membrane phospholipids and PTEN phospho-tail for binding to the C2 domain. These findings reveal a key aspect of PTEN’s regulation and suggest pharmacologic approaches for direct PTEN activation. DOI: PMID:23853711

Bolduc, David; Rahdar, Meghdad; Tu-Sekine, Becky; Sivakumaren, Sindhu Carmen; Raben, Daniel; Amzel, L Mario; Devreotes, Peter; Gabelli, Sandra B; Cole, Philip



Enzymatic and non-enzymatic mechanisms of dimesna metabolism.  


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

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



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

PubMed Central

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

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



Homogeneous, Heterogeneous, and Enzymatic Catalysis.  

ERIC Educational Resources Information Center

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)

Oyama, S. Ted; Somorjai, Gabor A.



Global Identification and Characterization of Both O-GlcNAcylation and Phosphorylation at the Murine Synapse*  

PubMed Central

O-linked N-acetylglucosamine (O-GlcNAc) is a dynamic, reversible monosaccharide modifier of serine and threonine residues on intracellular protein domains. Crosstalk between O-GlcNAcylation and phosphorylation has been hypothesized. Here, we identified over 1750 and 16,500 sites of O-GlcNAcylation and phosphorylation from murine synaptosomes, respectively. In total, 135 (7%) of all O-GlcNAcylation sites were also found to be sites of phosphorylation. Although many proteins were extensively phosphorylated and minimally O-GlcNAcylated, proteins found to be extensively O-GlcNAcylated were almost always phosphorylated to a similar or greater extent, indicating the O-GlcNAcylation system is specifically targeting a subset of the proteome that is also phosphorylated. Both PTMs usually occur on disordered regions of protein structure, within which, the location of O-GlcNAcylation and phosphorylation is virtually random with respect to each other, suggesting that negative crosstalk at the structural level is not a common phenomenon. As a class, protein kinases are found to be more extensively O-GlcNAcylated than proteins in general, indicating the potential for crosstalk of phosphorylation with O-GlcNAcylation via regulation of enzymatic activity. PMID:22645316

Trinidad, Jonathan C.; Barkan, David T.; Gulledge, Brittany F.; Thalhammer, Agnes; Sali, Andrej; Schoepfer, Ralf; Burlingame, Alma L.



Protein phosphorylation in stomatal movement.  


As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

Zhang, Tong; Chen, Sixue; Harmon, Alice C



Hydrolytic approach for production of deoxyribonucleoside- and ribonucleoside-5 -monophosphates and enzymatic synthesis of their polyphosphates.  


A new, simple, and ingenious method for enzymatic synthesis of deoxy- and ribonucleoside-5 -triphosphates (dNTP and NTP, respectively) has been developed. The method includes the following stages: hydrolysis of DNA with DNase and immobilized S1-nuclease, phosphorylation of the resulting deoxy- and ribonucleoside-5 -monophosphates (dNMP and NMP, respectively) with nucleotidyl kinase from Escherichia coli, and purification by chromatography of the synthesized dNTP and NTP. dNMP was phosphorylated using an ATP-regenerating system based on acetokinase from E. coli and lithium acetylphosphate. PMID:16457623

Bochkov, D V; Khomov, V V; Tolstikova, T G



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

PubMed Central

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

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



Enzymatic Synthesis of TDP-deoxysugars  

PubMed Central

Many biologically active bacterial natural products contain highly modified deoxysugar residues that are often critical for the activity of the parent compounds. Most of these deoxysugars are secondary metabolites that are biosynthesized in the form of nucleotide diphosphate (NDP) sugars prior to their transfer to natural product aglycones by glycosyltransferases. Over the past decade, many biosynthetic pathways that lead to the formation of these unusual sugars have been unraveled, and the mechanisms of many key enzymatic transformations involved in these pathways have been elucidated. However, obtaining workable quantities of NDP-deoxysugars for in vitro studies is often a difficult task. This limitation has hindered an in-depth investigation of the substrate specificity of deoxysugar biosynthetic enzymes, many of which are promiscuous with respect to their NDP-sugar substrates and are, thus, potentially useful catalysts for natural product glycoengineering. Presented in this review are procedures for the enzymatic synthesis and purification of a variety of NDP-deoxysugars, including some early intermediates in NDP-deoxysugar biosynthetic pathways, and highly modified NDP-deoxysugars that are late intermediates in their respective biosynthetic pathways. The procedures described herein could be used as general guidelines for the development of specific protocols for the synthesis of other NDP-deoxysugars. PMID:19362653

White-Phillip, Jessica; Thibodeaux, Christopher J.; Liu, Hung-wen



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

E-print Network

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 ...

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



Enzymatic Hydrolysis of Cellulosic Biomass  

SciTech Connect

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.

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



Phosphorylation of chicken growth hormone  

SciTech Connect

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

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



Emergence of pyridoxal phosphorylation through a promiscuous ancestor during the evolution of hydroxymethyl pyrimidine kinases.  


In the family of ATP-dependent vitamin kinases, several bifunctional enzymes that phosphorylate hydroxymethyl pyrimidine (HMP) and pyridoxal (PL) have been described besides enzymes specific towards HMP. To determine how bifunctionality emerged, we reconstructed the sequence of three ancestors of HMP kinases, experimentally resurrected, and assayed the enzymatic activity of their last common ancestor. The latter has ? 8-fold higher specificity for HMP due to a glutamine residue (Gln44) that is a key determinant of the specificity towards HMP, although it is capable of phosphorylating both substrates. These results show how a specific enzyme with catalytic promiscuity gave rise to current bifunctional enzymes. PMID:24952356

Castro-Fernandez, Victor; Bravo-Moraga, Felipe; Ramirez-Sarmiento, Cesar A; Guixe, Victoria



Enzymatic alcoholysis of palm kernel oil in n-hexane and SCCO 2  

Microsoft Academic Search

The use of biocatalysts in supercritical CO2 (SCCO2) has received widespread attention in recent years. Biocatalysts have the advantage of substrate specificity under mild reaction conditions and SCCO2 has several advantages over liquid solvents such as high solute diffusivities and low viscosity, which can accelerate mass transfer-limited enzymatic reactions. Concerning the enzymatic alcoholysis of vegetable oils, very little experimental data

D Oliveira; J. Vladimir Oliveira



STAT2 phosphorylation and signaling  

PubMed Central

STAT2 is an essential transcription factor in type I IFN mediated anti-viral and anti-proliferative signaling. STAT2 function is regulated by tyrosine phosphorylation, which is the trigger for STAT-dimerization, subsequent nuclear translocation, and transcriptional activation of IFN stimulated genes. Evidence of additional STAT2 phosphorylation sites has emerged as well as novel roles for STAT2 separate from the classical ISGF3-signaling. This review aims to summarize knowledge of phosphorylation-mediated STAT2-regulation and future avenues of related STAT2 research. PMID:24416652

Steen, Hĺkan C; Gamero, Ana M



Phosphorylation of Threonine 3  

PubMed Central

Huntingtin (Htt) is a widely expressed protein that causes tissue-specific degeneration when mutated to contain an expanded polyglutamine (poly(Q)) domain. Although Htt is large, 350 kDa, the appearance of amino-terminal fragments of Htt in extracts of postmortem brain tissue from patients with Huntington disease (HD), and the fact that an amino-terminal fragment, Htt exon 1 protein (Httex1p), is sufficient to cause disease in models of HD, points to the importance of the amino-terminal region of Htt in the disease process. The first exon of Htt encodes 17 amino acids followed by a poly(Q) repeat of variable length and culminating with a proline-rich domain of 50 amino acids. Because modifications to this fragment have the potential to directly affect pathogenesis in several ways, we have surveyed this fragment for potential post-translational modifications that might affect Htt behavior and detected several modifications of Httex1p. Here we report that the most prevalent modifications of Httex1p are NH2-terminal acetylation and phosphorylation of threonine 3 (pThr-3). We demonstrate that pThr-3 occurs on full-length Htt in vivo, and that this modification affects the aggregation and pathogenic properties of Htt. Thus, therapeutic strategies that modulate these events could in turn affect Htt pathogenesis. PMID:19710014

Aiken, Charity T.; Steffan, Joan S.; Guerrero, Cortnie M.; Khashwji, Hasan; Lukacsovich, Tamas; Simmons, Danielle; Purcell, Judy M.; Menhaji, Kimia; Zhu, Ya-Zhen; Green, Kim; LaFerla, Frank; Huang, Lan; Thompson, Leslie Michels; Marsh, J. Lawrence



Bioluminescence methods for enzymatic determinations  


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.

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



Multifractality in intracellular enzymatic reactions.  


Enzymatic kinetics adjust well to the Michaelis-Menten paradigm in homogeneous media with dilute, perfectly mixed reactants. These conditions are quite different from the highly structured cell plasm, so applications of the classic kinetics theory to this environment are rather limited. Cytoplasmic structure produces molecular crowding and anomalous diffusion of substances, modifying the mass action kinetic laws. The reaction coefficients are no longer constant but time-variant, as stated in the fractal kinetics theory. Fractal kinetics assumes that enzymatic reactions on such heterogeneous media occur within a non-Euclidian space characterized by a certain fractal dimension, this fractal dimension gives the dependence on time of the kinetic coefficients. In this work, stochastic simulations of enzymatic reactions under molecular crowding have been completed, and kinetic coefficients for the reactions, including the Michaelis-Menten parameter KM, were calculated. The simulations results led us to confirm the time dependence of michaelian kinetic parameter for the enzymatic catalysis. Besides, other chaos related phenomena were pointed out from the obtained KM time series, such as the emergence of strange attractors and multifractality. PMID:16256143

Aranda, Juan S; Salgado, Edgar; Muńoz-Diosdado, Alejandro



Oxidative and Photosynthetic Phosphorylation Mechanisms  

ERIC Educational Resources Information Center

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)

Wang, Jui H.



Phosphorylation Dynamics in Mammalian Cells  

Microsoft Academic Search

Living cells are auto-dynamic because of control systems operating in their periodic mode. They comprise diverse regulatory\\u000a networks and are thus multi-oscillators covering a wide range of characteristics. Phosphorylation reactions are involved in\\u000a virtually all aspects of cell function. Here, we outline a range of our studies on ATP and protein phosphorylation in order\\u000a to highlight certain features of ultradian

D. A. Gilbert; K. D. Hammond


Enzymatic resolution of alicyclic ?-lactams  

Microsoft Academic Search

Racemates of N-hydroxymethylated ?-lactams 4–6 were resolved through the lipase-catalyzed asymmetric acylation of the primary hydroxy group at the 6S stereogenic centre. High enantioselectivity (E>200) was observed when the enzymatic reactions were performed in acetone with lipase PS as catalyst and vinyl butyrate as acyl donor. The hydrolysis of the enantiomeric azetidinones 4a–6a and 4b–6b resulted in the enantiomerically pure

Judit Kámán; Enik? Forró; Ferenc Fülöp



Electrochemical enzymatic biosensors using carbon nanofiber nanoelectrode arrays  

NASA Astrophysics Data System (ADS)

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.

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



Blue Light–Dependent in Vivo and in Vitro Phosphorylation of Arabidopsis Cryptochrome 1  

PubMed Central

Cryptochromes are photolyase-like blue/UV-A light receptors that regulate various light responses in animals and plants. Arabidopsis cryptochrome 1 (cry1) is the major photoreceptor mediating blue light inhibition of hypocotyl elongation. The initial photochemistry underlying cryptochrome function and regulation remain poorly understood. We report here a study of the blue light–dependent phosphorylation of Arabidopsis cry1. Cry1 is detected primarily as unphosphorylated protein in etiolated seedlings, but it is phosphorylated in plants exposed to blue light. Cry1 phosphorylation increases in response to increased fluence of blue light, whereas the phosphorylated cry1 disappears rapidly when plants are transferred from light to dark. Light-dependent cry1 phosphorylation appears specific to blue light, because little cry1 phosphorylation is detected in seedlings treated with red light or far-red light, and it is largely independent from phytochrome actions, because no phytochrome mutants tested significantly affect cry1 phosphorylation. The Arabidopsis cry1 protein expressed and purified from insect cells is phosphorylated in vitro in a blue light–dependent manner, consistent with cry1 undergoing autophosphorylation. To determine whether cry1 phosphorylation is associated with its function or regulation, we isolated and characterized missense cry1 mutants that express full-length CRY1 apoprotein. Mutant residues are found throughout the CRY1 coding sequence, but none of these inactive cry1 mutant proteins shows blue light–induced phosphorylation. These results demonstrate that blue light–dependent cry1 phosphorylation is closely associated with the function or regulation of the photoreceptor and that the overall structure of cry1 is critical to its phosphorylation. PMID:14523249

Shalitin, Dror; Yu, Xuhong; Maymon, Maskit; Mockler, Todd; Lin, Chentao



Nucleoside phosphorylation by phosphate minerals.  


In the presence of formamide, crystal phosphate minerals may act as phosphate donors to nucleosides, yielding both 5'- and, to a lesser extent, 3'-phosphorylated forms. With the mineral Libethenite the formation of 5'-AMP can be as high as 6% of the adenosine input and last for at least 10(3) h. At high concentrations, soluble non-mineral phosphate donors (KH(2)PO(4) or 5'-CMP) afford 2'- and 2':3'-cyclic AMP in addition to 5'-and 3'-AMP. The phosphate minerals analyzed were Herderite Ca[BePO(4)F], Hureaulite Mn(2+)(5)(PO(3)(OH)(2)(PO(4))(2)(H(2)O)(4), Libethenite Cu(2+)(2)(PO(4))(OH), Pyromorphite Pb(5)(PO(4))(3)Cl, Turquoise Cu(2+)Al(6)(PO(4))(4)(OH)(8)(H(2)O)(4), Fluorapatite Ca(5)(PO(4))(3)F, Hydroxylapatite Ca(5)(PO(4))(3)OH, Vivianite Fe(2+)(3)(PO(4))(2)(H(2)O)(8), Cornetite Cu(2+)(3)(PO(4))(OH)(3), Pseudomalachite Cu(2+)(5)(PO(4))(2)(OH)(4), Reichenbachite Cu(2+)(5)(PO(4))(2)(OH)(4), and Ludjibaite Cu(2+)(5)(PO(4))(2)(OH)(4)). Based on their behavior in the formamide-driven nucleoside phosphorylation reaction, these minerals can be characterized as: 1) inactive, 2) low level phosphorylating agents, or 3) active phosphorylating agents. Instances were detected (Libethenite and Hydroxylapatite) in which phosphorylation occurs on the mineral surface, followed by release of the phosphorylated compounds. Libethenite and Cornetite markedly protect the beta-glycosidic bond. Thus, activated nucleic monomers can form in a liquid non-aqueous environment in conditions compatible with the thermodynamics of polymerization, providing a solution to the standard-state Gibbs free energy change (DeltaG degrees ') problem, the major obstacle for polymerizations in the liquid phase in plausible prebiotic scenarios. PMID:17412692

Costanzo, Giovanna; Saladino, Raffaele; Crestini, Claudia; Ciciriello, Fabiana; Di Mauro, Ernesto



A miniaturized assay for measuring small molecule phosphorylation in the presence of complex matrices.  


We describe here a simple, miniaturized radiation-based phosphorylation assay that can be used to monitor phosphorylation of a diverse range of small molecule substrates in the presence of purified and crude enzyme preparations. Ba(OH)2 and ZnSO4 are used to terminate phosphoryl transfer and to precipitate selectively the phosphorylated reaction product in a single step; non-phosphorylated substrate is removed by filtration prior to quantification. The key advantages over alternative radiation-based assays are that: (i) high-energy/short-lived radioactive emitters are not required; (ii) high-quality data can be obtained without the need for high radioactivity concentrations; and (iii) the assay is compatible with high-throughput applications. PMID:24333332

Spry, Christina; Saliba, Kevin J; Strauss, Erick



Oxidative Phosphorylation at the fin de siècle  

NSDL National Science Digital Library

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 5â²-triphosphate (ATP) synthase, to synthesize the energy carrier ATP.

Matti Saraste (European Molecular Biology Laboratory;)



Phosphorylation Alters the Interaction of the Arabidopsis Phosphotransfer Protein AHP1 with Its Sensor Kinase ETR1  

PubMed Central

The ethylene receptor ethylene response 1 (ETR1) and the Arabidopsis histidine-containing phosphotransfer protein 1 (AHP1) form a tight complex in vitro. According to our current model ETR1 and AHP1 together with a response regulator form a phosphorelay system controlling the gene expression response to the plant hormone ethylene, similar to the two-component signaling in bacteria. The model implies that ETR1 functions as a sensor kinase and is autophosphorylated in the absence of ethylene. The phosphoryl group is then transferred onto a histidine at the canonical phosphorylation site in AHP1. For phosphoryl group transfer both binding partners need to form a tight complex. After ethylene binding the receptor is switched to the non-phosphorylated state. This switch is accompanied by a conformational change that decreases the affinity to the phosphorylated AHP1. To test this model we used fluorescence polarization and examined how the phosphorylation status of the proteins affects formation of the suggested ETR1?AHP1 signaling complex. We have employed various mutants of ETR1 and AHP1 mimicking permanent phosphorylation or preventing phosphorylation, respectively. Our results show that phosphorylation plays an important role in complex formation as affinity is dramatically reduced when the signaling partners are either both in their non-phosphorylated form or both in their phosphorylated form. On the other hand, affinity is greatly enhanced when either protein is in the phosphorylated state and the corresponding partner in its non-phosphorylated form. Our results indicate that interaction of ETR1 and AHP1 requires that ETR1 is a dimer, as in its functional state as receptor in planta. PMID:21912672

Scharein, Benjamin; Groth, Georg



Strategies to Overcome the Enzymatic Barrier  

Microsoft Academic Search

Enzymatic degradation of various hydrophilic macromolecules including peptide- or protein drugs by enzymes present in the\\u000a gastrointestinal tract can be regarded as one main reason for their poor bioavailability after peroral administration. Within\\u000a the current chapter, strategies to overcome the so-called enzymatic barrier are described. Besides formulations that can protect\\u000a the drug from enzymatic digestion via, e.g., drug encapsulation and

Martin Werle; Hirofumi Takeuchi


Enzymatic reaction paths as determined by transition path sampling  

NASA Astrophysics Data System (ADS)

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.

Masterson, Jean Emily


Enzymatic transglycosylation for glycoconjugate synthesis  

PubMed Central

Summary Remarkable advances have been made in recent years in exploiting the transglycosylation activity of glycosidases and glycosynthase mutants for oligosaccharide and glycoconjugate synthesis. New glycosynthases were generated from retaining glycosidases, inverting glycosidases, and those that proceed in a mechanism of substrate-assisted catalysis. Directed evolution coupled with elegant screening methods has led to the discovery of an expanding number of glycosynthase mutants that show improved catalytic activity and/or altered substrate specificity. In particular, enzymatic transglycosylation strategy has been recently extended to the synthesis of complex glycoconjugates, including glycosphingolipids, N-glycoproteins, and other glycosylated natural products. PMID:19766528

Wang, Lai-Xi; Huang, Wei




SciTech Connect

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.




Physicochemical mechanisms of protein regulation by phosphorylation  

PubMed Central

Phosphorylation offers a dynamic way to regulate protein activity and subcellular localization, which is achieved through its reversibility and fast kinetics. Adding or removing a dianionic phosphate group somewhere on a protein often changes the protein’s structural properties, its stability and dynamics. Moreover, the majority of signaling pathways involve an extensive set of protein–protein interactions, and phosphorylation can be used to regulate and modulate protein–protein binding. Losses of phosphorylation sites, as a result of disease mutations, might disrupt protein binding and deregulate signal transduction. In this paper we focus on the effects of phosphorylation on protein stability, dynamics, and binding. We describe several physico-chemical mechanisms of protein regulation through phosphorylation and pay particular attention to phosphorylation in protein complexes and phosphorylation in the context of disorder–order and order–disorder transitions. Finally we assess the role of multiple phosphorylation sites in a protein molecule, their possible cooperativity and function. PMID:25147561

Nishi, Hafumi; Shaytan, Alexey; Panchenko, Anna R.



Method for the enzymatic production of hydrogen  


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.

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



Economics of enzymatic hydrolysis processes  

SciTech Connect

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

Wright, J.D.



The semi-phosphorylative Entner–Doudoroff pathway in hyperthermophilic archaea: a re-evaluation  

PubMed Central

Biochemical studies have suggested that, in hyperthermophilic archaea, the metabolic conversion of glucose via the ED (Entner–Doudoroff) pathway generally proceeds via a non-phosphorylative variant. A key enzyme of the non-phosphorylating ED pathway of Sulfolobus solfataricus, KDG (2-keto-3-deoxygluconate) aldolase, has been cloned and characterized previously. In the present study, a comparative genomics analysis is described that reveals conserved ED gene clusters in both Thermoproteus tenax and S. solfataricus. The corresponding ED proteins from both archaea have been expressed in Escherichia coli and their specificity has been identified, revealing: (i) a novel type of gluconate dehydratase (gad gene), (ii) a bifunctional 2-keto-3-deoxy-(6-phospho)-gluconate aldolase (kdgA gene), (iii) a 2-keto-3-deoxygluconate kinase (kdgK gene) and, in S. solfataricus, (iv) a GAPN (non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase; gapN gene). Extensive in vivo and in vitro enzymatic analyses indicate the operation of both the semi-phosphorylative and the non-phosphorylative ED pathway in T. tenax and S. solfataricus. The existence of this branched ED pathway is yet another example of the versatility and flexibility of the central carbohydrate metabolic pathways in the archaeal domain. PMID:15869466



Grb2 controls phosphorylation of FGFR2 by inhibiting receptor kinase and Shp2 phosphatase activity  

PubMed Central

Constitutive receptor tyrosine kinase phosphorylation requires regulation of kinase and phosphatase activity to prevent aberrant signal transduction. A dynamic mechanism is described here in which the adaptor protein, growth factor receptor–bound protein 2 (Grb2), controls fibroblast growth factor receptor 2 (FGFR2) signaling by regulating receptor kinase and SH2 domain–containing protein tyrosine phosphatase 2 (Shp2) phosphatase activity in the absence of extracellular stimulation. FGFR2 cycles between its kinase-active, partially phosphorylated, nonsignaling state and its Shp2-dephosphorylated state. Concurrently, Shp2 cycles between its FGFR2-phosphorylated and dephosphorylated forms. Both reciprocal activities of FGFR2 and Shp2 were inhibited by binding of Grb2 to the receptor. Phosphorylation of Grb2 by FGFR2 abrogated its binding to the receptor, resulting in up-regulation of both FGFR2’s kinase and Shp2’s phosphatase activity. Dephosphorylation of Grb2 by Shp2 rescued the FGFR2–Grb2 complex. This cycling of enzymatic activity results in a homeostatic, signaling-incompetent state. Growth factor binding perturbs this background cycling, promoting increased FGFR2 phosphorylation and kinase activity, Grb2 dissociation, and downstream signaling. Grb2 therefore exerts constitutive control over the mutually dependent activities of FGFR2 and Shp2. PMID:23420874

Lin, Chi-Chuan; Suen, Kin M.; Melo, Fernando A.; Levitt, James A; Suhling, Klaus



Aluminum interaction with human brain tau protein phosphorylation by various kinases  

SciTech Connect

Phosphorylation is an indispensable process for energy and signal transduction in biological systems. AlCl[sub 3] at 10 nM to 10 [mu]M range activated in-vitro [[gamma][sup [minus]32]P]ATP phosphorylation of the brain ([tau]) [Gamma] protein in both normal human or E.coli expressed [Gamma] forms; in the presence of the kinases P34,PKP, and PKC. However, higher concentrations of AlCl[sub 3] inhibited the [Gamma] phosphorylation with P34, PKP, and PKC to a maximum at 1 mM level. AlCl[sub 3] at 100 [mu]M to 500 [mu]M range induced non-enzymatic phosphorylation of [Gamma] with [gamma]-ATP, [gamma]-GTP, and [alpha]-GRP. AlCl[sub 3] activated histone phosphorylation by P34 in a similar pattern. The hyperphosphorylation of [Gamma] by Al[sup 3+] was accompanied in molecular shift and mobility retardation in SDS-PAGE. This may demonstrate the mechanism of the long term neurological effect of Al[sub 3+] in human brain leading to the formation of the neutrofibrillary tangles related to Alzeheimer's disease.

El-Sebae; Abou Zeid, M.M.; Saleh, M.A. (Texas Southern Univ., Houston, TX (United States). Environmental Chemistry and Toxicology Lab.); Abdel-Ghany, M.E.; Shalloway, D. (Cornell Univ., Ithaca, NY (United States). Section of Biochemistry, Mol, and Cell Biology); Blancato, J. (US EPA, Las Vegas, NV (United States). Environmental Monit. Systems Lab.)



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

E-print Network

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

Heller, Eric


Synthesis of ?- and ?-hydroxy ?-amino acids via enzymatic kinetic resolution and cyanate-to-isocyanate rearrangement.  


A new strategy for stereoselective preparation of all four isomers of ?- and ?-hydroxy ?-amino acids is presented. The developed procedure is based on enzymatic kinetic resolution and cyanate-to-isocyanate rearrangement as key steps. Stereocontrol is achieved by proper choice of the starting hydroxyacid, the course of kinetic resolution, and the stereospecific sigmatropic rearrangement step, which proceeds with full chirality transfer. PMID:25411803

Szcze?niak, Piotr; Pa?dzierniok-Holewa, Agnieszka; Klimczak, Urszula; Stecko, Sebastian



The biochemistry and control of enzymatic browning  

Microsoft Academic Search

Half of the world's fruit and vegetable crops is lost due to postharvest deteriorative reactions. Polyphenol oxidase (PPO), found in most fruit and vegetables, is responsible for enzymatic browning of fresh horticultural products, following bruising, cutting or other damage to the cell. Chemical methods for controlling enzymatic browning include the use of sodium bisulfite, ascorbic acid and\\/or packaging under controlled

M. Victoria Martinez; John R. Whitaker



Dispersing biofilms with engineered enzymatic bacteriophage  

E-print Network

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

Collins, James J.



Microsoft Academic Search

Enzymatic substrate regeneration is a tool to enhance the sensitivity of enzyme electrodes both for substrate analysis and immunoassays. The combination of immunoreactions and electrode based substrate recycling connects specific recognition of an analyte with highly sensitive detection. Most important for this field of application is the sensitivity, which permits to detect a label at very low concentration. Enzymatic substrate

Frieder W. Scheller; Christian G. Bauer; Alexander Makower; Ulla Wollenberger; Axel Warsinke; Frank F. Bier



Mycobacterium tuberculosis supports protein tyrosine phosphorylation  

PubMed Central

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

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



Mycobacterium tuberculosis supports protein tyrosine phosphorylation.  


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

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



Phosphorylation in the catalytic cleft stabilizes and attracts domains of a phosphohexomutase.  


Phosphorylation can modulate the activities of enzymes. The phosphoryl donor in the catalytic cleft of ?-D-phosphohexomutases is transiently dephosphorylated while the reaction intermediate completes a 180° reorientation within the cleft. The phosphorylated form of 52 kDa bacterial phosphomannomutase/phosphoglucomutase is less accessible to dye or protease, more stable to chemical denaturation, and widely stabilized against NMR-detected hydrogen exchange across the core of domain 3 to juxtaposed domain 4 (each by ?1.3 kcal/mol) and parts of domains 1 and 2. However, phosphorylation accelerates hydrogen exchange in specific regions of domains 1 and 2, including a metal-binding residue in the active site. Electrostatic field lines reveal attraction across the catalytic cleft between phosphorylated Ser-108 and domain 4, but repulsion when Ser-108 is dephosphorylated. Molecular dynamics (MD) simulated the dephosphorylated form to be expanded due to enhanced rotational freedom of domain 4. The contacts and fluctuations of the MD trajectories enabled correct simulation of more than 80% of sites that undergo either protection or deprotection from hydrogen exchange due to phosphorylation. Electrostatic attraction in the phosphorylated enzyme accounts for 1) domain 4 drawing closer to domains 1 and 3; 2) decreased accessibility; and 3) increased stability within these domains. The electrostriction due to phosphorylation may help capture substrate, whereas the opening of the cleft upon transient dephosphorylation allows rotation of the intermediate. The long-range effects of phosphorylation on hydrogen exchange parallel reports on protein kinases, suggesting a conceptual link among these multidomain, phosphoryl transfer enzymes. PMID:25606681

Xu, Jia; Lee, Yingying; Beamer, Lesa J; Van Doren, Steven R



Oxidative Phosphorylation at the fin de siècle  

NSDL National Science Digital Library

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 5â²-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.

Matti Saraste (European Molecular Biology Laboratory;)



Confident Phosphorylation Site Localization Using the Mascot Delta Score  

PubMed Central

Large scale phosphorylation analysis is more and more getting into focus of proteomic research. Although it is now possible to identify thousands of phosphorylated peptides in a biological system, confident site localization remains challenging. Here we validate the Mascot Delta Score (MD-score) as a simple method that achieves similar sensitivity and specificity for phosphosite localization as the published Ascore, which is mainly used in conjunction with Sequest. The MD-score was evaluated using liquid chromatography-tandem MS data of 180 individually synthesized phosphopeptides with precisely known phosphorylation sites. We tested the MD-score for a wide range of commonly available fragmentation methods and found it to be applicable throughout with high statistical significance. However, the different fragmentation techniques differ strongly in their ability to localize phosphorylation sites. At 1% false localization rate, the highest number of correctly assigned phosphopeptides was achieved by higher energy collision induced dissociation in combination with an Orbitrap mass analyzer followed very closely by low resolution ion trap spectra obtained after electron transfer dissociation. Both these methods are significantly better than low resolution spectra acquired after collision induced dissociation and multi stage activation. Score thresholds determined from simple calibration functions for each fragmentation method were stable over replicate analyses of the phosphopeptide set. The MD-score outperforms the Ascore for tyrosine phosphorylated peptides and we further show that the ability to call sites correctly increases with increasing distance of two candidate sites within a peptide sequence. The MD-score does not require complex computational steps which makes it attractive in terms of practical utility. We provide all mass spectra and the synthetic peptides to the community so that the development of present and future localization software can be benchmarked and any laboratory can determine MD-scores and localization probabilities for their individual analytical set up. PMID:21057138

Savitski, Mikhail M.; Lemeer, Simone; Boesche, Markus; Lang, Manja; Mathieson, Toby; Bantscheff, Marcus; Kuster, Bernhard



BglF, the sensor of the E. coli bgl system, uses the same site to phosphorylate both a sugar and a regulatory protein.  

PubMed Central

The Escherichia coli BglF protein is a sugar permease that is a member of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). It catalyses transport and phosphorylation of beta-glucosides. In addition to its ability to phosphorylate its sugar substrate, BglF has the unusual ability to phosphorylate and dephosphorylate the transcriptional regulator BglG according to beta-glucoside availability. By controlling the phosphorylation state of BglG, BglF controls the dimeric state of BglG and thus its ability to bind RNA and antiterminate transcription of the bgl operon. BglF has two phosphorylation sites. The first site accepts a phosphoryl group from the PTS protein HPr; the phosphoryl group is then transferred to the second phosphorylation site, which can deliver it to the sugar. We provide both in vitro and in vivo evidence that the same phosphorylation site on BglF, the second one, is in charge not only of sugar phosphorylation but also of BglG phosphorylation. Possible mechanisms that ensure correct phosphoryl delivery to the right entity, sugar or protein, depending on environmental conditions, are discussed. PMID:9303306

Chen, Q; Arents, J C; Bader, R; Postma, P W; Amster-Choder, O



Phosphorylated nano-diamond/ Polyimide Nanocomposites  

NASA Astrophysics Data System (ADS)

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.

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



Insulin-stimulated phosphorylation of calmodulin.  

PubMed Central

Calmodulin is phosphorylated in vitro by the insulin-receptor tyrosine kinase and a variety of serine/threonine kinases. Here we report that insulin stimulates the phosphorylation of calmodulin on average 3-fold in intact rat hepatocytes. Although calmodulin is constitutively phosphorylated, insulin increases phosphate incorporation into serine, threonine and tyrosine residues. We demonstrate that casein kinase II, an insulin-sensitive kinase, phosphorylates calmodulin in vitro on serine/thyronine residues (Thr-79, Ser-81, Ser-101 and Thr-117). The ability of the insulin receptor to phosphorylate calmodulin that has been pre-phosphorylated by casein kinase II is enhanced up to 35-fold, and the sites of phosphorylation on calmodulin are shifted from tyrosine to threonine and serine. These observations, obtained with a new specific monoclonal antibody to calmodulin, confirm that insulin stimulates calmodulin phosphorylation in intact cells. The observation that calmodulin is phosphorylated in vivo, coupled with the recent demonstration that phosphocalmodulin exhibits altered biological activity, strongly suggests that phosphorylation of calmodulin is a critical component of intracellular signalling. Images Fig. 1. Fig. 3. PMID:1520270

Sacks, D B; Davis, H W; Crimmins, D L; McDonald, J M



Hydrogen transfer in SAM-mediated enzymatic radical reactions.  


S-adenosylmethionine (SAM) plays an essential role in a variety of enzyme-mediated radical reactions. One-electron reduction of SAM is currently believed to generate the C5'-desoxyadenosyl radical, which subsequently abstracts a hydrogen atom from the actual substrate in a catalytic or a non-catalytic fashion. Using a combination of theoretical and experimental bond dissociation energy (BDE) data, the energetics of these radical processes have now been quantified. SAM-derived radicals are found to react with their respective substrates in an exothermic fashion in enzymes using SAM in a stoichiometric (non-catalytic) way. In contrast, the catalytic use of SAM appears to be linked to a sequence of moderately endothermic and exothermic reaction steps. The use of SAM in spore photoproduct lyase (SPL) appears to fit neither of these general categories and appears to constitute the first example of a SAM-initiated radical reaction propagated independently of the cofactor. PMID:23139189

Hioe, Johnny; Zipse, Hendrik



In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.  


Extracellular stimuli are transduced inside the cell by posttranslational modifications (PTMs), such as phosphorylation, of proteins in signaling networks. Insight into the structure of these networks requires quantification of PTM levels in individual cells. Fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to image PTM levels in situ. FLIM on cell arrays that express fluorescent protein fusions can quantify tyrosine phosphorylation patterns in large networks in individual cells. We identified tyrosine kinase substrates by imaging their phosphorylation levels after inhibition of protein tyrosine phosphatases. Analysis of the correlation between protein phosphorylation and expression levels at single cell resolution allowed us to identify positive feedback motifs. Using FLIM on cell arrays (CA-FLIM), we uncovered components that transduce signals from epidermal growth factor receptor. PMID:20453867

Grecco, Hernán E; Roda-Navarro, Pedro; Girod, Andreas; Hou, Jian; Frahm, Thomas; Truxius, Dina C; Pepperkok, Rainer; Squire, Anthony; Bastiaens, Philippe I H



Effect of resveratrol on cultured skin fibroblasts from patients with oxidative phosphorylation defects.  


Few therapeutic options are available to patients with oxidative phosphorylation disorders. Administering pharmacological agents that are able to stimulate mitochondrial biogenesis have been put forward as a possible treatment, yet the approach remains in need of thorough testing. We investigated the effect of resveratrol in an in vitro setting. Mitochondrial enzymatic activities were tested in cultured skin fibroblasts from patients harboring a nuclear defect in either complex II or complex IV (n?=?11), and in fibroblasts from healthy controls (n?=?11). In the latter, preincubation with resveratrol resulted in a significant increase of citrate synthase, complex II and complex IV enzyme activity. In patients with complex II or complex IV deficiency, however, activity of the deficient complex could not be substantially augmented, and response was dependent upon the residual activity. We conclude that resveratrol is not capable of normalizing oxidative phosphorylation activities in deficient cell lines. PMID:23620374

De Paepe, Boel; Vandemeulebroecke, Katrien; Smet, Joél; Vanlander, Arnaud; Seneca, Sara; Lissens, Willy; Van Hove, Johan Lk; Deschepper, Ellen; Briones, Paz; Van Coster, Rudy



Phosphorylation Regulates SIRT1 Function  

PubMed Central

Background SIR2 is an NAD+-dependent deacetylase [1]–[3] implicated in the regulation of lifespan in species as diverse as yeast [4], worms [5], and flies [6]. We previously reported that the level of SIRT1, the mammalian homologue of SIR2 [7], [8], is coupled to the level of mitotic activity in cells both in vitro and in vivo [9]. Cells from long-lived mice maintained SIRT1 levels of young mice in tissues that undergo continuous cell replacement by proliferating stem cells. Changes in SIRT1 protein level were not associated with changes in mRNA level, suggesting that SIRT1 could be regulated post-transcriptionally. However, other than a recent report on sumoylation [10] and identification of SIRT1 as a nuclear phospho-protein by mass spectrometry [11], post-translational modifications of this important protein have not been reported. Methodology/Principal Findings We identified 13 residues in SIRT1 that are phosphorylated in vivo using mass spectrometry. Dephosphorylation by phosphatases in vitro resulted in decreased NAD+-dependent deacetylase activity. We identified cyclinB/Cdk1 as a cell cycle-dependent kinase that forms a complex with and phosphorylates SIRT1. Mutation of two residues phosphorylated by Cyclin B/Cdk1 (threonine 530 and serine 540) disturbs normal cell cycle progression and fails to rescue proliferation defects in SIRT1-deficient cells [12], [13]. Conclusions/Significance Pharmacological manipulation of SIRT1 activity is currently being tested as a means of extending lifespan in mammals. Treatment of obese mice with resveratrol, a pharmacological activator of SIRT1, modestly but significantly improved longevity and, perhaps more importantly, offered some protection against the development of type 2 diabetes mellitus and metabolic syndrome [14]–[16]. Understanding the endogenous mechanisms that regulate the level and activity of SIRT1, therefore, has obvious relevance to human health and disease. Our results identify phosphorylation by cell cycle dependent kinases as a major mechanism controlling the level and function of this sirtuin and complement recent reports of factors that inhibit [17], [18] and activate [19] SIRT1 by protein-protein interactions. PMID:19107194

Sasaki, Tsutomu; Maier, Bernhard; Koclega, Katarzyna D.; Chruszcz, Maksymilian; Gluba, Wendy; Stukenberg, P. Todd; Minor, Wladek; Scrable, Heidi



Prebiotic phosphorylation of nucleosides in formamide  

NASA Technical Reports Server (NTRS)

Results are presented for an experimental study intended to assess phosphorylation under neither aqueous nor dry thermal conditions. Instead, phosphorylations were attempted in possible nonaqueous prebiotic solvents. Formamide appeared to be the most obvious candidate for phosphorylation studies. Three main classes of phosphorylated products were formed in formamide solution: adenosine monophosphates, cyclic adenosine phosphate, and adenosine diphosphates. Experiments were designed to investigate the extent of phosphorylation of nucleosides in formamide, the relative amounts of nucleoside monophosphate, diphosphates and cyclic phosphate formed and the relative effectiveness of different sources of phosphate as phosphorylating agents in formamide. Reaction variables were temperature, nature of the phosphate or condensed phosphate, nucleoside, concentration of reactants and possible effects of additives. Product identification was based on qualitative and quantitative thin layer chromatography.

Schoffstall, A. M.




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...


Activation of Escherichia coli antiterminator BglG requires its phosphorylation  

PubMed Central

Transcriptional antiterminator proteins of the BglG family control the expression of enzyme II (EII) carbohydrate transporters of the bacterial phosphotransferase system (PTS). In the PTS, phosphoryl groups are transferred from phosphoenolpyruvate (PEP) via the phosphotransferases enzyme I (EI) and HPr to the EIIs, which phosphorylate their substrates during transport. Activity of the antiterminators is negatively controlled by reversible phosphorylation catalyzed by the cognate EIIs in response to substrate availability and positively controlled by the PTS. For the Escherichia coli BglG antiterminator, two different mechanisms for activation by the PTS were proposed. According to the first model, BglG is activated by HPr-catalyzed phosphorylation at a site distinct from the EII-dependent phosphorylation site. According to the second model, BglG is not activated by phosphorylation, but solely through interaction with EI and HPr, which are localized at the cell pole. Subsequently BglG is released from the cell pole to the cytoplasm as an active dimer. Here we addressed this discrepancy and found that activation of BglG requires phosphorylatable HPr or the HPr homolog FruB in vivo. Further, we uniquely demonstrate that purified BglG protein becomes phosphorylated by FruB as well as by HPr in vitro. Histidine residue 208 in BglG is essential for this phosphorylation. These data suggest that BglG is in fact activated by phosphorylation and that there is no principal difference between the PTS-exerted mechanisms controlling the activities of BglG family proteins in Gram-positive and Gram-negative bacteria. PMID:22984181

Rothe, Fabian M.; Bahr, Thomas; Stülke, Jörg; Rak, Bodo; Görke, Boris



[Enzymatic catalysis in non-aqueous solvents].  


It is well known that non-aqueous enzymatic catalysis has emerged as an important area of enzyme engineering with the advantages of higher substrate solubility, increased stereoselectivity, modified substrate specificity and suppression of unwanted water-dependent side reactions. As a result, non-aqueous enzymatic catalysis has been applied in the biocatalytic synthesis of important pharmaceuticals and nutriceuticals. With the advancement of non-aqueous enzymatic catalysis in recent years, the efforts have been centered on the discovery and modification of solvent-tolerant biocatalysts for non-aqueous environments. Additionally, with the inevitable trends of green chemistry and sustainable development, green solvents have been utilized for increased number of enzymatic reactions to replace conventional organic solvents. In this review, modification, immobilization and mutagenesis of various enzymes for non-aqueous catalysis are discussed. Recent progress of non-aqueous enzymatic catalysis in solvent-free environments, reverse micelles, supercritical liquid and ionic liquid are also presented. In particular, while direct evolution, high-throughput screening and site-directed mutagenesis are combined as powerful tools for protein engineering, vapor/solid/ice water mixture, sticky solid-state liquid crystal and high density salt suspension are the future directions for solvent engineering in order to broaden the utility and elevate the efficiency of non-aqueous enzymatic catalysis. PMID:20352953

Wang, Lili; Chen, Yijun



FRAP-Dependent Serine Phosphorylation of IRS-1 Inhibits IRS-1 Tyrosine Phosphorylation  

Microsoft Academic Search

We have previously shown that interferon-? (IFN?)-dependent tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) is impaired by serine phosphorylation of IRS-1 due to the reduced ability of serine phosphorylated IRS-1 to serve as a substrate for Janus kinase 1 (JAK1). Here we report that FKBP12–rapamycin-associated protein (FRAP) is a physiologic IRS-1 kinase that blocks IFN? signaling by serine phosphorylating IRS-1.

Matthew E. Hartman; Montserrat Villela-Bach; Jie Chen; Gregory G. Freund



Relationships between histone phosphorylation and cell proliferation  

SciTech Connect

From studies with various Peromyscus cell lines, correlations were made which led to the proposal that H2A phosphorylation is most active in constitutive heterochromatin. Recent studies on the two H2A variants found in these cells have revealed that the high level of H2A phosphorylation associated with heterochromatin is not the result of an increase in H2A phosphorylation rate or an increase in the number of phosphorylation sites, but rather, is due to an increase in the proportion of one of the H2A variants which is more highly phosphorylated than the other. If H2A phosphorylation is necessary for the constitutive heterochromatin state, it is reasonable that the cell would accomplish the generation of this structure by permanently installing a more highly phosphorylated H2A in the heterochromatin nucleosome rather than by trying to modulate the phosphorylation rate in such a condensed structure. The proposal that histone phosphorylation is involved with the condensed structures of chromatin is based primarily on correlations between histone phosphorylation measurements and cellular phenomena. One proof that this concept is correct ultimately rests in the ability to demonstrate these correlations in isolated chromosomes and chromatin fractions. This demonstration is presently limited by the excessive dephosphorylation of histones which occurs during the isolation of chromosomes and chromatin fractions. Thus, the demonstration of an effective inhibitor of histone dephosphorylation which is compatible with the isolation of nuclear structures and chromatin fractions having native morphologies is essential for future studies on the biological function of histone phosphorylation. (ERB)

Gurley, L.R.; D'Anna, J.A.; Halleck, M.S.; Barham, S.S.; Walters, R.A.; Jett, J.H.; Tobey, R.A.



1776 Brief Communication Short 5 -phosphorylated double-stranded RNAs induce RNA  

E-print Network

effect on the were actually transferred. The dsRNA induced a strong silencing potential, indicating1776 Brief Communication Short 5 -phosphorylated double-stranded RNAs induce RNA interference quence-specific RNA degradation in an in vitro system as well as in insect and mammalian tissue culture

Delidakis, Christos


Cancer signaling: when phosphorylation meets methylation.  


The propagation of kinase-mediated phosphorylation signals is central to the oncogenic activity of the RAS-MAPK pathway in human cancers. A recent study shows that protein lysine methylation controls the phosphorylation status of a key component of the RAS-MAPK pathway to enable oncogenic KRAS in cancer progression. PMID:25104733

Ying, Haoqiang; DePinho, Ronald A



Systematic Discovery of In Vivo Phosphorylation Networks  

Microsoft Academic Search

SUMMARY Protein kinases control cellular decision pro- cesses by phosphorylating specific substrates. Thousands of in vivo phosphorylation sites have been identified, mostly by proteome- wide mapping. However, systematically match- ing these sites to specific kinases is presently infeasible, due to limited specificity of consen- sus motifs, and the influence of contextual factors, such as protein scaffolds, localization, and expression, on

Rune Linding; Lars Juhl Jensen; Gerard J. Ostheimer; Marcel A. T. M. van Vugt; Claus Jřrgensen; Ioana M. Miron; Francesca Diella; Karen Colwill; Lorne Taylor; Kelly Elder; Pavel Metalnikov; Vivian Nguyen; Adrian Pasculescu; Jing Jin; Jin Gyoon Park; Leona D. Samson; James R. Woodgett; Peer Bork; Michael B. Yaffe; Tony Pawson



Global analysis of phosphorylation networks in humans  

PubMed Central

Phosphorylation-mediated signaling plays a crucial role in nearly every aspect of cellular physiology. A recent study based on protein microarray experiments identified a large number of kinase-substrate relationships (KSRs), and built a comprehensive and reliable phosphorylation network in humans. Analysis of this network, in conjunction with additional resources, revealed several key features. First, comparison of the human and yeast phosphorylation networks uncovered an evolutionarily conserved signaling backbone dominated by kinase-to-kinase relationships. Second, although most of the KSRs themselves are not conserved, the functions enriched in the substrates for a given kinase are often conserved. Third, the prevalence of kinase-transcription factor regulatory modules suggests that phosphorylation and transcriptional regulatory networks are inherently wired together to form integrated regulatory circuits. Overall, the phosphorylation networks described in this work promise to offer new insights into the properties of kinase signaling pathways, at both the global and the protein levels. PMID:23524292

Hu, Jianfei; Rho, Hee-Sool; Newman, Robert H.; Hwang, Woochang; Neiswinger, John; Zhu, Heng; Zhang, Jin; Qian, Jiang



Lignin hydrolysis and phosphorylation mechanism during phosphoric acid-acetone pretreatment: a DFT study.  


The study focused on the structural sensitivity of lignin during the phosphoric acid-acetone pretreatment process and the resulting hydrolysis and phosphorylation reaction mechanisms using density functional theory calculations. The chemical stabilities of the seven most common linkages (?-O-4, ?-?, 4-O-5, ?-1, 5-5, ?-O-4, and ?-5) of lignin in H3PO4, CH3COCH3, and H2O solutions were detected, which shows that ?-O-4 linkage and ?-O-4 linkage tend to break during the phosphoric acid-acetone pretreatment process. Then ?-O-4 phosphorylation and ?-O-4 phosphorylation follow a two-step reaction mechanism in the acid treatment step, respectively. However, since phosphorylation of ?-O-4 is more energetically accessible than phosphorylation of ?-O-4 in phosphoric acid, the phosphorylation of ?-O-4 could be controllably realized under certain operational conditions, which could tune the electron and hole transfer on the right side of ?-O-4 in the H2PO4- functionalized lignin. The results provide a fundamental understanding for process-controlled modification of lignin and the potential novel applications in lignin-based imprinted polymers, sensors, and molecular devices. PMID:25529020

Qin, Wu; Wu, Lingnan; Zheng, Zongming; Dong, Changqing; Yang, Yongping



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

NASA Astrophysics Data System (ADS)

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.

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



Review: Enzymatic Hydrolysis of Cellulosic Biomass  

SciTech Connect

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.

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



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


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

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



Phosphorylation by PKA potentiates retinoic acid receptor ? activity by means of increasing interaction with and phosphorylation by cyclin H/cdk7  

PubMed Central

Nuclear retinoic acid receptors (RARs) work as ligand-dependent heterodimeric RAR/retinoid X receptor transcription activators, which are targets for phosphorylations. The N-terminal activation function (AF)-1 domain of RAR? is phosphorylated by the cyclin-dependent kinase (cdk) 7/cyclin H complex of the general transcription factor TFIIH and the C-terminal AF-2 domain by the cAMP-dependent protein kinase A (PKA). Here, we report the identification of a molecular pathway by which phosphorylation by PKA propagates cAMP signaling from the AF-2 domain to the AF-1 domain. The first step is the phosphorylation of S369, located in loop 9–10 of the AF-2 domain. This signal is transferred to the cyclin H binding domain (at the N terminus of helix 9 and loop 8–9), resulting in enhanced cyclin H interaction and, thereby, greater amounts of RAR? phosphorylated at S77 located in the AF-1 domain by the cdk7/cyclin H complex. This molecular mechanism relies on the integrity of the ligand-binding domain and the cyclin H binding surface. Finally, it results in higher DNA-binding efficiency, providing an explanation for how cAMP synergizes with retinoic acid for transcription. PMID:16769902

Gaillard, Emilie; Bruck, Nathalie; Brelivet, Yann; Bour, Gaétan; Lalevée, Sébastien; Bauer, Annie; Poch, Olivier; Moras, Dino; Rochette-Egly, Cécile



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

PubMed Central

Structurally distinguishable mutants of human epidermal growth factor receptor (EGFR) were used to investigate the mechanism of EGFR autophosphorylation. Mutant receptors generated by site-directed mutagenesis were expressed in transfected NIH 3T3 cells lacking endogenous receptors. After coincubation of cell lysates in the presence or absence of EGF, receptor immunoprecipitates were incubated with [gamma-32P]ATP. A kinase-negative mutant EGFR (K721A), in which Lys-721 in the ATp binding site was replaced by an alanine residue, was shown to be phosphorylated in an EGF-dependent manner by an enzymatically active EGFR deletion mutant lacking two autophosphorylation sites. A mutant EGFR lacking the EGF-binding domain as well as the phosphorylation sites also phosphorylated the kinase-negative mutant. In both cases the kinase-negative mutant K721A was phosphorylated on sites virtually identical to the sites that are autophosphorylated by wild-type recombinant or native human EGFRs. With four different site-specific anti-EGFR antibodies, it was shown that deletion mutants devoid of epitopes recognized by the antibodies were coimmunoprecipitated together with wild-type or mutant receptors recognized by the antibodies. This indicates that EGFR oligomers were preserved during immunoprecipitation. On the basis of these results, we propose that autophosphorylation of solubilized EGFR is mediated by intermolecular cross-phosphorylation, probably facilitated by receptor oligomerization. Images PMID:2915986

Honegger, A M; Kris, R M; Ullrich, A; Schlessinger, J



Enzymatic hydrolysis of cellulose: theory and applications  

SciTech Connect

A large amount of research has been published on the theory of enzymatic hydrolysis and the various microbial, and other, sources of the enzymes. The present report endeavors to supplement this information by emphasizing insofar as possible the status of the technology and of potential industrial processes for production of sugars from cellulose. A substantial research effort on cellulose conversion has been underway in the authors' laboratories at the University of California at Berkeley over the past ten years. This report is based in part upon this background of experience, and experimental data from relatively recent studies are presented in certain sections to make the information as timely and useful as possible. Because of current interest in production of ethanol a section is included which summarizes various methods for high productivity fermentation systems. Chapter titles are: Theory of enzymatic hydrolysis; production of cellulase and xylanase; hydrolysis of agricultural residues, enzymatic hydrolysis processes, high productivity ethanol fermentation; and ethanol economics. (DMC)

Wilke, C.R.; Maiorella, B.; Sciamanna, A.; Tangnu, K.; Wiley, D.; Wong, H.



An infrared radiation based thermal biosensor for enzymatic biochemical reactions.  


In this paper, a thermal biosensor based on the infrared radiation energy is proposed for calorimetric measurement of biochemical reactions. Having a good structure design combined with MEMS technology as well as employing the Si /SiGe quantum well sensing material with a high TCR and low 1/f noise, the sensor shows potentials to be high sensitive and real-time. The urea enzymatic reaction was tested to verify the performance of sensor, which demonstrates a linear detection range from 0.5mM to 150mM and a relative standard deviation less than 1%. For the sensor fabrication, wafer-level transfer bonding is a key process, which makes the integration of quantum well material and a free standing structure possible. It reduces the heat loss from the sensor to the surrounding environment. PMID:23365944

Zhang, Lei; Dong, Tao; Zhao, Xinyan; Yang, Zhaochu; Pires, Nuno M M



The negative impact of ?-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation  

PubMed Central

A decline in ?-ketoglutarate dehydrogenase complex (KGDHC) activity has been associated with neurodegeneration. Provision of succinyl-CoA by KGDHC is essential for generation of matrix ATP (or GTP) by substrate-level phosphorylation catalyzed by succinyl-CoA ligase. Here, we demonstrate ATP consumption in respiration-impaired isolated and in situ neuronal somal mitochondria from transgenic mice with a deficiency of either dihydrolipoyl succinyltransferase (DLST) or dihydrolipoyl dehydrogenase (DLD) that exhibit a 20–48% decrease in KGDHC activity. Import of ATP into the mitochondrial matrix of transgenic mice was attributed to a shift in the reversal potential of the adenine nucleotide translocase toward more negative values due to diminished matrix substrate-level phosphorylation, which causes the translocase to reverse prematurely. Immunoreactivity of all three subunits of succinyl-CoA ligase and maximal enzymatic activity were unaffected in transgenic mice as compared to wild-type littermates. Therefore, decreased matrix substrate-level phosphorylation was due to diminished provision of succinyl-CoA. These results were corroborated further by the finding that mitochondria from wild-type mice respiring on substrates supporting substrate-level phosphorylation exhibited ?30% higher ADP-ATP exchange rates compared to those obtained from DLST+/? or DLD+/? littermates. We propose that KGDHC-associated pathologies are a consequence of the inability of respiration-impaired mitochondria to rely on “in-house” mitochondrial ATP reserves.—Kiss, G., Konrad, C., Doczi, J., Starkov, A. A., Kawamata, H., Manfredi, G., Zhang, S. F., Gibson, G. E., Beal, M. F., Adam-Vizi, V., Chinopoulos, C. The negative impact of ?-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation. PMID:23475850

Kiss, Gergely; Konrad, Csaba; Doczi, Judit; Starkov, Anatoly A.; Kawamata, Hibiki; Manfredi, Giovanni; Zhang, Steven F.; Gibson, Gary E.; Beal, M. Flint; Adam-Vizi, Vera; Chinopoulos, Christos



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

PubMed Central

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

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



Structural Perspective on Enzymatic Halogenation  

PubMed Central

Simple halogen substituents frequently afford key structural features that account for the potency and selectivity of natural products, including antibiotics and hormones. For example, when a single chlorine atom on the antibiotic vancomycin is replaced by hydrogen, the resulting antibacterial activity decreases by up to 70% (HarrisC. M.; KannanR.; KopeckaH.; HarrisT. M.J. Am. Chem. Soc.1985, 107, 6652?6658). This Account analyzes how structure underlies mechanism in halogenases, the molecular machines designed by nature to incorporate halogens into diverse substrates. Traditional synthetic methods of integrating halogens into complex molecules are often complicated by a lack of specificity and regioselectivity. Nature, however, has developed a variety of elegant mechanisms for halogenating specific substrates with both regio- and stereoselectivity. An improved understanding of the biological routes toward halogenation could lead to the development of novel synthetic methods for the creation of new compounds with enhanced functions. Already, researchers have co-opted a fluorinase from the microorganism Streptomyces cattleya to produce 18F-labeled molecules for use in positron emission tomography (PET) (DengH.; CobbS. L.; GeeA. D.; LockhartA.; MartarelloL.; McGlincheyR. P.; O’HaganD.; OnegaM.Chem. Commun.2006, 652?654). Therefore, the discovery and characterization of naturally occurring enzymatic halogenation mechanisms has become an active area of research. The catalogue of known halogenating enzymes has expanded from the familiar haloperoxidases to include oxygen-dependent enzymes and fluorinases. Recently, the discovery of a nucleophilic halogenase that catalyzes chlorinations has expanded the repertoire of biological halogenation chemistry (DongC.; HuangF.; DengH.; SchaffrathC.; SpencerJ. B.; O’HaganD.; NaismithJ. H.Nature2004, 427, 561?56514765200). Structural characterization has provided a basis toward a mechanistic understanding of the specificity and chemistry of these enzymes. In particular, the latest crystallographic snapshots of active site architecture and halide binding sites have provided key insights into enzyme catalysis. Herein is a summary of the five classes of halogenases, focusing on the three most recently discovered: flavin-dependent halogenases, non-heme iron-dependent halogenases, and nucleophilic halogenases. Further, the potential roles of halide-binding sites in determining halide selectivity are discussed, as well as whether or not binding-site composition is always a seminal factor for selectivity. Expanding our understanding of the basic chemical principles that dictate the activity of the halogenases will advance both biology and chemistry. A thorough mechanistic analysis will elucidate the biological principles that dictate specificity, and the application of those principles to new synthetic techniques will expand the utility of halogenations in small-molecule development. PMID:18774824



Phosphorylation of the hepatitis delta virus antigens.  

PubMed Central

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

Bichko, V; Barik, S; Taylor, J



ATM-mediated phosphorylation of the chromatin remodeling enzyme BRG1 modulates DNA double-strand break repair.  


ATP-dependent chromatin remodeling complexes such as SWI/SNF (SWItch/Sucrose NonFermentable) have been implicated in DNA double-strand break (DSB) repair and damage responses. However, the regulatory mechanisms that control the function of chromatin remodelers in DNA damage response are largely unknown. Here, we show that ataxia telangiectasia mutated (ATM) mediates the phosphorylation of BRG1, the catalytic ATPase of the SWI/SNF complex that contributes to DSB repair by binding ?-H2AX-containing nucleosomes via interaction with acetylated histone H3 and stimulating ?-H2AX formation, at Ser-721 in response to DNA damage. ATM-mediated phosphorylation of BRG1 occurs rapidly and transiently after DNA damage. Phosphorylated BRG1 binds ?-H2AX-containing nucleosomes to form the repair foci. The Ser-721 phosphorylation of BRG1 is critical for binding ?-H2AX-containing nucleosomes and stimulating ?-H2AX formation and DSB repair. BRG1 binds to acetylated H3 peptides much better after phosphorylation at Ser-721 by DNA damage. However, the phosphorylation of Ser-721 does not significantly affect the ATPase and transcriptional activities of BRG1. These results, establishing BRG1 as a novel and functional ATM substrate, suggest that the ATM-mediated phosphorylation of BRG1 facilitates DSB repair by stimulating the association of this remodeler with ?-H2AX nucleosomes via enhancing the affinity to acetylated H3. Our work also suggests that the mechanism of BRG1 stimulation of DNA repair is independent of the remodeler's enzymatic or transcriptional activities. PMID:24413084

Kwon, S-J; Park, J-H; Park, E-J; Lee, S-A; Lee, H-S; Kang, S W; Kwon, J



Phosphorylation based insulation devices design and implementation  

E-print Network

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 ...

Rivera Ortiz, Phillip M. (Phillip Michael)



The Chemical Biology of Protein Phosphorylation  

PubMed Central

The explosion of scientific interest in protein kinase-mediated signaling networks has led to the infusion of new chemical methods and their applications related to the analysis of phosphorylation pathways. We highlight some of these chemical biology approaches across three areas. First, we discuss the development of chemical tools to modulate the activity of protein kinases to explore kinase mechanisms and their contributions to phosphorylation events and cellular processes. Second, we describe chemical techniques developed in the past few years to dissect the structural and functional effects of phosphate modifications at specific sites in proteins. Third, we cover newly developed molecular imaging approaches to elucidate the spatiotemporal aspects of phosphorylation cascades in live cells. Exciting advances in our understanding of protein phosphorylation have been obtained with these chemical biology approaches, but continuing opportunities for technological innovation remain. PMID:19489734

Tarrant, Mary Katherine; Cole, Philip A.



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...


Starch: chemistry, microstructure, processing and enzymatic degradation  

Technology Transfer Automated Retrieval System (TEKTRAN)

Starch is recognized as one of the most abundant and important commodities containing value added attributes for a vast number of industrial applications. Its chemistry, structure, property and susceptibility to various chemical, physical and enzymatic modifications offer a high technological value ...


Original article Enzymatic equipment of Ascosphaera apis  

E-print Network

Original article Enzymatic equipment of Ascosphaera apis and the development of infection — The process of infection of honey bee brood by the fungus Ascosphaera apis was ini- tiated the fungus pierces the cuticle. Ascosphaera apis / Apis mellifera / larval cuticle / penetration / infection

Paris-Sud XI, Université de


Heavy metal pollution and soil enzymatic activity  

Microsoft Academic Search

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

Germund Tyler



Frank Westheimer's Early Demonstration of Enzymatic Specificity  

ERIC Educational Resources Information Center

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…

Ault, Addison



Tyrosine Phosphorylation of Botulinum Neurotoxin Protease Domains  

PubMed Central

Botulinum neurotoxins are most potent of all toxins. Their N-terminal light chain domain (Lc) translocates into peripheral cholinergic neurons to exert its endoproteolytic action leading to muscle paralysis. Therapeutic development against these toxins is a major challenge due to their in vitro and in vivo structural differences. Although three-dimensional structures and reaction mechanisms are very similar, the seven serotypes designated A through G vastly vary in their intracellular catalytic stability. To investigate if protein phosphorylation could account for this difference, we employed Src-catalyzed tyrosine phosphorylation of the Lc of six serotypes namely LcA, LcB, LcC1, LcD, LcE, and LcG. Very little phosphorylation was observed with LcD and LcE but LcA, LcB, and LcG were maximally phosphorylated by Src. Phosphorylation of LcA, LcB, and LcG did not affect their secondary and tertiary structures and thermostability significantly. Phosphorylation of Y250 and Y251 made LcA resistant to autocatalysis and drastically reduced its kcat/Km for catalysis. A tyrosine residue present near the essential cysteine at the C-terminal tail of LcA, LcB, and LcG was readily phosphorylated in vitro. Inclusion of a competitive inhibitor protected Y426 of LcA from phosphorylation, shedding light on the role of the C-terminus in the enzyme’s substrate or product binding. PMID:22675300

Toth, Stephen; Brueggmann, Ernst E.; Oyler, George A.; Smith, Leonard A.; Hines, Harry B.; Ahmed, S. Ashraf



Crystal Structure of a Phosphorylated Smad2  

Microsoft Academic Search

Ligand-induced phosphorylation of the receptor-regulated Smads (R-Smads) is essential in the receptor Ser\\/Thr kinase-mediated TGF-? signaling. The crystal structure of a phosphorylated Smad2, at 1.8 Ĺ resolution, reveals the formation of a homotrimer mediated by the C-terminal phosphoserine (pSer) residues. The pSer binding surface on the MH2 domain, frequently targeted for inactivation in cancers, is highly conserved among the Co-

Jia-Wei Wu; Min Hu; Jijie Chai; Joan Seoane; Morgan Huse; Carey Li; Daniel J. Rigotti; Saw Kyin; Tom W. Muir; Robert Fairman; Joan Massagué; Yigong Shi



Phosphorylation regulates myo-inositol-3-phosphate synthase: a novel regulatory mechanism of inositol biosynthesis.  


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 (32)Pi 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

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



Protein phosphorylation in neurodegeneration: friend or foe?  

PubMed Central

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

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



Phosphorylation of human skeletal muscle myosin  

SciTech Connect

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.

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



Phosphorylation of Tau by Fyn: Implications for Alzheimer's Disease  

Microsoft Academic Search

The abnormal phosphorylation of tau protein on serines and threonines is a hallmark characteristic of the neurofibrillary tangles of Alzheimer's disease (AD). The discovery that tau could be phosphorylated on tyrosine and evidence that A signal transduction involved tyrosine phosphorylation led us to question whether tyrosine phosphorylation of tau occurred during the neurodegenerative process. In this study we determined that

Gloria Lee; Ramasamy Thangavel; Vandana M. Sharma; Joel M. Litersky; Kiran Bhaskar; Sandy M. Fang; Lana H. Do; Athena Andreadis; Gary Van Hoesen; Hanna Ksiezak-Reding



In silico modeling of the Menkes copper-translocating P-type ATPase 3rd metal binding domain predicts that phosphorylation regulates copper-binding.  


The Menkes (ATP7A) P(1B)-type ATPase is a transmembrane copper-translocating protein. It contains six similar high-affinity metal-binding domains (MBDs) in the N-terminal cytoplasmic tail that are important for sensing intracellular copper and regulating ATPase function through the transfer of copper between domains. Molecular characterization of copper-binding and transfer is predominantly dependent on NMR structures derived from E. coli expression systems. A limitation of these models is the exclusion of post-translational modifications. We have previously shown that the third copper-binding domain, MBD3, uniquely contains two phosphorylated residues: Thr-327, which is phosphorylated only in the presence of elevated copper; and Ser-339, which is constitutively phosphorylated independent of copper levels. Here, using molecular dynamic simulations, we have incorporated these phosphorylated residues into a model based on the NMR structures of MBD3. Our data suggests that constitutively phosphorylated Ser-339, which is in a loop facing the copper-binding site, may facilitate the copper transfer process by exposing the CxxC copper-binding region of MBD3. Copper-induced phosphorylation of Thr327 is predicted to stabilize this change in conformation. This offers new molecular insights into how cell signaling (phosphorylation) can affect MBD structure and dynamics and how this may in turn affect copper-binding and thus copper-translocation functions of ATP7A. PMID:21258844

Veldhuis, N A; Kuiper, M J; Dobson, R C J; Pearson, R B; Camakaris, J



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Non-enzymatic and enzymatic lipid peroxidation of microsomes and nuclei obtained from rat liver.  


The present study investigates in a experimental system in vitro the relationship between the non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation in rat liver microsomes and nuclei. Chemiluminescence was measured as cpm/mg protein during 180 min at 37 degrees C. Approximately 50-55% of the fatty acids located in rat liver microsomes and nuclei are polyunsaturated with a prevalence of C18:2 n6 and C20:4 n6. The values of total light emission during the non-enzymatic and enzymatic lipid peroxidation were highest in microsomes than in nuclei. A significant decrease of C20:4 n6 and C22:6 n3 in rat liver microsomes and nuclei was observed during the lipid ascorbate-Fe2+-dependent peroxidation, whereas a significant decrease of C20:4 n6 in rat liver microsomes was observed during enzymatic lipid peroxidation. Over the time course studies, analysis of chemiluminescence in microsomes and nuclei demonstrated that the lipid peroxidation in the presence of ascorbate-Fe2+ reach a maximum at 50 and 30 min, respectively, whereas in the presence of NADPH it reachs a maximum at 20 min in both organelles. In liver microsomes and nuclei the peroxidizability index (pi) which indicates the degree of vulnerability to degradation of a selected membrane showed statistically significant differences between control versus ascorbate-Fe2+ when microsomes or nuclei were compared. Our results indicate that non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation are operative in rat liver microsomes and nuclei but the sensitivities of both organelles to lipid peroxidation evidenced by chemiluminescence was greater in the presence of ascorbate-Fe2+ when compared with NADPH. PMID:15543928

Marmunti, Mónica; Gavazza, Mariana; Catalá, Angel



Synthesis of dolichol derivatives in trypanosomatids. Characterization of enzymatic patterns.  


We have previously described that in certain parasitic protozoa, namely the trypanosomatids, the dolichol-P-P-linked oligosaccharides synthesized in vivo and transferred to protein are devoid of glucose residues and contain 6, 7, or 9 mannose units depending on the species. We have now conducted a cell-free characterization of the enzymatic patterns responsible for these phenotypes. Microsomes from Trypanosoma cruzi, Crithidia fasciculata, Leishmania enriettii, and Blastocrithidia culicis were found to synthesize dolichol-P-[14C]Man but not dolichol-P-[14C]Glc when incubated with rat liver dolichol-P and GDP-[14C]Man or UDP-[14C]Glc, thus providing for an explanation to the absence of glucosylated dolichol-P-P derivatives. Formation of dolichol-P-P-oligosaccharides was assayed in incubation mixtures containing rat liver dolichol-P, GDP-[14C]Man, microsomes, and unlabeled Man5-8GlcNAc2-P-P-dolichol from bovine liver. Membranes from species synthesizing dolichol-P-P-linked Man6GlcNAc2 or Man7GlcNAc2 in vivo were found to synthesize the same compounds but not the higher homologues in the cell-free assay. Species forming Man9GlcNAc2-P-P-dolichol in vivo were found to synthesize lipid-linked Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 in vitro. It is concluded that there are at least three and probably four different dolichol-P-Man-dependent enzymatic activities involved in the synthesis of dolichol-P-P-linked Man9GlcNAc2 and that microorganisms not forming that compound are devoid of all mannosyltransferases responsible for the addition of the missing residues and not only of the enzyme involved in the synthesis of the homologue higher than the oligosaccharide occurring in vivo by a single mannose unit. PMID:3112153

de la Canal, L; Parodi, A J



Enzymatic intracrine regulation of white adipose tissue  

PubMed Central

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

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



Enzymatic intracrine regulation of white adipose tissue.  


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

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



Enzymatic glucose biosensors based on nanomaterials.  


: Glucose biosensors have an important place in the diagnosis of diabetes as well as in various food and biotechnological processes. Recent advances in nanomaterials have directly improved enzymatic glucose biosensors owing to their distinguished structural and physiochemical properties. Here, we review the recent developments in electrochemical and fluorescent glucose biosensors based on nanomaterials. New technologies that combine nanomaterials with glucose-sensing enzymes will result in promising glucose biosensors with high specificity and sensitivity. PMID:23824144

Lim, Butaek; Kim, Young-Pil



Structural characterization of cellulose with enzymatic treatment  

NASA Astrophysics Data System (ADS)

Different cellulosic materials were treated with different extraceller microbial enzymes. Changes in structure and properties of the cellulose caused by enzymatic treatment depend on the composition, the type of enzyme and the type of cellulosic materials. Both endoglucanase and crude cellulase have pronounced effects on the degree of polymerization and the structure of cellulose. The variation of supramolecular structure was found to wide variations in conformation caused by the change of the hydrogen bonding energy.

Cao, Yu; Tan, Huimin



Enzymatic solubilization of nitrogenous constituents of carrots  

E-print Network

H. Effect of Incubation Time. . . . , . . Effect of Incubation Temperature. . Summary 3 3 3 6 7 8 9 9 10 10 EXPERIMENTAL Raw Materials. Analysis of Raw Materials. . . Enzymes Enzyme Concentration H. o Incubation Time. Temperature.... Preparation of Material for Enzymatic Treatment 12 12 12 13 13 14 14 14 14 Chapter Page Residue Assay Procedure. . . Statistical Plan. . 15 17 IV RESULTS AND DISCUSSION. Analysis of Raw Materials. . . . . . . Enzymati c Treatments Effect...

Curry, James Cannon



Enzymatic transformation of nonfood biomass to starch.  


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

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



Enzymatic transformation of nonfood biomass to starch  

PubMed Central

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

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



Phosphorylated silk fibroin matrix for methotrexate release.  


Silk-based matrix was produced for delivery of a model anticancer drug, methotrexate (MTX). The calculation of net charge of silk fibroin and MTX was performed to better understand the electrostatic interactions during matrix formation upon casting. Silk fibroin films were cast at pH 7.2 and pH 3.5. Protein kinase A was used to prepare phosphorylated silk fibroin. The phosphorylation content of matrix was controlled by mixing at specific ratios the phosphorylated and unphosphorylated solutions. In vitro release profiling data suggest that the observed interactions are mainly structural and not electrostatical. The release of MTX is facilitated by use of proteolytic enzymes and higher pHs. The elevated ?-sheet content and crystallinity of the acidified-cast fibroin solution seem not to favor drug retention. All the acquired data underline the prevalence of structural interactions over electrostatical interactions between methotrexate and silk fibroin. PMID:25435334

Volkov, Vadim; Sárria, Marisa P; Gomes, Andreia C; Cavaco-Paulo, Artur



Cold enzymatic bleaching of fluid whey.  


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

Campbell, R E; Drake, M A



Focus Issue: Systems Analysis of Protein Phosphorylation  

NSDL National Science Digital Library

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

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



An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway  

PubMed Central

Generation and turnover of phosphoinositides (PIs) must be coordinated in a spatial- and temporal-restricted manner. The small GTPase Rab5 interacts with two PI 3-kinases, Vps34 and PI3K?, suggesting that it regulates the production of 3-PIs at various stages of the early endocytic pathway. Here, we discovered that Rab5 also interacts directly with PI 5- and PI 4-phosphatases and stimulates their activity. Rab5 regulates the production of phosphatidylinositol 3-phosphate (PtdIns[3]P) through a dual mechanism, by directly phosphorylating phosphatidylinositol via Vps34 and by a hierarchical enzymatic cascade of phosphoinositide-3-kinase? (PI3K?), PI 5-, and PI 4-phosphatases. The functional importance of such an enzymatic pathway is demonstrated by the inhibition of transferrin uptake upon silencing of PI 4-phosphatase and studies in weeble mutant mice, where deficiency of PI 4-phosphatase causes an increase of PtdIns(3,4)P2 and a reduction in PtdIns(3)P. Activation of PI 3-kinase at the plasma membrane is accompanied by the recruitment of Rab5, PI 4-, and PI 5-phosphatases to the cell cortex. Our data provide the first evidence for a dual role of a Rab GTPase in regulating both generation and turnover of PIs via PI kinases and phosphatases to coordinate signaling functions with organelle homeostasis. PMID:16103228

Shin, Hye-Won; Hayashi, Mitsuko; Christoforidis, Savvas; Lacas-Gervais, Sandra; Hoepfner, Sebastian; Wenk, Markus R.; Modregger, Jan; Uttenweiler-Joseph, Sandrine; Wilm, Matthias; Nystuen, Arne; Frankel, Wayne N.; Solimena, Michele; De Camilli, Pietro; Zerial, Marino



Intracellular phosphorylation of vitellogenin in the liver of estrogen-stimulated Xenopus laevis  

SciTech Connect

A procedure was developed for the preparation of rough and smooth microsomes from small quantities of liver obtained from estrogen-stimulated Xenopus laevis females. Morphological, biochemical, and enzymatic characterization of these fractions indicates that excellent separation of rough microsomes from smooth microsomes is achieved. In addition, pulse-chase experiments demonstrate that rough and smooth microsomes each exhibit the predictable patterns of incorporation characteristic of secretory protein synthesis and intracellular translocation. This procedure was combined with suitable incubation conditions for pulse-chase experiments which demonstrate the subcellular sites of vitellogenin phosphorylation. The data presented indicate that approximately 70% of the phosphate residues are covalently attached to vitellogenin during its intracellular translocation through the smooth microsomes, while the rough microsomes can account for the remainder of the total incorporated phosphate.

Gottlieb, T.A.; Wallace, R.A.



Herpes Simplex Virus 1 Protein Kinase Us3 Phosphorylates Viral dUTPase and Regulates Its Catalytic Activity in Infected Cells  

PubMed Central

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). In this study, a large-scale phosphoproteomic analysis of titanium dioxide affinity chromatography-enriched phosphopeptides from HSV-1-infected cells using high-accuracy mass spectrometry (MS) and subsequent analyses showed that Us3 phosphorylated HSV-1-encoded dUTPase (vdUTPase) at serine 187 (Ser-187) in HSV-1-infected cells. Thus, the following observations were made. (i) In in vitro kinase assays, Ser-187 in the vdUTPase domain was specifically phosphorylated by Us3. (ii) Phosphorylation of vdUTPase Ser-187 in HSV-1-infected cells was detected by phosphate-affinity polyacrylamide gel electrophoresis analyses and was dependent on the kinase activity of Us3. (iii) Replacement of Ser-187 with alanine (S187A) in vdUTPase and an amino acid substitution in Us3 that inactivated its kinase activity significantly downregulated the enzymatic activity of vdUTPase in HSV-1-infected cells, whereas a phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type enzymatic activity of vdUTPase. (iv) The vdUTPase S187A mutation as well as the kinase-dead mutation in Us3 significantly reduced HSV-1 replication in human neuroblastoma SK-N-SH cells at a multiplicity of infection (MOI) of 5 but not at an MOI of 0.01, whereas the phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type viral replication at an MOI of 5. In contrast, these mutations had no effect on HSV-1 replication in Vero and HEp-2 cells. Collectively, our results suggested that Us3 phosphorylation of vdUTPase Ser-187 promoted HSV-1 replication in a manner dependent on cell types and MOIs by regulating optimal enzymatic activity of vdUTPase. PMID:24173231

Kato, Akihisa; Tsuda, Shumpei; Liu, Zhuoming; Kozuka-Hata, Hiroko; Oyama, Masaaki



Comparative performance of enzymatic and combined alkaline-enzymatic pretreatments on methane production from ensiled sorghum forage.  


This study investigated the effect of enzymatic and combined alkaline-enzymatic pretreatments on chemical composition and methane production from ensiled sorghum forage. Four commercial enzymatic preparations were tested and the two yielding the highest sugars release were added to evaluate any hydrolytic effect on both untreated and alkaline pretreated samples. In the combined alkaline-enzymatic pretreatment trials, the highest sugar release was found with Primafast and BGL preparations (added at a final concentration 0.12 and 0.20 mL/g TS, respectively), with a total monomeric content of 12 and 6.5 g/L. Fibre composition analysis confirmed that the combined alkaline-enzymatic pretreatment led to cellulose (up to 32 %) and hemicelluloses (up to 56 %) solubilisation, compared to the enzymatic pretreatment alone. BMP tests were performed on both untreated and pretreated samples, and time courses of methane production were fitted. Both enzymatic and combined alkaline-enzymatic pretreatment led to a methane production increase (304 and 362 mL CH4/g VS), compared to that of untreated sorghum (265 mL CH4/g VS), as  +15 and  +37 %, respectively. Moreover, higher specific methane production rates, compared to that of untreated sorghum (20.31 mL CH4/g VS/d), were obtained by applying the enzymatic and combined alkaline-enzymatic pretreatment (33.94 and 31.65 mL CH4/g VS/d), respectively. PMID:24962776

Rollini, Manuela; Sambusiti, Cecilia; Musatti, Alida; Ficara, Elena; Retinň, Isabella; Malpei, Francesca



Distinct DNA damage determines differential phosphorylation of Chk2.  


Checkpoint kinase 2 (Chk2) has been implicated in DNA damage signaling. By using BJ human fibroblasts, HCT116 colorectal cancer cells and HeLa cervical cancer cells, we further detailed phosphorylation kinetics of Chk2 under treatment with neocarcinostatin (NCS) or doxorubicin (Dox). After NCS treatment, phosphorylation of Chk2 Thr68 occurs in 3 min, followed by phosphorylation of Ser19 and Ser33/35. In ATM deficient fibroblasts, NCS does not induce phosphorylation of NBS1 Ser343 and Chk2 Ser19 and Ser33/35, however Chk2 Thr68 is still phosphorylated, indicating that ATM is essential for phosphorylation of these residues when treated with NCS. By using Chk2-deficient HCT116 cells re-expressing phospho-mutant Chk2 (T68A), we found that inhibition of Thr68 phosphorylation enhances Ser19 phosphorylation in NCS treated cells. Interestingly, in contrast to NCS, Dox does not induce Ser33/35 phosphorylation in HeLa and HCT116 cells. Phosphorylation of Thr68 is sustained until 3 to 4 hours, and phosphorylation of Ser19 occurs 70 to 80 min after Dox treatment. These results demonstrate that Chk2 s involved in the early stages of DNA damage response. Differential phosphorylation kinetics of these residues suggests that DNA damage determines intermolecular and intramolecular interaction of Chk2, which may regulate phosphorylation. PMID:25535901

Ouchi, Mutsuko; Ouchi, Toru



Proteomics Analysis Identifies Phosphorylation-dependent -Synuclein  

E-print Network

to familial Parkinson disease (Thomas, B., and Beal, M. F. (2007) Parkinson's disease. Hum. Mol. Genet. 16, R bodies, a pathological hallmark of Parkinson disease, and is mostly phosphorylated at Ser-129 (Anderson linked to familial Parkinson disease (PD)1 (2, 44). The presence of large amounts of -synuclein

Baker, Chris I.


Temporal Phosphorylation of the Drosophila Period Protein  

Microsoft Academic Search

The period gene (per) is required for Drosophila melanogaster to manifest circadian ( ≈24 hr) rhythms. We report here that per protein (PER) undergoes daily oscillations in apparent molecular mass as well as abundance. The mobility changes are largely or exclusively due to multiple phosphorylation events. The temporal profile of the classic short-period form of PER (PERS) is altered in

Isaac Edery; Laurence J. Zwiebel; Marie E. Dembinska; Michael Rosbash



Effects of validamycin on some enzymatic activities in soil  

Microsoft Academic Search

There have been few investigations of the possible effects of validamycin on the enzymatic activities in soil. The objective\\u000a of this present work was to study the short-term influence of enzymatic activity in validamycin treated soils. The results\\u000a showed that high dose of validamycin introduced into soil had an significant effect on soil enzymatic activities, the degree\\u000a of inhibition or

Haifeng Qian; Baolan Hu; Zhiye Wang; Xi Xu; Tao Hong



Phenindione interference in enzymatic creatinine assay - A case report.  


Enzymatic creatinine assays are considered superior to Jaffe assays due to greater analytical specificity. We report a case of phenindione interference with an enzymatic assay resulting in significant misclassification in a patient with chronic kidney disease (CKD). Analysis of creatinine values of a further 36 patients who were treated with phenindione showed significant negative interference of phenindione with the Roche enzymatic creatinine assay. PMID:24361058

Natarajan, Balan; Hart, Tanya; Smith, Sophy; Parry, Robin Geoffrey



Stereoselective synthesis of 2-deoxy-2-fluoroarabinofuranosyl-alpha-1-phosphate and its application to the synthesis of 2'-deoxy-2'-fluoroarabinofuranosyl purine nucleosides by a chemo-enzymatic method.  


Stereoselective introduction of a phosphate moiety into 2-deoxy-2-fluoroarabinofuranose derivatives at the anomeric position was investigated by two methods. One involved a stereoselective hydrolysis of 1-bromo-derivative, and the consecutive phosphorylation of 2-deoxy-2-fluoro-alpha-D-arabinofuranose via a phosphoramidite derivative. The other method involved stereoselective alpha-phosphorylation of the 1-bromo-derivative at the 1-position. The resulting alpha-1-phosphate was utilized to prepare 2'-deoxy-2'-fluoroarabinofuranosyl purine nucleosides by an enzymatic glycosylation reaction. This chemo-enzymatic method will be applicable to the synthesis of some 2'F-araNs, and three important 2'F-araNs were actually obtained in 30-40% yields from 1,3,5-tri-O-benzoyl-2-deoxy-2-fluoro-alpha-D-arabinose with high purity. PMID:20183578

Yamada, Kohei; Matsumoto, Noritake; Hayakawa, Hiroyuki



Continuous enzymatic liquefaction of starch for saccharification  

SciTech Connect

A process was explored for continuous enzymatic liquefaction of corn starch at high concentration and subsequent saccharification to glucose. The process appears to be quite efficient for conversion of starch to glucose and enzymatic liquefaction and should be readily adaptable to industrial fermentation processes. Preliminary work indicated that milled corn or other cereal grains also can be suitably converted by such a process. Essentially, the process involved incorporation of a thermostable, bacterial alpha-amylase for liquefaction and, subsequently, of a glucoamylase into the continuous mixer under conditions conductive to rapid enzymatic hydrolyses. Also studied was the effect on substrate liquefaction of variables such as starch concentration (40-70%), level of alpha-amylase (0.14-0.4%, dry starch basis), temperature (70-100 degrees C), pH (5.8-7.1), and residence time (6 and 12 minutes). The degree of liquefaction was assessed by determining 1) the Brookfield viscosity, 2) the amount of reducing groups, and 3) the rate and extent of glucose formed after glucoamylase treatment. Best liquefaction processing conditions were achieved by using 50-60% starch concentration, at 95 degrees C, with 0.4% alpha-amylase, and a 6 minute residence period in the mixer. Under these conditions, rates and extents of glucose obtained after glucoamylase treatment approached those obtained in longer laboratory batch liquefactions. The amount of glucose formed in 24 hours with the use of 0.4% glucoamylase was 86% of theory after a 6-min continuous liquefaction, compared to 90% for a 30-min laboratory batch liquefaction (95 degrees C, 0.4% alpha-amylase). (Refs. 15).

Carr, M.E.; Black, L.T.; Bagby, M.O.



Large-scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis  

PubMed Central

Protein phosphorylation regulates a wide range of cellular processes. Here, we report the proteome-wide mapping of in vivo phosphorylation sites in Arabidopsis by using complementary phosphopeptide enrichment techniques coupled with high-accuracy mass spectrometry. Using unfractionated whole cell lysates of Arabidopsis, we identified 2597 phosphopeptides with 2172 high-confidence, unique phosphorylation sites from 1346 proteins. The distribution of phosphoserine, phosphothreonine, and phosphotyrosine sites was 85.0, 10.7, and 4.3%. Although typical tyrosine-specific protein kinases are absent in Arabidopsis, the proportion of phosphotyrosines among the phospho-residues in Arabidopsis is similar to that in humans, where over 90 tyrosine-specific protein kinases have been identified. In addition, the tyrosine phosphoproteome shows features distinct from those of the serine and threonine phosphoproteomes. Taken together, we highlight the extent and contribution of tyrosine phosphorylation in plants. PMID:18463617

Sugiyama, Naoyuki; Nakagami, Hirofumi; Mochida, Keiichi; Daudi, Arsalan; Tomita, Masaru; Shirasu, Ken; Ishihama, Yasushi



Protein Synthesis Initiation Factors: Phosphorylation and Regulation  

SciTech Connect

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.

Karen S. Browning



Enzymatic Catalytic Beds For Oxidation Of Alcohols  

NASA Technical Reports Server (NTRS)

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.

Jolly, Clifford D.; Schussel, Leonard J.



RNA structural analysis by enzymatic digestion.  


Enzymatic probing is a rapid, straightforward method for determining which regions of a folded RNA are structurally constrained. It can be carried out using very small amounts of material, and is especially suitable for short RNAs. Here we report a protocol that we have found to be useful and readily adaptable to the evaluation of RNAs up to 150-200 nucleotides in length. Considerations for optimization are also included. In brief, the method includes folding end-labeled RNA into its native conformation, partial digestion with structure-sensitive nucleases, and identification of the cleavage sites by electrophoretic separation of the cleavage fragments. PMID:24136597

Biondi, Elisa; Burke, Donald H



Enzymatic kinetic resolution of silybin diastereoisomers.  


In nature, the flavonolignan silybin (1) occurs as a mixture of two diastereomers, silybin A and silybin B, which in a number of biological assays exhibit different activities. A library of hydrolases (lipases, esterases, and proteases) was tested for separating the silybin A and B diastereomers by selective transesterification or by stereoselective alcoholysis of 23-O-acetylsilybin (2). Novozym 435 proved to be the most suitable enzyme for the preparative production of both optically pure silybins A and B by enzymatic discrimination. Gram amounts of the optically pure substances can be produced within one week, and the new method is robust and readily scalable to tens of grams. PMID:20297826

Monti, Daniela; Gazák, Radek; Marhol, Petr; Biedermann, David; Purchartová, Katerina; Fedrigo, Mirko; Riva, Sergio; Kren, Vladimír



In vitro rescue study of a malignant familial hypertrophic cardiomyopathy phenotype by pseudo-phosphorylation of myosin regulatory light chain.  


Pseudo-phosphorylation of cardiac myosin regulatory light chain (RLC) has never been examined as a rescue method to alleviate a cardiomyopathy phenotype brought about by a disease causing mutation in the myosin RLC. This study focuses on the aspartic acid to valine substitution (D166V) in the myosin RLC shown to be associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC). The mutation has also been demonstrated to cause severe functional abnormalities in transgenic mice expressing D166V in the heart. To explore this novel rescue strategy, pseudo-phosphorylation of D166V was used to determine whether the D166V-induced detrimental phenotype could be brought back to the level of wild-type (WT) RLC. The S15D substitution at the phosphorylation site of RLC was inserted into the recombinant WT and D166V mutant to mimic constitutively phosphorylated RLC proteins. Non-phosphorylatable (S15A) constructs were used as controls. A multi-faceted approach was taken to determine the effect of pseudo-phosphorylation on the ability of myosin to generate force and motion. Using mutant reconstituted porcine cardiac muscle preparations, we showed an S15D-induced rescue of both the enzymatic and binding properties of D166V-myosin to actin. A significant increase in force production capacity was noted in the in vitro motility assays for S15D-D166V vs. D166V reconstituted myosin. A similar pseudo-phosphorylation induced effect was observed on the D166V-elicited abnormal Ca(2+) sensitivity of force in porcine papillary muscle strips reconstituted with phosphomimic recombinant RLCs. Results from this study demonstrate a novel in vitro rescue strategy that could be utilized in vivo to ameliorate a malignant cardiomyopathic phenotype. We show for the first time that pseudo-RLC phosphorylation can reverse the majority of the mutation-induced phenotypes highlighting the importance of RLC phosphorylation in combating cardiac disease. PMID:24374283

Muthu, Priya; Liang, Jingsheng; Schmidt, William; Moore, Jeffrey R; Szczesna-Cordary, Danuta



The properties and kinetics of enzymatic reaction in the process of the enzymatic extraction of fish oil  

Microsoft Academic Search

The technology of enzymatic extraction of fish oil has many advantages, such as moderate operating conditions, lower energy\\u000a consumption and high efficiency. Moreover, it could reduce the breakage for the functional component of fish oil. In enzymatic\\u000a extraction of oil, the investigation of the property of enzymatic reaction is propitious to improve the enzymolysis efficiency.\\u000a In this study, the 1398

Junqing Qian; Hongyong Zhang; Qiyuan Liao



The importance of intrinsic disorder for protein phosphorylation  

E-print Network

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 often affect protein function. For example, serine phosphorylation of the peptide corresponding

Radivojac, Predrag


Enzymatic deconstruction of xylan for biofuel production  

PubMed Central

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




Enzymatic activities in coniferous leaf litter  

SciTech Connect

Assays for measuring the activities of cellulase, xylanase, mannase, amylase, ..beta..-glucosidase, invertase, and protease employing buffered suspensions of ground coniferous and deciduous leaf litter exhibited zero-order kinetics. Only a small percentage of the whole-litter activities of invertase, ..beta..-glucosidase, and protease were extractable into 0.05M potassium acetate, pH 5.0; however, extractable activities of cellulase and xylanase represented from 39 to 174% of the whole-litter activities indicating their soluble exocellar nature. Extractable protease and amylase activities were best correlated with the average daily rates of CO/sub 2/ evolution in a group of 90 leaf litter samples equally representing 18 coniferous species. Enzymatic activities were readily detectable in extracts of all samples but classification of the samples by species provided little differentiation in the distribution of either enzymatic activities or rates of CO/sub 2/ evolution. Mannase, cellulase, and xylanase activities were well-correlated with each other in all samples. Assays attempting to measure a pool of readily-metabolizable substances in litter by extractable reducing substances, ninhydrin-positive substances, glucose, and phenolics failed to show correlation coefficients >0.41 with rates of CO/sub 2/ evolution. Addition of D-(+)-catechin to litter extracts, up to levels equivalent to those observed in the group of samples, did not inhibit any carbohydrase thus suggesting the lack of inhibition of litter-decomposing enzymes by the concentrations of phenolics present in these coniferous leaf litters.

Spalding, B.P.



Polymerization of glucans by enzymatically active membranes.  


Conventional enzyme membrane reactors are not appropriate for a continuous synthesis of macromolecules and simultaneous product release. By immobilizing the enzyme in sufficiently large pores of a membrane an ensemble of miniaturized bioreactors is created. Product molecules are continuously removed from the enzyme by the flow of the reaction mixture across the membrane. Additionally, by varying the flow rate, it ought to be possible to influence the substrate as well as the enzyme-product residence times and thereby the product macromolecule's size. In this paper we present the first results of experiments involving enzymatic 1,4-alpha-glucan synthesis, using sucrose as substrate, maltooligosaccharides (DP 3-6) as primers, and membrane-immobilized amylosucrase. Epoxy groups for a covalent enzyme immobilization were generated on polypropylene microfiltration membranes by heterogeneous photoinitiated graft polymerization of glycidyl methacrylate. The influence of primer concentration and flow rate through the enzyme-membrane on amylosucrase activity, molecule growth, and coupling efficiency for glucose (% of coupled glucose versus free glucose) were investigated. The enzymatically mediated chain elongation of maltooligosaccharides by the successive addition of glucose units was achieved for the first time in a transmembrane process utilizing amylosucrase membranes. PMID:12363346

Becker, Margot; Provart, Nicholas; Lehmann, Ingeburg; Ulbricht, Mathias; Hicke, Hans-Georg



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

SciTech Connect

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.

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



Regulation of gamma-glutamylcysteine synthetase by protein phosphorylation.  

PubMed Central

We previously reported that the activity of gamma-glutamylcysteine synthetase (GCS; EC, the rate-limiting enzyme in GSH synthesis, can be acutely inhibited approximately 20-40% by agonists of various signal transduction pathways in rat hepatocytes [Lu, Kuhlenkamp, Garcia-Ruiz and Kaplowitz (1991) J. Clin. Invest. 88, 260-269]. We have now examined the possibility that GCS is phosphorylated directly by activation of protein kinase A (PKA), protein kinase C (PKC) and Ca2+/calmodulin-dependent kinase II (CMK). Phosphorylation of GCS was studied using both purified rat kidney GCS and cultured rat hepatocytes by immunoprecipitating the reaction product with specific rabbit anti-(rat GCS heavy subunit) (anti-GCS-HS) antibodies. All three kinases, PKA, PKC and CMK, phosphorylated rat kidney GCS-HS in a Mg(2+)-concentration-dependent manner, with the highest degree of phosphorylation occurring at 20 mM Mg2+. The maximum incorporation of phosphate in mol/mol of GCS was 1.17 for PKA, 0.70 for PKC and 0.62 for CMK. The degree of phosphorylation was correlated with the degree of loss of GCS activity, and no additional inhibition occurred when GCS was phosphorylated by all three kinases, suggesting that the kinases phosphorylated the same site(s). Phosphoamino analysis showed that all three kinases phosphorylated serine and threonine residues. Two-dimensional phosphopeptide mapping demonstrated that all three kinases phosphorylated the same five peptides, both PKA and PKC phosphorylated two other peptides, and only PKA phosphorylated one additional peptide. Phosphorylation of GCS decreased its Vmax for cysteine and glutamate without changing its K(m). Finally, treatment of cultured rat hepatocytes with dibutyryl cAMP and phenylephrine significantly increased the phosphorylation of GCS, suggesting a potentially important physiological role. In summary, we have demonstrated that GCS is phosphorylated and suggest that phosphorylation/dephosphorylation may regulate GCS activity. PMID:8947504

Sun, W M; Huang, Z Z; Lu, S C



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


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

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



Enhancing phosphorylation cascades by anomalous diffusion  

NASA Astrophysics Data System (ADS)

A key event in many cellular signaling cascades is the multiple phosphorylation of proteins by specialized kinases. A prototypical example is the mitogen-activated protein kinase (MAPK) that alters the cell's gene transcription after having been phosphorylated twice by the same kinase. Here, we show that anomalous diffusion, induced, for example, by cytoplasmic crowding, can significantly improve the activation of MAPK. Our results on anomalous diffusion with the characteristics of fractional Brownian motion and obstructed diffusion compare favorably to very recent biochemical data on MAPK activation at varying degrees of cytoplasmic crowding. Our results predict any Michaelis-Menten scheme in which a substrate is modified by the same enzyme several times to show an increased performance due to anomalous diffusion when dissociation rates of the intermediate enzyme-substrate complexes are high while the irreversible catalytic step is slow. Thus, crowding-induced anomalous diffusion can strongly alter the behavior of many cellular signaling pathways.

Hellmann, M.; Heermann, D. W.; Weiss, M.



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


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

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



RESEARCH Open Access Comparison of enzymatic reactivity of corn stover  

E-print Network

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

California at Riverside, University of


RESEARCH Open Access Enzymatic-and temperature-sensitive controlled  

E-print Network

imaging can expose the patient to relatively high doses of ionizing radiation. Magnetic resonance imaging increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer plaques. Background Enzymatic activity is understood to be a hallmark of var- ious diseases, including


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...


Sizing up single-molecule enzymatic conformational dynamics.  


Enzymatic reactions and related protein conformational dynamics are complex and inhomogeneous, playing crucial roles in biological functions. The relationship between protein conformational dynamics and enzymatic reactions has been a fundamental focus in modern enzymology. It is extremely difficult to characterize and analyze such complex dynamics in an ensemble-averaged measurement, especially when the enzymes are associated with multiple-step, multiple-conformation complex chemical interactions and transformations. Beyond the conventional ensemble-averaged studies, real-time single-molecule approaches have been demonstrated to be powerful in dissecting the complex enzymatic reaction dynamics and related conformational dynamics. Single-molecule enzymology has come a long way since the early demonstrations of the single-molecule spectroscopy studies of enzymatic dynamics about two decades ago. The rapid development of this fundamental protein dynamics field is hand-in-hand with the new development of single-molecule imaging and spectroscopic technology and methodology, theoretical model analyses, and correlations with biological preparation and characterization of the enzyme protein systems. The complex enzymatic reactions can now be studied one molecule at a time under physiological conditions. Most exciting developments include active manipulation of enzymatic conformational changes and energy landscape to regulate and manipulate the enzymatic reactivity and associated conformational dynamics, and the new advancements have established a new stage for studying complex protein dynamics beyond by simply observing but by actively manipulating and observing the enzymatic dynamics at the single-molecule sensitivity temporally and spatially. PMID:24306450

Lu, H Peter



Solid polymer electrolyte from phosphorylated chitosan  

SciTech Connect

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.

Fauzi, Iqbal, E-mail:; Arcana, I Made, E-mail: [Inorganic and Physical Chemistry Research Groups, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Jl. Ganesha 10, Bandung 40132 (Indonesia)



Regulation of Smad Activity by Phosphorylation  

Microsoft Academic Search

Transforming growth factor-? (TGF-?) regulates a wide variety of biological activities. Smad proteins play a major role in\\u000a transducing the TGF-? family signal at the cell surface into gene regulation in the nucleus. In response to TGF-?, Smad2 and\\u000a Smad3 are activated through phosphorylation by the TGF-? receptor at the C-tail SSXS motif. The activated Smad2 and Smad3\\u000a then form

Fang Liu


Pretreatment and enzymatic hydrolysis of lignocellulosic biomass  

NASA Astrophysics Data System (ADS)

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

Corredor, Deisy Y.


Phosphorylation Stoichiometries of Human Eukaryotic Initiation Factors  

PubMed Central

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

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



Multistep phosphorylation systems: tunable components of biological signaling circuits  

PubMed Central

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

Valk, Evin; Venta, Rainis; Örd, Mihkel; Faustova, Ilona; Kőivomägi, Mardo; Loog, Mart



Multistep phosphorylation systems: tunable components of biological signaling circuits.  


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

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



Fungal biodegradation and enzymatic modification of lignin  

PubMed Central

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

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



In Plants, 3-O-Methylglucose Is Phosphorylated by Hexokinase But Not Perceived as a Sugar1  

PubMed Central

In plants, sugars are the main respiratory substrates and important signaling molecules in the regulation of carbon metabolism. Sugar signaling studies suggested that sugar sensing involves several key components, among them hexokinase (HXK). Although the sensing mechanism of HXK is unknown, several experiments support the hypothesis that hexose phosphorylation is a determining factor. Glucose (Glc) analogs transported into cells but not phosphorylated are frequently used to test this hypothesis, among them 3-O-methyl-Glc (3-OMG). The aim of the present work was to investigate the effects and fate of 3-OMG in heterotrophic plant cells. Measurements of respiration rates, protein and metabolite contents, and protease activities and amounts showed that 3-OMG is not a respiratory substrate and does not contribute to biosynthesis. Proteolysis and lipolysis are induced in 3-OMG-fed maize (Zea mays L. cv DEA) roots in the same way as in sugar-starved organs. However, contrary to the generally accepted idea, phosphorous and carbon nuclear magnetic resonance experiments and enzymatic assays prove that 3-OMG is phosphorylated to 3-OMG-6-phosphate, which accumulates in the cells. Insofar as plant HXK is involved in sugar sensing, these findings are discussed on the basis of the kinetic properties because the catalytic efficiency of HXK isolated from maize root tips is five orders of magnitude lower for 3-OMG than for Glc and Man. PMID:12586906

Cortčs, Sandra; Gromova, Marina; Evrard, Adeline; Roby, Claude; Heyraud, Alain; Rolin, Dominique B.; Raymond, Philippe; Brouquisse, Renaud M.



?-cyclodextrin assistant flavonoid glycosides enzymatic hydrolysis  

PubMed Central

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

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



Puzzling over protein cysteine phosphorylation--assessment of proteomic tools for S-phosphorylation profiling.  


Cysteine phosphorylation has recently been discovered in both prokaryotic and eukaryotic systems, and is thought to play crucial roles in signaling and regulation of cellular responses. This article explores the topics of chemical stability of this type of structural modification and the resulting issues regarding affinity enrichment of S-phosphopeptides and their mass spectrometry-based detection in the course of general proteomics studies. Together, this work suggests that the current advances in phosphoproteomic methodologies provide adequate tools for investigating protein cysteine phosphorylation and appear to be immediately available for practical implementation. The article provides useful information necessary for designing experiments in the emerging cysteine phosphoproteomics. The examples of methodological proposals for S-linked phosphorylation detection are included herein in order to stimulate development of new approaches by the phosphoproteomic community. PMID:25011562

Buchowiecka, A K



Mitotic phosphorylation of histone H3 threonine 80.  


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

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



Mitotic phosphorylation of histone H3 threonine 80  

PubMed Central

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

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



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


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

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



High-yield enzymatic bioconversion of hydroquinone to ?-arbutin, a powerful skin lightening agent, by amylosucrase.  


?-Arbutin (?-Ab) is a powerful skin whitening agent that blocks epidermal melanin biosynthesis by inhibiting the enzymatic oxidation of tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA). ?-Ab was effectively synthesized from hydroquinone (HQ) by enzymatic biotransformation using amylosucrase (ASase). The ASase gene from Deinococcus geothermalis (DGAS) was expressed and efficiently purified from Escherichia coli using a constitutive expression system. The expressed DGAS was functional and performed a glycosyltransferase reaction using sucrose as a donor and HQ as an acceptor. The presence of a single HQ bioconversion product was confirmed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The HQ bioconversion product was isolated by silica gel open column chromatography and its chemical structure determined by 1H and 13C nuclear magnetic resonance (NMR). The product was determined to be hydroquinone-O-?-D-glucopyranoside with a glucose molecule linked to HQ through an ?-glycosidic bond. However, the production yield of the transfer reaction was significantly low (1.3%) due to the instability of HQ in the reaction mixture. The instability of HQ was considerably improved by antioxidant agents, particularly ascorbic acid, implying that HQ is labile to oxidation. A maximum yield of HQ transfer product of 90% was obtained at a 10:1 molar ratio of donor (sucrose) and acceptor (HQ) molecules in the presence of 0.2 mM ascorbic acid. PMID:22314516

Seo, Dong-Ho; Jung, Jong-Hyun; Ha, Suk-Jin; Cho, Hyun-Kug; Jung, Dong-Hyun; Kim, Tae-Jip; Baek, Nam-In; Yoo, Sang-Ho; Park, Cheon-Seok



Effect of Ultrasonic Frequency on Enzymatic Hydrolysis of Cellulose  

NASA Astrophysics Data System (ADS)

The effect of ultrasonic frequency on the enzymatic hydrolysis of cellulose was examined. As the cellulose and enzyme, needle unbleached kraft pulp and cellulase were used. In the cases of the horn-type transducer at 20 kHz and the plate-type transducer at 28 kHz, the enzymatic hydrolysis was accelerated by ultrasonic irradiation. Total sugar concentration linearly increased with ultrasonic intensity. On the other hand, in the case of the plate-type transducer at 500 kHz, the enzymatic hydrolysis was inhibited. Total sugar concentration decreased with increasing ultrasonic intensity.

Keiji Yasuda,; Daiki Kato,; Zheng Xu,; Makiko Sakka,; Kazuo Sakka,



Can You Hear Me Now? Regulating Transcriptional Activators by Phosphorylation  

NSDL National Science Digital Library

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

Kevin H. Gardner (University of Texas Southwestern Medical Center;Departments of Biochemistry and Pharmacology REV); Marc Montminy (The Salk Institute for Biological Studies; REV)



Enzymatic detection of troponin C and melittin bee  

NASA Astrophysics Data System (ADS)

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

Jeetender, Amritsar; Stiharu, Ion; Packirisamy, Muthukumaran



Selenium mediated arsenic toxicity modifies cytotoxicity, reactive oxygen species and phosphorylated proteins.  


The effect of selenium on modulating arsenic cytotoxicity is well known in mammals, but not well understood. Cell cytotoxicity and reactive oxygen species (ROS) changes were performed in combinations of As(III) and selenomethionine (SeMet) toxic mixes on, HEK293, human kidney cells. Cell growth is readily restored from 20% to 60% when switching from 30 ?M As(III) as toxin to a mix of 30 ?M As(III) and 100 ?M SeMet. As(III) alone triggers ROS formation, primarily hydrogen peroxide, in a concentration dependent manner as observed through changes in the fluorescence from 2',7'-dichlorofluorescein diacetate. Importantly, SeMet induces lower ROS levels at the same concentrations used to modulate As(III) cytotoxicity (IC50). Elevated ROS is important to As(III) cytotoxicity and minimizing it is essential to the SeMet modulating function. Changes in cell signaling, through analysis of signaling changes via differential protein phosphorylation to uncover molecular level changes occurring in HEK293 human kidney cells as SeMet modulates the As(III) cytotoxicity. To discover changes in the phosphoproteome, cells were incubated under three conditions: 30 ?M As(III), 100 ?M SeMet, and 30 ?M As(III) + 100 ?M SeMet. After total protein isolation the three samples were separated into fractions using size exclusion chromatography by detecting (31)P(+). Each sample was analyzed for the phosphorylated peptides by enzymatic digestion, selective enrichment of phosphorylated peptides via TiO2, followed by nanoLC-ESIMS. Phosphorylated proteins unique to the As(III)-SeMet mixture were then identified. The molecular level changes to the cells show uniquely that the As(III)-SeMet mixture details proteins involved in ROS detoxification, cell cycle arrest, and protein/DNA damage. This study shows that SeMet not only lowers the total amount of ROS in a cell but also confers upon HEK293 cells the ability to detoxify. Thus, SeMet is not only a potent antioxidant in this system, but induces molecular changes that confer survival. PMID:23503360

Chitta, Karnakar R; Landero Figueroa, Julio A; Caruso, Joseph A; Merino, Edward J



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

E-print Network

ERK Nuclear Translocation Is Dimerization-independent but Controlled by the Rate of Phosphorylation, Universite´ de Nice, Centre A. Lacassagne, 06189 Nice, France Upon activation, ERKs translocate from the molecular mechanisms that regulate ERK nuclear translocation are not fully understood. We have used a mouse

Rieger, Bernd


Glycogen Synthase Kinase 3? Is Tyrosine Phosphorylated by PYK2  

Microsoft Academic Search

Glycogen synthase kinase 3? (GSK3?) is a Ser\\/Thr kinase that is involved in numerous cellular activities. GSK3? is activated by tyrosine phosphorylation. However, very little is known about the tyrosine kinases that are responsible for phosphorylating GSK3?. In this report, we investigated the ability of the calcium-dependent tyrosine kinase, proline-rich tyrosine kinase 2 (PYK2) to tyrosine phosphorylate GSK3?. In transfected

Judith A. Hartigan; Wen-Cheng Xiong; Gail V. W. Johnson



Evolutionary Constraints of Phosphorylation in Eukaryotes, Prokaryotes, and Mitochondria*  

PubMed Central

High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the evolution of life. PMID:20688971

Gnad, Florian; Forner, Francesca; Zielinska, Dorota F.; Birney, Ewan; Gunawardena, Jeremy; Mann, Matthias



Growth of calcium phosphate on phosphorylated chitin fibres  

Microsoft Academic Search

Calcium phosphate growth on chitin phosphorylated fibres was studied using scanning electron microscopy and energy dispersive X-ray analysis (SEM, EDX), micro-Fourier transform infrared spectroscopy (FTIR), and solid state magic angle spinning nuclear magnetic resonance (MAS NMR) techniques. The C6 chemical shift positions of 13C MAS NMR in the chitin fibres phosphorylated using urea and H3PO4 are obvious indicating that phosphorylation




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

SciTech Connect

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.

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



Evidence for an ectophosvitin kinase activity that phosphorylates a 123 kDa endogenous substrate on a human colonic adenocarcinoma cell (HT 29).  


When HT 29 cells grown as a monolayer were incubated in a synthetic medium in presence of 0.1 microM [gamma 32P]-ATP, the radioactivity was incorporated predominantly into three major endogenous polypeptides of 123 kDa, 50 kDa and 46 kDa. The radioactive proteins could be detected as soon as 30 s after the addition of the labelled ATP. When exogenous substrates such as casein or phosvitin were added in the synthetic medium, these proteins became phosphorylated. The phosvitin-kinase activity was released in the culture medium following an incubation of the cells with phosvitin. Depletion of the enzymatic activity from the cell surface as well as competition between phosvitin and endogenous substrates led specifically to the inhibition of the 123 kDa polypeptide phosphorylation. At low density, endogenous phosphorylation increased with the cell number, whereas on the contrary it decreased at high cell density. We concluded that the surface of HT 29 cells expressed several protein kinase activities. We have characterized one of them as an ectophosvitin kinase which phosphorylated specifically a 123 kDa polypeptide and whose expression or accessibility varied according to cell density. PMID:3622524

Fantini, J; Muller, J M; Abadie, B; el Battari, A; Marvaldi, J; Tirard, A



Localization of the phosphorylations of polyomavirus large T antigen.  

PubMed Central

Polyomavirus large T antigen is phosphorylated on both serine and threonine residues at a ratio of approximately 6 to 1. This phosphorylation could be resolved into a series of nine Staphylococcus aureus V8 phosphopeptides. All of these were found in an N-terminal chymotryptic fragment with a molecular weight of 57,000. A C-terminal formic acid fragment of 50,000-molecular-weight lacked phosphate. Therefore, unlike simian virus 40 large T antigen, polyomavirus large T antigen has no significant C-terminal phosphorylation. Limited V8 and hydroxylamine cleavage showed that the phosphorylations can be localized to two different portions of the molecule. A significant fraction of the phosphate was localized in the N-terminal portion of the molecule before residue 183. Within this region V8 peptides 4, 8, and 9 represented phosphorylations that were more proximal, while peptides 1, 2, and 3 included more distal phosphorylations. None of these phosphorylations appeared analogous to those of simian virus 40 large T antigen. V8 phosphopeptides 5 and 7 were more distal and could be distinguished in biological experiments from the N-terminal phosphorylations. Formic acid mapping suggested that much, if not all, of this phosphorylation is located between residues 257 and 285. Images PMID:3029410

Bockus, B J; Schaffhausen, B



Tropomyosin Ser-283 pseudo-phosphorylation slows myofibril relaxation  

PubMed Central

Tropomyosin (Tm) is a central protein in the Ca2+ 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 from insect cells. Purified Tm’s regulate thin filaments similar to that reported for muscle purified Tm. Steady-state Ca2+ binding to troponin C (TnC) in reconstituted thin filaments did not differ between the 3 Tm’s, however disassociation of Ca2+ from filaments containing pseudo-phosphorylated Tm was slowed compared to WT 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

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.



Rho-associated kinase phosphorylates MARCKS in human neuronal cells.  


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

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



Prebiotic Phosphorylation Reactions on the Early Earth  

NASA Astrophysics Data System (ADS)

Phosphorus (P) is an essential element for life. It occurs in living beings in the form of phosphate, which is ubiquitous in biochemistry, chiefly in the form of C-O-P (carbon, oxygen and phosphorus), C-P, or P-O-P linkages to form life. Within prebiotic chemistry, several key questions concerning phosphorus chemistry have developed: what were the most likely sources of P on the early Earth? How did it become incorporated into the biological world to form the P compounds that life employs today? Can meteorites be responsible for the delivery of P? What were the most likely solvents on the early Earth and out of those which are favorable for phosphorylation? Or, alternatively, were P compounds most likely produced in relatively dry environments? What were the most suitable temperature conditions for phosphorylation? A route to efficient formation of biological P compounds is still a question that challenges astrobiologists. This article discusses these important issues related to the origin of biological P compounds.

Gull, Maheen



Regulation of cardiac C-protein phosphorylation  

SciTech Connect

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 Iso and 17% in hearts exposed to Iso plus 1 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.

Titus, F.L.



Enzymatic purification of polyunsaturated fatty acids.  


Polyunsaturated fatty acids (PUFAs) have various physiological functions. Of these, ethyl eicosapentaenoate is industrially purified and used as a medicine. Other PUFAs, such as docosahexaenoic acid (DHA), gamma-linolenic acid (GLA), and arachidonic acid (AA), are also expected to be used as pharmaceutical agents; however, their industrial purification processes have not been established. Because PUFAs are highly unstable against heat and oxidation, we attempted to purify them by taking advantage of their enzymatic reactions. When free fatty acids (FFAs) originating from PUFA-containing oil were selectively esterified with lauryl alcohol (LauOH) using a lipase acting on a desired PUFA very weakly, the PUFA was efficiently enriched in the FFA fraction. In addition, when selective alcoholysis of ethyl esters originating from PUFA-containing oil with LauOH was carried out, the PUFA ethyl ester (EtPUFA) was enriched to a desired purity in the unreacted ethyl ester fraction. These reaction mixtures contain LauOH, PUFA (EtPUFA), and lauryl esters, and their molecular weights are different from one another. Hence, PUFA or EtPUFA can be easily separated by conventional distillation. Selective esterification increased the purity of DHA, GLA, and n-6 PUFAs rich in AA to 91, 98, and 96 wt%, respectively. Selective alcoholysis was also effective for increasing the purity of ethyl docosahexaenoate to 90 wt%. PMID:16233035

Shimada, Y; Sugihara, A; Tominaga, Y



Enzymatically Degradable Mussel-Inspired Adhesive Hydrogel  

PubMed Central

Mussel-inspired adhesive hydrogels represent innovative candidate medical sealants or glues. In the present work, we describe an enzyme-degradable mussel-inspired adhesive hydrogel formulation, achieved by incorporating minimal elastase substrate peptide Ala-Ala into the branched poly(ethylene glycol) (PEG) macromonomer structure. The system takes advantage of neutrophil elastase expression upregulation and secretion from neutrophils upon recruitment to wounded or inflamed tissue. By integrating adhesive degradation behaviors that respond to cellular cues, we expand the functional range of our mussel-inspired adhesive hydrogel platforms. Rapid (<1 min) and simultaneous gelation and adhesion of the proteolytically active, catechol-terminated precursor macromonomer was achieved by addition of sodium periodate oxidant. Rheological analysis and equilibrium swelling studies demonstrated that the hydrogel is appropriate for soft tissue-contacting applications. Notably, hydrogel storage modulus (G?) achieved values on the order of 10 kPa, and strain at failure exceeded 200% strain. Lap shear testing confirmed the material’s adhesive behavior (shear strength: 30.4 ± 3.39 kPa). Although adhesive hydrogel degradation was not observed during short-term (27 h) in vitro treatment with neutrophil elastase, in vivo degradation proceeded over several months following dorsal subcutaneous implantation in mice. This work represents the first example of an enzymatically degradable mussel-inspired adhesive and expands the potential biomedical applications of this family of materials. PMID:22059927



Enzymatic hydrolysis of spent coffee ground.  


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

Jooste, T; García-Aparicio, M P; Brienzo, M; van Zyl, W H; Görgens, J F



Natural Product Sugar Biosynthesis and Enzymatic Glycodiversification**  

PubMed Central

Many biologically active small molecule natural products produced by microorganisms derive their activities from sugar substituents. Changing the structures of these sugars can have a profound impact on the biological properties of the parent compounds. This realization has inspired attempts to derivatize the sugar moieties of these natural products through exploitation of the sugar biosynthetic machinery. This approach requires an understanding of the biosynthetic pathway of each target sugar and detailed mechanistic knowledge of the key enzymes. Scientists have begun to unravel the biosynthetic logic behind the assembly of many glycosylated natural products, and have found that a core set of enzyme activities is mixed and matched to synthesize the diverse sugar structures observed in nature. Remarkably, many of these sugar biosynthetic enzymes and glycosyltransferases also exhibit relaxed substrate specificity. The promiscuity of these enzymes has prompted efforts to modify the sugar structures and/or alter the glycosylation patterns of natural products via metabolic pathway engineering and/or enzymatic glycodiversification. In applied biomedical research, these studies will enable the development of new glycosylation tools and generate novel glycoforms of secondary metabolites with useful biological activity. PMID:19058170

Thibodeaux, Christopher J.; Melançon, Charles E.; Liu, Hung-wen



Two Pdk1 phosphorylation sites on the plant cell death suppressor Adi3 contribute to substrate phosphorylation  

PubMed Central

The tomato AGC kinase Adi3 is phosphorylated by Pdk1 for activation of its cell death suppression activity. The Pdk1 phosphorylation site for activation of Adi3 is at Ser539. However, there is at least one additional Pdk1 phosphorylation site on Adi3 that has an unknown function. Here we identify an Arabidopsis thaliana sequence homologue of Adi3 termed AGC1-3. Two Pdk1 phosphorylation sites were identified on AGC1-3, activation site Ser596 and Ser269, and by homology Ser212 on Adi3 was identified as a second Pdk1 phosphorylation site. While Ser212 is not required for Adi3 autophosphorylation, Ser212 was shown to be required for full phosphorylation of the Adi3 substrate Gal83. PMID:23507047

Gray, Joel W.; Nelson Dittrich, Anna C.; Chen, Sixue; Avila, Julian; Giavalisco, Patrick; Devarenne, Timothy P.



Regulation of the activity of the Bacillus subtilis antiterminator LicT by multiple PEP-dependent, enzyme I- and HPr-catalysed phosphorylation.  


The transcriptional antiterminator LicT regulates the induction and carbon catabolite repression of the Bacillus subtilis bglPH operon. LicT is inactive in mutants affected in one of the two general components of the phosphoenolpyruvate (PEP):glycose phosphotransferase system, enzyme I or histidine-containing protein (HPr). We demonstrate that LicT becomes phosphorylated in the presence of PEP, enzyme I and HPr. The phosphoryl group transfer between HPr and LicT is reversible. Phosphorylation of LicT with PEP, enzyme I and HPr led to the appearance of three additional LicT bands on polyacrylamide-urea gels. These bands probably correspond to one-, two- and threefold phosphorylated LicT. After phosphorylation of LicT with [32P]-PEP, enzyme I and HPr, proteolytic digestion of [32P]-P-LicT, separation of the peptides by reverse-phase chromatography, mass spectrometry and N-terminal sequencing of radiolabelled peptides, three histidyl residues were found to be phosphorylated in LicT. These three histidyl residues (His-159, His-207 and His-269) are conserved in most members of the BglG/SacY family of transcriptional antiterminators. Phosphorylation of LicT in the presence of serylphosphorylated HPr (P-Ser-HPr) was much slower compared with its phosphorylation in the presence of HPr. The slower phosphorylation in the presence of P-Ser-HPr leading to reduced LicT activity is presumed to play a role in a recently described LicT-mediated CcpA-independent carbon catabolite repression mechanism operative for the bglPH operon. PMID:10048041

Lindner, C; Galinier, A; Hecker, M; Deutscher, J



Enzymatic biodegradable micro-reactors for therapeutic applications.  


Poly(epsilon-caprolactone) is a well known biocompatible polymer, widely used as drug immobilization systems. In this work poly(epsilon-caprolactone) microparticles with average size between 5 and 25 microm have been prepared by O/W emulsion evaporation method. Inside the microparticles, we have encapsulated Glucose Oxidase with the aim of preparing micro-reactors for enzymatic therapy. These microparticles were structurally characterized and its enzymatic activity analyzed in order to improve the enzyme entrapment. Thus, at the optimum synthesis conditions the enzyme entrapped in the microparticles showed an enzymatic activity of (29.9 +/- 2.1)% comparing with the same amount of free enzyme. Moreover the microparticles maintained a (70.4 +/- 3.2)% of their initial enzymatic activity after placing them in buffer solution for two weeks. PMID:17318827

Rubio-Retama, J; Marińo, F Tamimi; López-Cabarcos, E



Cellulase-lignin interactions in the enzymatic hydrolysis of lignocellulose.  

E-print Network

??Lignin, a major non-carbohydrate polymer in lignocellulosic plant biomass, restricts the action of hydrolytic enzymes in the enzymatic hydrolysis of lignocellulosic feedstocks. Non-productive enzyme adsorption… (more)

Rahikainen, Jenni



Colorimetric enzymatic activity assay based on noncrosslinking aggregation of gold nanoparticles induced by adsorption of substrate peptides.  


The mechanisms of colorimetric assays based on aggregation of gold nanoparticles (GNPs) have been separated into two categories, crosslinking, and noncrosslinking aggregation. The noncrosslinking aggregation has recently been emerging as a simple and rapid mechanism and has been applied to enzymatic activity assays and DNA detection. We report here the detailed study of an enzymatic activity assay for protein kinases based on noncrosslinking aggregation. The principle of the assay is to detect kinase activity by utilizing the difference of coagulating ability of a cationic substrate peptide and its phosphorylated form toward GNPs with anionic surface charge. The critical coagulation concentrations (CCCs) of the peptides were about 10(3) times lower than those of the metal cations with the same cationic charges. The multivalent coordination bonds of the functional groups of the peptides with the GNP surface will strongly support the adsorption of the peptide on the GNP surface. The effect of the GNP size (10, 20, 40, 60 nm) on the dynamic range of OD before and after aggregation was studied. The dynamic range became a maximum for 20 nm GNP among those studied. The difference of CCC between the phosphorylated and nonphosphorylated peptides was governed by (1) the ratio between the peptide concentration and the surface area concentration of GNP and (2) the net charge of the peptides. When the assay system was applied to the activity assessment of protein kinase A, the dynamic range of OD was largest for 20 nm GNPs. However, when the peptide concentration was lowered, the largest 60 nm GNP was advantageous because of its smaller specific surface area. PMID:18680343

Oishi, Jun; Asami, Yoji; Mori, Takeshi; Kang, Jeong-Hun; Niidome, Takuro; Katayama, Yoshiki



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


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

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



[Phosphorylation as a method of regulating inorganic pyrophosphatase activity in E. coli. I. Phosphorylation and enzyme activation induced by ATP].  


ATP phosphorylates the regulatory center of E. coli inorganic pyrophosphatase with the resultant 1,5-fold increase in the activity of the enzyme. The maximal incorporation of the ATP gamma-group into pyrophosphatase is 3 moles per mole of the protein. Pi likewise phosphorylates the enzyme regulatory center and lowers the pyrophosphatase activity by 10-15%. The ATP- and Pi-mediated phosphorylation processes are interrelated; ATP prevents phosphorylation by Pi and brings about rapid dephosphorylation of Pi-modified protein. PMID:3006699

Vener, A V; Ichetovkina, L E; Nazarova, T I; Avaeva, S M



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


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

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



Promotion of PDGF-induced endothelial cell migration by phosphorylated VASP depends on PKA anchoring via AKAP  

Microsoft Academic Search

Vasodilator-stimulated phosphoprotein (VASP), an important substrate of PKA, plays a critical role in remodeling of actin\\u000a cytoskeleton and actin-based cell motility. However, how PKA accurately transfers extracellular signals to VASP and then how\\u000a phosphorylation of VASP regulates endothelial cell migration have not been clearly defined. Protein kinase A anchoring proteins\\u000a (AKAPs) are considered to regulate intracellular-specific signal targeting of PKA

Deling Zhang; Jingping Ouyang; Nian Wang; Yahui Zhang; Jinghua Bie; Yemin Zhang



Casein kinase II-mediated phosphorylation of general repressor Maf1 triggers RNA polymerase III activation  

PubMed Central

Maf1 protein is a global negative regulator of RNA polymerase (Pol) III transcription conserved from yeast to man. We report that phosphorylation of Maf1 by casein kinase II (CK2), a highly evolutionarily conserved eukaryotic kinase, is required for efficient Pol III transcription. Both recombinant human and yeast CK2 were able to phosphorylate purified human or yeast Maf1, indicating that Maf1 can be a direct substrate of CK2. Upon transfer of Saccharomyces cerevisiae from repressive to favorable growth conditions, CK2 activity is required for the release of Maf1 from Pol III bound to a tRNA gene and for subsequent activation of tRNA transcription. In a yeast strain lacking Maf1, CK2 inhibition showed no effect on tRNA synthesis, confirming that CK2 activates Pol III via Maf1. Additionally, CK2 was found to associate with tRNA genes, and this association is enhanced in absence of Maf1, especially under repressive conditions. These results corroborate the previously reported TFIIIB–CK2 interaction and indicate an important role of CK2-mediated Maf1 phosphorylation in triggering Pol III activation. PMID:21383183

Graczyk, Damian; D?bski, Janusz; Muszy?ska, Gra?yna; Bretner, Maria; Lefebvre, Olivier; Boguta, Magdalena



[Enzymatic hydrolysis of cellulose in reverse micelles].  


Several types of surfactants were adopted to construct reverse micelles, in order to investigate the characteristics of cellulose hydrolysis, we used the carboxymethyl cellulose as substrate. The electrical conductivity was measured to determine the maximum water solubilization W0( W0 = [H2O]/[SA] ) of CTAB, SDS, Tween-80 and rhamnolipid reverse micellar systems were 15.2, 20.1, 2.3 and 40.3. In this condition we studied the effects of surfactants concentrations and cellulose dosage on the enzymatic hydrolysis of reverse micelle,and compared with aqueous systems. It was shown by the results that when the cellulose dosage was 0.15 FPU/g substrate, the maximum yield of reducing sugar in reverse micelles was obtained at 1 cmc of CTAB, SDS, Tween-80 and rhamnolipid, in which the rhamnolipid yield was the highest of 198.03 mg substrate. When the concentrations of CTAB, SDS, Tween-80 and rhamnolipid were 1 cmc, the productions of reverse micelles systems were higher than that of aqueous systems of 34.36%, 21.24%, 11.44% and 34.62%. In the optimum conditions of the surfactant concentration, taking the saving cost and sugar yield into consideration, the cellulose dosage of 5 FPU substrate was the most suitable. The reducing sugar's yield of biosurfactant rhamnolipid reverse micellar system was higher than those of three chemical surfactant systems, it was shown that the adoption of biosurfactant has technologically promising prospect in constructing reverse micelles and enhancing the stability of reverse micelles. PMID:21072947

Wang, Wei-Wei; Yuan, Xing-Zhong; Zeng, Guang-Ming; Liang, Yun-Shan; Chao, Yang



Quantification of Protein Phosphorylation by Liquid Chromatography-Mass Spectrometry  

E-print Network

that affect protein-protein interactions. The ability to link protein phosphorylation and the regulationQuantification of Protein Phosphorylation by Liquid Chromatography-Mass Spectrometry Michael J. Matthews*,,§, Cell and Molecular Biology Program and Departments of Molecular Physiology and Biophysics

Vigoreaux, Jim O.


Phosphorylation of Human CTP Synthetase 1 by Protein Kinase A  

E-print Network

Phosphorylation of Human CTP Synthetase 1 by Protein Kinase A IDENTIFICATION OF Thr455 AS A MAJOR SITE OF PHOSPHORYLATION* Received for publication,November 29, 2006, and in revised form, December 21 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr455

Chen, Kuang-Yu


Turnover of protein phosphorylation evolving under stabilizing selection  

PubMed Central

Most proteins are regulated by posttranslational modifications and changes in these modifications contribute to evolutionary changes as well as to human diseases. Phosphorylation of serines, threonines, and tyrosines are the most common modifications identified to date in eukaryotic proteomes. While the mode of action and the function of most phosphorylation sites remain unknown, functional studies have shown that phosphorylation affects protein stability, localization and ability to interact. Two broad modes of action have been described for protein phosphorylation. The first mode corresponds to the canonical and qualitative view whereby single phosphorylation sites act as molecular switches that either turn on or off specific protein functions through direct or allosteric effects. The second mode is more akin to a rheostat than a switch. In this case, a group of phosphorylation sites in a given protein region contributes collectively to the modification of the protein, irrespective of the precise position of individual sites, through an aggregate property. Here we discuss these two types of regulation and examine how they affect the rate and patterns of protein phosphorylation evolution. We describe how the evolution of clusters of phosphorylation sites can be studied under the framework of complex traits evolution and stabilizing selection. PMID:25101120

Landry, Christian R.; Freschi, Luca; Zarin, Taraneh; Moses, Alan M.



RESEARCH ARTICLE Phosphorylation of Rat Aquaporin-4 at Ser111  

E-print Network

RESEARCH ARTICLE Phosphorylation of Rat Aquaporin-4 at Ser111 Is Not Required for Channel Gating gating of the channel itself. Protein kinase (PK)-dependent phosphorylation of Ser111 has been reported, questioned based on the crystal structure of the human AQP4. Our study aimed to resolve if Ser111 was indeed

de Groot, Bert


Michael Lampson, Research Proposal Measuring phosphorylation dynamics in living cells  

E-print Network

Michael Lampson, Research Proposal 1 Measuring phosphorylation dynamics in living cells: from local. Measurements of phosphorylation dynamics in living cells can provide a powerful representation of the state of localized peptide substrates in living cells. This proposal is motivated by specific questions related

Kim, Junhyong


Tyrosine phosphorylation enhances activity of pneumococcal autolysin LytA.  


Tyrosine phosphorylation has long been recognized as a crucial post-translational regulatory mechanism in eukaryotes. However, only in the past decade has recognition been given to the crucial importance of bacterial tyrosine phosphorylation as an important regulatory feature of pathogenesis. This study describes the effect of tyrosine phosphorylation on the activity of a major virulence factor of the pneumococcus, the autolysin LytA, and a possible connection to the Streptococcus pneumoniae capsule synthesis regulatory proteins (CpsB, CpsC and CpsD). We show that in vitro pneumococcal tyrosine kinase, CpsD, and the protein tyrosine phosphatase, CpsB, act to phosphorylate and dephosphorylate LytA. Furthermore, this modulates LytA function in vitro with phosphorylated LytA binding more strongly to the choline analogue DEAE. A phospho-mimetic (Y264E) mutation of the LytA phosphorylation site displayed similar phenotypes as well as an enhanced dimerization capacity. Similarly, tyrosine phosphorylation increased LytA amidase activity, as evidenced by a turbidometric amidase activity assay. Similarly, when the phospho-mimetic mutation was introduced in the chromosomal lytA of S. pneumoniae, autolysis occurred earlier and at an enhanced rate. This study thus describes, to our knowledge, the first functional regulatory effect of tyrosine phosphorylation on a non-capsule-related protein in the pneumococcus, and suggests a link between the regulation of LytA-dependent autolysis of the cell and the biosynthesis of capsular polysaccharide. PMID:25288646

Standish, Alistair J; Whittall, Jonathan J; Morona, Renato



Initiation of TCR Phosphorylation and Signal Transduction  

PubMed Central

Recent data with CD8+ T cells show that the initial phase of T cell receptor (TCR) binding to MHC–peptide (MHCp) is quickly followed by a second, stronger, binding phase representing the binding of CD8 to the MHCp. This second phase requires signaling by a Src-family kinase such as Lck. These data point out two aspects of the initial stage of TCR signaling that have not yet been clearly resolved. Firstly, how and by which Src-family kinase, is the initial phosphorylation of CD3? accomplished, given that the Lck associated with the co-receptors (CD4 or CD8) is not yet available. Secondly, what is the mechanism by which the co-receptor is brought close to the bound TCR before the co-receptor binds to MHCp? PMID:22566861

Gascoigne, Nicholas R. J.; Casas, Javier; Brzostek, Joanna; Rybakin, Vasily



Phosphorylated ?-Synuclein in Parkinson’s Disease  

PubMed Central

Phosphorylated ?-synuclein (PS-129), a protein implicated in the pathogenesis of Parkinson’s disease (PD), was identified by mass spectrometry in human cerebrospinal fluid (CSF). A highly sensitive and specific assay was established and used to measure PS-129, along withtotal ?-synuclein, in the CSF of patients with PD, other parkinsonian disorders such as multiple system atrophy (MSA) and progressive supranuclear palsy (PSP), and healthy individuals (a total of ~600 samples). PS-129 CSF concentrations correlated weakly with PD severity and, when combined with total ?-synuclein CSF concentrations, contributed to distinguishing PD from MSA and PSP. Further rigorous validation in independent cohorts of patients, especially those where samples have been collected longitudinally, will determine whether PS-129 CSF concentrations will be useful for diagnosing PD and for monitoring PD severity and progression. PMID:22344688

Wang, Yu; Shi, Min; Chung, Kathryn A.; Zabetian, Cyrus P.; Leverenz, James B.; Berg, Daniela; Srulijes, Karin; Trojanowski, John Q.; Lee, Virginia M.-Y.; Siderowf, Andrew D.; Hurtig, Howard; Litvan, Irene; Schiess, Mya C.; Peskind, Elaine R.; Masuda, Masami; Hasegawa, Masato; Lin, Xiangmin; Pan, Catherine; Galasko, Douglas; Goldstein, David S.; Jensen, Poul Henning; Yang, Hui; Cain, Kevin C.; Zhang, Jing



Tau Phosphorylation and ?-Calpain Activation Mediate the Dexamethasone-Induced Inhibition on the Insulin-Stimulated Akt Phosphorylation  

PubMed Central

Evidence has suggested that insulin resistance (IR) or high levels of glucocorticoids (GCs) may be linked with the pathogenesis and/or progression of Alzheimer's disease (AD). Although studies have shown that a high level of GCs results in IR, little is known about the molecular details that link GCs and IR in the context of AD. Abnormal phosphorylation of tau and activation of ?-calpain are two key events in the pathology of AD. Importantly, these two events are also related with GCs and IR. We therefore speculate that tau phosphorylation and ?-calpain activation may mediate the GCs-induced IR. Akt phosphorylation at Ser-473 (pAkt) is commonly used as a marker for assessing IR. We employed two cell lines, wild-type HEK293 cells and HEK293 cells stably expressing the longest human tau isoform (tau-441; HEK293/tau441 cells). We examined whether DEX, a synthetic GCs, induces tau phosphorylation and ?-calpain activation. If so, we examined whether the DEX-induced tau phosphorylation and ?-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. The results showed that DEX increased tau phosphorylation and induced tau-mediated ?-calpain activation. Furthermore, pre-treatment with LiCl prevented the effects of DEX on tau phosphorylation and ?-calpain activation. Finally, both LiCl pre-treatment and calpain inhibition prevented the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. In conclusion, our study suggests that the tau phosphorylation and ?-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. PMID:22536436

Liu, Yudong; Su, Ying; Sun, Shenggang; Wang, Tao; Qiao, Xian; Run, Xiaoqin; Liang, Zhihou



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


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

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



Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adenosinetriphosphatase  

SciTech Connect

The roles of the phosphorylation (phosphorylated enzyme intermediate) and nucleotide binding domains in calcium transport were studied by comparing acetyl phosphate and ATP as substrates for the Ca/sup 2 +/-ATPase of sarcoplasmic reticulum vesicles. The authors found that the maximal level of phosphoenzyme obtained with either substrate is approximately 4 nmol/mg of protein, corresponding to the stoichiometry of catalytic sites in their preparation. The initial burst of phosphoenzyme formation observed in the transient state, following addition of either substrate, is accompanied by internalization of 2 mol of calcium per mole of phosphoenzyme. The internalized calcium is then translocated with a sequential pattern, independent of the substrate used. Following a rate-limiting step, the phosphoenzyme undergoes hydrolytic cleavage and proceeds to the steady-state activity which is soon back inhibited by the rise of Ca/sup 2 +/ concentration in the lumen of the vesicles. When the back inhibition is released by the addition of oxalate, substrate utilization and calcium transport occur with a ratio of 1:2, independent of the substrate and its concentration. When the nucleotide binding site is derivatized with FITP, the enzyme can still utilize acetyl phosphate (but not ATP) for calcium transport. These observations demonstrate that the basic coupling mechanism of catalysis and calcium transport involves the phosphorylation and calcium binding domains, and not the nucleotide binding domain. On the other hand, occupancy of the FITC-sensitive nucleotide site is involved in kinetic regulation not only with respect to utilization of substrate for the phosphoryl transfer reaction but also for subsequent steps related to calcium translocation and phosphoenzyme turnover.

Teruel, J.A.; Inesi, G.



In vivo regulatory phosphorylation of the phosphoenolpyruvate carboxylase AtPPC1 in phosphate-starved Arabidopsis thaliana  

PubMed Central

PEPC [PEP(phosphoenolpyruvate) carboxylase] is a tightly controlled cytosolic enzyme situated at a major branchpoint in plant metabolism. Accumulating evidence indicates important functions for PEPC and PPCK (PEPC kinase) in plant acclimation to nutritional Pi deprivation. However, little is known about the genetic origin or phosphorylation status of native PEPCs from ?Pi (Pi-deficient) plants. The transfer of Arabidopsis suspension cells or seedlings to ?Pi growth media resulted in: (i) the marked transcriptional upregulation of genes encoding the PEPC isoenzyme AtPPC1 (Arabidopsis thaliana PEPC1), and PPCK isoenzymes AtPPCK1 and AtPPCK2; (ii) >2-fold increases in PEPC specific activity and in the amount of an immunoreactive 107-kDa PEPC polypeptide (p107); and (iii) In vivo p107 phosphorylation as revealed by immunoblotting of clarified extracts with phosphosite-specific antibodies to Ser-11 (which could be reversed following Pi resupply). Approx. 1.3 mg of PEPC was purified 660-fold from ?Pi suspension cells to apparent homogeneity with a specific activity of 22.3 units · mg?1 of protein. Gel filtration, SDS/PAGE and immunoblotting demonstrated that purified PEPC exists as a 440-kDa homotetramer composed of identical p107 subunits. Sequencing of p107 tryptic and Asp-N peptides by tandem MS established that this PEPC is encoded by AtPPC1. Pi-affinity PAGE coupled with immunoblotting indicated stoichiometric phosphorylation of the p107 subunits of AtPPC1 at its conserved Ser-11 phosphorylation site. Phosphorylation activated AtPPC1 at pH 7.3 by lowering its Km(PEP) and its sensitivity to inhibition by L-malate and L-aspartate, while enhancing activation by glucose 6-phosphate. Our results indicate that the simultaneous induction and In vivo phosphorylation activation of AtPPC1 contribute to the metabolic adaptations of ?Pi Arabidopsis. PMID:19228119

Gregory, Allison L.; Hurley, Brenden A.; Tran, Hue T.; Valentine, Alexander J.; She, Yi-Min; Knowles, Vicki L.; Plaxton, William C.



Phosphorylation of the antiviral protein interferon-inducible transmembrane protein 3 (IFITM3) dually regulates its endocytosis and ubiquitination.  


Interferon-inducible transmembrane protein 3 (IFITM3) is essential for innate defense against influenza virus in mice and humans. IFITM3 localizes to endolysosomes where it prevents virus fusion, although mechanisms controlling its trafficking to this cellular compartment are not fully understood. We determined that both mouse and human IFITM3 are phosphorylated by the protein-tyrosine kinase FYN on tyrosine 20 (Tyr(20)) and that mouse IFITM3 is also phosphorylated on the non-conserved Tyr(27). Phosphorylation led to a cellular redistribution of IFITM3, including plasma membrane accumulation. Mutation of Tyr(20) caused a similar redistribution of IFITM3 and resulted in decreased antiviral activity against influenza virus, whereas Tyr(27) mutation of mouse IFITM3 showed minimal effects on localization or activity. Using FYN knockout cells, we also found that IFITM3 phosphorylation is not a requirement for its antiviral activity. Together, these results indicate that Tyr(20) is part of an endocytosis signal that can be blocked by phosphorylation or by mutation of this residue. Further mutagenesis narrowed this endocytosis-controlling region to four residues conforming to a YXX? (where X is any amino acid and ? is Val, Leu, or Ile) endocytic motif that, when transferred to CD4, resulted in its internalization from the cell surface. Additionally, we found that phosphorylation of IFITM3 by FYN and mutagenesis of Tyr(20) both resulted in decreased IFITM3 ubiquitination. Overall, these results suggest that modification of Tyr(20) may serve in a cellular checkpoint controlling IFITM3 trafficking and degradation and demonstrate the complexity of posttranslational regulation of IFITM3. PMID:24627473

Chesarino, Nicholas M; McMichael, Temet M; Hach, Jocelyn C; Yount, Jacob S



Sirt3 Regulates Metabolic Flexibility of Skeletal Muscle Through Reversible Enzymatic Deacetylation  

PubMed Central

Sirt3 is an NAD+-dependent deacetylase that regulates mitochondrial function by targeting metabolic enzymes and proteins. In fasting mice, Sirt3 expression is decreased in skeletal muscle resulting in increased mitochondrial protein acetylation. Deletion of Sirt3 led to impaired glucose oxidation in muscle, which was associated with decreased pyruvate dehydrogenase (PDH) activity, accumulation of pyruvate and lactate metabolites, and an inability of insulin to suppress fatty acid oxidation. Antibody-based acetyl-peptide enrichment and mass spectrometry of mitochondrial lysates from WT and Sirt3 KO skeletal muscle revealed that a major target of Sirt3 deacetylation is the E1? subunit of PDH (PDH E1?). Sirt3 knockout in vivo and Sirt3 knockdown in myoblasts in vitro induced hyperacetylation of the PDH E1? subunit, altering its phosphorylation leading to suppressed PDH enzymatic activity. The inhibition of PDH activity resulting from reduced levels of Sirt3 induces a switch of skeletal muscle substrate utilization from carbohydrate oxidation toward lactate production and fatty acid utilization even in the fed state, contributing to a loss of metabolic flexibility. Thus, Sirt3 plays an important role in skeletal muscle mitochondrial substrate choice and metabolic flexibility in part by regulating PDH function through deacetylation. PMID:23835326

Jing, Enxuan; O’Neill, Brian T.; Rardin, Matthew J.; Kleinridders, André; Ilkeyeva, Olga R.; Ussar, Siegfried; Bain, James R.; Lee, Kevin Y.; Verdin, Eric M.; Newgard, Christopher B.; Gibson, Bradford W.; Kahn, C. Ronald



Tyrosine phosphorylation events during coxsackievirus B3 replication.  

PubMed Central

In order to study cellular and viral determinants of pathogenicity, interactions between coxsackievirus B3 (CVB3) replication and cellular protein tyrosine phosphorylation were investigated. During CVB3 infection of HeLa cells, distinct proteins become phosphorylated on tyrosine residues, as detected by the use of antiphosphotyrosine Western blotting. Two proteins of 48 and 200 kDa showed enhanced tyrosine phosphorylation 4 to 5 h postinfection (p.i.), although virus-induced inhibition of cellular protein synthesis had already occurred 3 to 4 h p.i. Subcellular fractionation experiments revealed distinct localization of tyrosine-phosphorylated proteins of 48 and 200 kDa in the cytosol and membrane fractions of infected cells, respectively. In addition, in Vero cells infected with CVB3, echovirus (EV)11, or EV12, increased tyrosine phosphorylation of a 200-kDa protein was detected 6 h p.i. Herbimycin A, a specific inhibitor of Src-like protein tyrosine kinases, was shown to inhibit virus-induced tyrosine phosphorylations and to reduce the production of progeny virions. In contrast, in cells treated with the inhibitors staurosporine and calphostin C, the synthesis of progeny virions was not affected. Immunoprecipitation experiments suggested that the tyrosine-phosphorylated 200-kDa protein in CVB3-infected cells is of cellular origin. In summary, these investigations have begun to unravel the effect of CVB3 as well as EV11 and EV12 replication on cellular tyrosine phosphorylation and support the importance of tyrosine phosphorylation events for effective virus replication. Such cellular phosphorylation events triggered in the course of enterovirus infection may enhance virus replication. PMID:8985388

Huber, M; Selinka, H C; Kandolf, R



Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5  

PubMed Central

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



Nitric Oxide-Induced Calcium Release: Activation of Type 1 Ryanodine Receptor, a Calcium Release Channel, through Non-Enzymatic Post-Translational Modification by Nitric Oxide  

PubMed Central

Nitric oxide (NO) is a typical gaseous messenger involved in a wide range of biological processes. In our classical knowledge, effects of NO are largely achieved by activation of soluble guanylyl cyclase to form cyclic guanosine-3?, 5?-monophosphate. However, emerging evidences have suggested another signaling mechanism mediated by NO: “S-nitrosylation” of target proteins. S-nitrosylation is a covalent addition of an NO group to a cysteine thiol/sulfhydryl (RSH), and categorized into non-enzymatic post-translational modification (PTM) of proteins, contrasted to enzymatic PTM of proteins, such as phosphorylation mediated by various protein kinases. Very recently, we found novel intracellular calcium (Ca2+) mobilizing mechanism, NO-induced Ca2+ release (NICR) in cerebellar Purkinje cells. NICR is mediated by type 1 ryanodine receptor (RyR1), a Ca2+ release channel expressed in endoplasmic-reticular membrane. Furthermore, NICR is indicated to be dependent on S-nitrosylation of RyR1, and involved in synaptic plasticity in the cerebellum. In this review, molecular mechanisms and functional significance of NICR, as well as non-enzymatic PTM of proteins by gaseous signals, are described. PMID:24130553

Kakizawa, Sho



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

PubMed Central

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

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



Modulation of repulsive forces between neurofilaments by sidearm phosphorylation  

E-print Network

are sedimented into a gelatinous pellet, pellet volume falls with increasing ionic strength and enzymatic reduces pellet volume. Further, atomic force microscopy imaging of isolated mammalian NFs reveals robust; Intermediate filaments; Atomic force microscopy; Polymer brush; Polyelectrolyte; Cell mechanics; Unstructured

Kumar, Sanjay


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

PubMed Central

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

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



Mini-review: androgen receptor phosphorylation in prostate cancer  

PubMed Central

Androgen receptor (AR) plays an important role in the tumorigenesis and progression of prostate cancer (PCa), and is the primary therapeutic target for PCa treatment. AR activity can be regulated via phosphorylation at multiple phosphorylation sites within the protein. Modifications by phosphorylation alter AR function, including its cellular localization, stability and transcriptional activity, ultimately leading to changes in cancer cell biology and disease progression. Here we present a brief overview of AR phosphorylation sites in PCa, focusing on functional roles of phospho-AR (p-AR) species, relevance in PCa disease progression, and potential as biomarkers and/or therapeutic targets through the use of kinase inhibitors. Additionally, recent evidence has shown the important role of AR activity in the cancer associated stroma on PCa growth and progression. The phosphorylation status of epithelial and stromal AR may be distinct; however, the current data available on stromal AR phosphorylation is limited. Further research will determine global view on the synergistic effects of phosphorylation across multiple AR sites in both epithelial and stromal cells and validate whether together they can be used as prognostic markers and/or effective therapeutic targets for PCa. PMID:25374897

Daniels, Garrett; Pei, Zhiheng; Logan, Susan K; Lee, Peng



Role of Ser129 phosphorylation of ?-synuclein in melanoma cells  

PubMed Central

Summary ?-Synuclein, a protein central to Parkinson's disease, is frequently expressed in melanoma tissues, but not in non-melanocytic cutaneous carcinoma and normal skin. Thus, ?-synuclein is not only related to Parkinson's disease, but also to melanoma. Recently, epidemiologists reported co-occurrence of melanoma and Parkinson's disease in patients, suggesting that these diseases could share common pathogenetic components and that ?-synuclein might be one of these. In Parkinson's disease, phosphorylation of ?-synuclein at Ser129 plays an important role in the pathobiology. However, its role in melanoma is not known. Here, we show the biological relevance of Ser129 phosphorylation in human melanoma cells. First, we have identified an antibody that reacts with Ser129-unphosphorylated ?-synuclein but not with Ser129-phosphorylated ?-synuclein. Using this and other antibodies to ?-synuclein, we investigated the role of Ser129 phosphorylation in human melanoma SK-MEL28 and SK-MEL5 cells. Our immunofluorescence microscopy showed that the Ser129-phosphorylated form, but not the Ser129-unphosphorylated form, of ?-synuclein localizes to dot-like structures at the cell surface and the extracellular space. Furthermore, immuno-electron microscopy showed that the melanoma cells release microvesicles in which Ser129-phosphorylated ?-synuclein localizes to the vesicular membrane. Taken together, our studies suggest that the phosphorylation of Ser129 leads to the cell surface translocation of ?-synuclein along the microtubule network and its subsequent vesicular release in melanoma cells. PMID:23203798

Lee, Byung Rho; Matsuo, Yasuhiro; Cashikar, Anil G.; Kamitani, Tetsu



A Systems Model of Phosphorylation for Inflammatory Signaling Events  

PubMed Central

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

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



Ionizing radiation causes increased tau phosphorylation in primary neurons.  


Radiotherapy is the major treatment modality for primary and metastatic brain tumors which involves the exposure of brain to ionizing radiation. Ionizing radiation can induce various detrimental pathophysiological effects in the adult brain, and Alzheimer's disease and related neurodegenerative disorders are considered to be late effects of radiation. In this study, we investigated whether ionizing radiation causes changes in tau phosphorylation in cultured primary neurons similar to that in Alzheimer's disease. We demonstrated that exposure to 0.5 or 2 Gy ? rays causes increased phosphorylation of tau protein at several phosphorylation sites in a time- and dose-dependent manner. Consistently, we also found ionizing radiation causes increased activation of GSK3?, c-Jun N-terminal kinase and extracellular signal-regulated kinase before radiation-induced increase in tau phosphorylation. Specific inhibitors of these kinases almost fully blocked radiation-induced tau phosphorylation. Our studies further revealed that oxidative stress plays an important role in ionizing radiation-induced tau phosphorylation, likely through the activation of c-Jun N-terminal kinase and extracellular signal-regulated kinase, but not GSK3?. Overall, our studies suggest that ionizing radiation may cause increased risk for development of Alzheimer's disease by promoting abnormal tau phosphorylation. PMID:24861936

Li, Li; Wang, Wenzhang; Welford, Scott; Zhang, Teng; Wang, Xinglong; Zhu, Xiongwei



Phosphorylation of murine mammary tumor virus precursor polypeptides.  

PubMed Central

Phosphorylation of the murine mammary tumor virus (MuMTV) structural proteins was studied in an MuMTV-infected epithelial cell line derived from a BALB/cf C3H mouse mammary tumor. Immunoprecipitation of 32P-labeled cell extracts with monospecific anti-p27 serum revealed that phosphorylation occurred at the stage of the core-protein polyprotein precursor prp75. Two forms of phosphorylated prp75 were found: one migrating with an apparent molecular weight of 80,000, and the other with a molecular weight of 76,000. The 80,000-molecular-weight species was found to be the most heavily phosphorylated. In addition, a relatively stable phosphorylated processing intermediate of 34,000 molecular weight was observed as well. Tryptic peptide mapping analysis of the 32P-labeled viral proteins indicated a precursor product relationship between the intracellular phosphorylated, high-molecular-weight peptides and the mature MuMTV phosphoproteins p23 and p27. Phosphopeptide analysis also suggested that phosphorylation of the viral proteins occurred in discrete steps and that the attached phosphate groups were conserved throughout the processing steps. Images PMID:225520

Racevskis, J; Sarkar, N H



Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells  

PubMed Central

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

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



Phosphorylation at serine 331 is required for Aurora B activation.  


Aurora B kinase activity is required for successful cell division. In this paper, we show that Aurora B is phosphorylated at serine 331 (Ser331) during mitosis and that phosphorylated Aurora B localizes to kinetochores in prometaphase cells. Chk1 kinase is essential for Ser331 phosphorylation during unperturbed prometaphase or during spindle disruption by taxol but not nocodazole. Phosphorylation at Ser331 is required for optimal phosphorylation of INCENP at TSS residues, for Survivin association with the chromosomal passenger complex, and for complete Aurora B activation, but it is dispensable for Aurora B localization to centromeres, for autophosphorylation at threonine 232, and for association with INCENP. Overexpression of Aurora B(S331A), in which Ser331 is mutated to alanine, results in spontaneous chromosome missegregation, cell multinucleation, unstable binding of BubR1 to kinetochores, and impaired mitotic delay in the presence of taxol. We propose that Chk1 phosphorylates Aurora B at Ser331 to fully induce Aurora B kinase activity. These results indicate that phosphorylation at Ser331 is an essential mechanism for Aurora B activation. PMID:22024163

Petsalaki, Eleni; Akoumianaki, Tonia; Black, Elizabeth J; Gillespie, David A F; Zachos, George



Phosphorylation at serine 331 is required for Aurora B activation  

PubMed Central

Aurora B kinase activity is required for successful cell division. In this paper, we show that Aurora B is phosphorylated at serine 331 (Ser331) during mitosis and that phosphorylated Aurora B localizes to kinetochores in prometaphase cells. Chk1 kinase is essential for Ser331 phosphorylation during unperturbed prometaphase or during spindle disruption by taxol but not nocodazole. Phosphorylation at Ser331 is required for optimal phosphorylation of INCENP at TSS residues, for Survivin association with the chromosomal passenger complex, and for complete Aurora B activation, but it is dispensable for Aurora B localization to centromeres, for autophosphorylation at threonine 232, and for association with INCENP. Overexpression of Aurora BS331A, in which Ser331 is mutated to alanine, results in spontaneous chromosome missegregation, cell multinucleation, unstable binding of BubR1 to kinetochores, and impaired mitotic delay in the presence of taxol. We propose that Chk1 phosphorylates Aurora B at Ser331 to fully induce Aurora B kinase activity. These results indicate that phosphorylation at Ser331 is an essential mechanism for Aurora B activation. PMID:22024163

Petsalaki, Eleni; Akoumianaki, Tonia; Black, Elizabeth J.; Gillespie, David A.F.



[Regulation of pollen tube growth by reversible protein phosphorylation].  


The tip-growth of pollen tube is regulated by diverse signaling and metabolic processes, including Rop GTPase signaling pathway, phosphatidylinositol signaling pathway, Ca(2+) signaling, actin dynamics, vesicular trafficking, and cell wall re-modulation. These processes are regulated by reversible protein phosphorylation events: (1) The activities of Rop regulatory proteins (GEF, GDI, and GAP) that regulate Rop GTPase are variable under different protein phosphorylation states. In addition, various protein kinases function as the downstream effectors of Rop GTPase, and participate in the downstream pathways of Rop signaling. (2) Reversible protein phosphorylation can activate/inactivate the plasma membrane (PM) Ca(2+) channels and/or Ca(2+) pump, as well as control the release of intracellular Ca(2+), thereby regulating the formation of Ca(2+) gradient in pollen tube tip. In addition, protein kinases function as Ca(2+) sensors and phosphorylate the target proteins involved in the downstream regulatory pathways of Ca(2+) signaling. (3) The dynamics of actin polymerization and depolymerization are regulated by reversible phosphorylation of actin binding proteins (e.g., ADF and profilin). (4) Reversible protein phosphorylation regulates the activities of endo/exocytosis-related proteins and PM phospholipid metabolism that are involved in the membrane trafficking. (5) Reversible protein phosphorylation participates in the pollen tube cell wall re-modulation through regulating the function and subcellular distribution of cytoplasmic serine/threonine protein kinase and sucrose synthase. (6) The activities of transcription regulatory protein and eukaryotic translation initiation factor are regulated by protein phosphorylation, modulating the RNA transcription and protein synthesis. In this review, we present an overview of the functions of protein reversible phosphorylation in the aforementioned processes during the pollen tube tip-growth. PMID:25143274

Suo, Jinwei; Dai, Shaojun



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

PubMed Central

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

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



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

NASA Astrophysics Data System (ADS)

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.

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



Plasmon-Enhanced Enzymatic Reactions: A Study of Nanoparticle-Enzyme Distance- and Nanoparticle Loading-Dependent Enzymatic Activity.  


A detailed investigation of the dependence of the efficiency of plasmon-enhanced enzymatic reactions on the distance between silver island films (SIFs) and horse radish peroxidase (HRP) enzyme and on the loading of SIFs on glass surfaces is presented. Three different extent of loading of SIFs on glass slides were used: 1) low, 2) medium and 3) high, which was characterized by using optical absorption spectroscopy and scanning electron microscopy. Streptavidin-linked HRP enzyme was deposited onto SIFs and glass slides by using three different strategies: strategy 1: biotin-avidin protein assay (distance between SIFs and HRP = 4-8 nm), strategy 2: self assembled monolayers (SAMs) (1-5 nm), strategy 3: polymer layer (1-5 nm). The efficiency of enzymatic conversion of O-phenylenediamine dihydrochloride (OPD) to a colored product by HRP on SIFs and glass surfaces was assessed by optical absorption spectroscopy. The distance between SIFs and HRP and the extent of loading of SIFs on the glass surfaces were shown to have significant effect on the efficiency of plasmon-enhanced enzymatic reactions. In this regard, up to an %250 increase in enzymatic conversion of OPD was observed from SIFs with high loading using strategy 1. In addition, we have studied the potential of repeated use of SIFs in plasmon-enhanced enzymatic reactions. PMID:21949594

Abel, Biebele; Akinsule, Alice; Andrews, Canisha; Aslan, Kadir



Phosphorylation of skeletal muscle microsomes by acetylphosphate.  


The phosporyl group of acetylphosphate is transferred to the membranal protein of the sarcoplasmic vesicles during active calcium transport. Although the phosphoprotein formed cannot be distinguished from that obtained in the presence of ATP, the conditions for ATP and acetylphosphate hydrolysis are different from each other. PMID:11945452

Friedman, Z; Makinose, M



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


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

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



Cloning and expression of the enzymatic region of Streptococcal hyaluronidase  

PubMed Central

Objective(s): Streptococcus pyogenes produces extracellular hyaluronidase enzyme. This enzyme is directly associated with the spread of the organism during infection. The objective of the present study was to clone and express the nucleotide sequence of the enzyme which is involved in hyaluronidase enzymatic activity. Materials and Methods: The enzymatic region of hyaluronidase gene was detected by bioinformatics method. The PCR method was used to amplify enzymatic region of hyaluronidase gene from chromosomal DNA of Streptococcus pyogenes. The eluted product was cloned into the prokaryotic expression vector pET32a which was digested by BamHI and HindIII restriction endonuclease enzymes. The target protein was expressed in the Escherichia coli. The bacteria including pET32a-hylA (hylA is abbreviation of Streptococcus pyogenes hyaluronidase gene and hylA is abbreviation of Streptococcus pyogenes hyaluronidase protein) plasmids were induced by IPTG and analyzed by SDS-PAGE. The enzymatic evaluation and antigenicity was finally studied. Results: Enzymes digestion analysis, sequencing results showed that the target gene (1296 base pair) was inserted correctly into the recombinant vector. The expressed protein (65 KDa) was purified successfully via affinity chromatography. Data also indicated that enzymatic region of hyaluronidase protein from Streptococcus pyogenes was recognized in all 5 patient's sera. Conclusion: In general, it is possible to produce the enzymatic regions of the Streptococcus pyogenes hyaluronidase in E. coli. The antigenic property of the produced protein is well retained. Considering the product's domestic demand and also low efficiency of production and pathogenicity of Streptococcus species, it is possible to produce it as recombinant product.

Mirjamali, Nafiseh Al-Sadat; Soufian, Safieh; Molaee, Neda; Abbasian, Shabnam Sadoogh; Abtahi, Hamid



Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant of MurA.  


MurA (UDP-GlcNAc enolpyruvyl transferase), the first enzyme in bacterial peptidoglycan biosynthesis, catalyzes the enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the 3'-OH of UDP-GlcNAc by an addition-elimination mechanism that proceeds through a tetrahedral ketal intermediate. The crystal structure of the Cys115-to-Ala (C115A) mutant of Escherichia coli MurA complexed with a fluoro analogue of the tetrahedral intermediate revealed the absolute configuration of the adduct and the stereochemical course of the reaction. The fluorinated adduct was generated in a preincubation of wild-type MurA with (Z)-3-fluorophosphoenolpyruvate (FPEP) and UDP-GlcNAc and purified after enzyme denaturation. The fluorine substituent stabilizes the tetrahedral intermediate toward decomposition by a factor of 10(4)-10(6), facilitating manipulation of the adduct. The C115A mutant of MurA was utilized to avoid the microheterogeneity that arises in the wild-type MurA from the attack of Cys115 on C-2 of FPEP in competition with the formation of the fluorinated adduct. The crystal structure of the complex was determined to 2.8 A resolution, and the absolute configuration at C-2 of the adduct was found to be 2R. Thus, addition of the 3'-OH of UDP-GlcNAc is to the 2-si face of FPEP, corresponding to the 2-re face of PEP. Given the previous observation that, in D2O, the addition of D+ to C-3 of PEP proceeds from the 2-si face [Kim, D. H., Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381], the addition across the double bond of PEP is anti. Also, because the overall stereochemical course has been shown to be either anti/syn or syn/anti [Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 7329-7337], it now follows that the stereochemistry of elimination of H+ from C-3 and Pi from C-2 of the tetrahedral intermediate of the reaction is syn. PMID:9485407

Skarzynski, T; Kim, D H; Lees, W J; Walsh, C T; Duncan, K



Rosamines Targeting the Cancer Oxidative Phosphorylation Pathway  

PubMed Central

Reprogramming of energy metabolism is pivotal to cancer, so mitochondria are potential targets for anticancer therapy. A prior study has demonstrated the anti-proliferative activity of a new class of mitochondria-targeting rosamines. This present study describes in vitro cytotoxicity of second-generation rosamine analogs, their mode of action, and their in vivo efficacies in a tumor allografted mouse model. Here, we showed that these compounds exhibited potent cytotoxicity (average IC50<0.5 µM), inhibited Complex II and ATP synthase activities of the mitochondrial oxidative phosphorylation pathway and induced loss of mitochondrial transmembrane potential. A NCI-60 cell lines screen further indicated that rosamine analogs 4 and 5 exhibited potent antiproliferative effects with Log10GI50?=??7 (GI50?=?0.1 µM) and were more effective against a colorectal cancer sub-panel than other cell lines. Preliminary in vivo studies on 4T1 murine breast cancer-bearing female BALB/c mice indicated that treatment with analog 5 in a single dosing of 5 mg/kg or a schedule dosing of 3 mg/kg once every 2 days for 6 times (q2d×6) exhibited only minimal induction of tumor growth delay. Our results suggest that rosamine analogs may be further developed as mitochondrial targeting agents. Without a doubt proper strategies need to be devised to enhance tumor uptake of rosamines, i.e. by integration to carrier molecules for better therapeutic outcome. PMID:24622277

Lim, Siang Hui; Wu, Liangxing; Kiew, Lik Voon; Chung, Lip Yong; Burgess, Kevin; Lee, Hong Boon



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

PubMed Central

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

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



Enzymatic Processing of Bioactive Glycosides from Natural Sources  

NASA Astrophysics Data System (ADS)

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.

Weignerová, Lenka; K?en, Vladimír


Protein microchips : use for immunoassay and enzymatic reactions.  

SciTech Connect

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.

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



Study on beta-galactosidase enzymatic activity of herbal yogurt.  


Different types of herbal yogurts were developed by mixing standardized milk with pretreated herbs, namely tulsi leaf (Ocimum sanctum), pudina leaf (Mentha arvensis) and coriander leaf (Coriandrum sativum), with leaves separately and a 1:1 (v/v) mixture of the strains of lactic starter cultures---Lactobacillus acidophilus (NCIM 2903) and Lactobacillus plantarum (NCIM 2083)-followed by incubation at 40 degrees C for 6 h. The beta-galactosidase enzymatic activity of the abovementioned herbal yogurts was determined and interestingly noted to exhibit higher enzymatic activity compared with the control yogurt (without any herbs). Among all herbal yogurts, tulsi yogurt had the maximum beta-galactosidase activity. PMID:17852503

Chowdhury, Banani Ray; Chakraborty, Runu; Raychaudhuri, Utpal



Transfer factor.  


Transfer factor, a natural substance of the immune system, was discovered in 1949. More than 3,000 scientific articles have established it as an effective treatment for many diseases, usually those related to the immune system. In China, more than six million people have used transfer factor as a prophylaxis for hepatitis. Information on ordering articles on transfer factor, olive leaf extract, and coconut oil is included. PMID:11366549



Cell survival after UV radiation stress in the unicellular chlorophyte Dunaliella tertiolecta is mediated by DNA repair and MAPK phosphorylation.  


Ultraviolet radiation (UVR) induces damage in a variety of organisms, and cells may adapt by developing repair or tolerance mechanisms to counteract such damage; otherwise, the cellular fate is cell death. Here, the effect of UVR-induced cell damage and the associated signalling and repair mechanisms by which cells are able to survive was studied in Dunaliella tertiolecta. UVR did not cause cell death, as shown by the absence of SYTOX Green-positive labelling cells. Ultrastructure analysis by transmission electron microscopy demonstrated that the cells were alive but were subjected to morphological changes such as starch accumulation, chromatin disaggregation, and chloroplast degradation. This behaviour paralleled a decrease in F(v)/F(m) and the formation of cyclobutane-pyrimidine dimers, showing a 10-fold increase at the end of the time course. There was a high accumulation of the repressor of transcriptional gene silencing (ROS1), as well as the cell proliferation nuclear antigen (PCNA) in UVR-treated cells, revealing activation of DNA repair mechanisms. The degree of phosphorylation of c-Jun N-terminal kinase (JNK) and p38-like mitogen-activated protein kinases was higher in UVR-exposed cells; however, the opposite occurred with the phosphorylated extracellular signal-regulated kinase (ERK). This confirmed that both JNK and p38 need to be phosphorylated to trigger the stress response, as well as the fact that cell division is arrested when an ERK is dephosphorylated. In parallel, both DEVDase and WEHDase caspase-like enzymatic activities were active even though the cells were not dead, suggesting that these proteases must be considered within a wider frame of stress proteins, rather than specifically being involved in cell death in these organisms. PMID:22859678

García-Gómez, Candela; Parages, María L; Jiménez, Carlos; Palma, Armando; Mata, M Teresa; Segovia, María



Heat Transfer  

NSDL National Science Digital Library

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

Integrated Teaching and Learning Program,


Methods for generating phosphorylation site-specific immunological reagents  


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.

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



A phosphorylation-dependent intramolecular interaction regulates the membrane association  

E-print Network

of phosphorylated C-terminal residues, were not included in the crystal structure. We report that membrane binding stress (18­20) by disulfide linkage of catalytic cysteine 124 with cysteine 71 (18). E3 ubiquitin ligase

Devreotes, Peter


Phosphorylation of a neuronal-specific beta-tubulin isotype  

SciTech Connect

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.

Diaz-Nido, J.; Serrano, L.; Lopez-Otin, C.; Vandekerckhove, J.; Avila, J. (Universidad Autonoma, Canto Blanco, Madrid (Spain))



Enrichment of phosphorylated peptides and proteins by selective precipitation methods.  


Protein phosphorylation is one of the most prominent post-translational modifications involved in the regulation of cellular processes. Fundamental understanding of biological processes requires appropriate bioanalytical methods for selectively enriching phosphorylated peptides and proteins. Most of the commonly applied enrichment approaches include chromatographic materials including Fe(3+)-immobilized metal-ion affinity chromatography or metal oxides. In the last years, the introduction of several non-chromatographic isolation technologies has increasingly attracted the interest of many scientists. Such approaches are based on the selective precipitation of phosphorylated peptides and proteins by applying various metal cations. The excellent performance of precipitation-based enrichment methods can be explained by the absence of any stationary phase, resin or sorbent, which usually leads to unspecific binding. This review provides an overview of recently published methods for the selective precipitation of phosphorylated peptides and proteins. PMID:25587840

Rainer, Matthias; Bonn, Günther K



The effects of connexin phosphorylation on gap junctional communication  

Microsoft Academic Search

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

Paul D. Lampe; Alan F. Lau



Circadian clocks and phosphorylation: Insights from computational modeling  

Microsoft Academic Search

Circadian clocks are based on a molecular mechanism regulated at the transcriptional, translational and post-translational\\u000a levels. Recent experimental data unravel a complex role of the phosphorylations in these clocks. In mammals, several kinases\\u000a play differential roles in the regulation of circadian rhythmicity. A dysfunction in the phosphorylation of one clock protein\\u000a could lead to sleep disorders such as the Familial

Jean-Christophe Leloup



Regulation of mitochondrial aconitase by phosphorylation in diabetic rat heart  

Microsoft Academic Search

.  Mitochondrial dysfunction and protein kinase C (PKC) activation are consistently found in diabetic cardiomyopathy but their\\u000a relationship remains unclear. This study identified mitochondrial aconitase as a downstream target of PKC activation using\\u000a immunoblotting and mass spectrometry, and then characterized phosphorylation-induced changes in its activity in hearts from\\u000a type 1 diabetic rats. PKC?2 co-immunoprecipitated with phosphorylated aconitase from mitochondria isolated from

G. Lin; R. W. Brownsey; K. M. MacLeod



Fission Yeast Rad52 Phosphorylation Restrains Error Prone Recombination Pathways  

PubMed Central

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

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



Sequence, Structure, and Network Evolution of Protein Phosphorylation  

NSDL National Science Digital Library

With the increasing amount of information about the phosphoproteomes of diverse organisms, it is now possible to begin to evaluate this information in the context of evolution. Work described at the inaugural Keystone Symposium on “The Evolution of Protein Phosphorylation” covered a wide range of eukaryotic and prokaryotic organisms, revealing insights into the evolution of protein phosphorylation at the sequence, network, and structural levels.

Chris Soon Heng Tan (Mount Sinai Hospital;Samuel Lunenfeld Research Institute REV)



Phosphorylation Negatively Regulates the Function of Coactivator PC4  

Microsoft Academic Search

Human positive cofactor 4 (PC4) mediates activator-dependent transcription by RNA polymerase II, apparently through interactions with transcriptional activators and the basal transcription machinery. We report here that PC4 function is modulated by in vivo phosphorylation. Proteinprotein interaction studies and in vitro transcription assays demonstrate that only the nonphosphorylated form of PC4 is functionally active. Although recombinant PC4 can be phosphorylated

Hui Ge; Yingming Zhao; Brian T. Chait; Robert G. Roeder



Regulation of influenza a virus nucleoprotein oligomerization by phosphorylation.  


In the influenza virus ribonucleoprotein complex, the oligomerization of the nucleoprotein is mediated by an interaction between the tail-loop of one molecule and the groove of the neighboring molecule. In this study, we show that phosphorylation of a serine residue (S165) within the groove of influenza A virus nucleoprotein inhibits oligomerization and, consequently, ribonucleoprotein activity and viral growth. We propose that nucleoprotein oligomerization in infected cells is regulated by reversible phosphorylation. PMID:25355893

Turrell, Lauren; Hutchinson, Edward C; Vreede, Frank T; Fodor, Ervin



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

SciTech Connect

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.

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



An increase in apparent affinity for sucrose of mung bean sucrose synthase is caused by in vitro phosphorylation or directed mutagenesis of Ser11.  


A mutational analysis of mung bean (Vigna radiata Wilczek) sucrose synthase was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli, in which two different acidic amino acid residues (Asp or Glu) were introduced at Ser11 (S11D, S11E). Only the wild-type enzyme (Ser11) was phosphorylated in vitro by a Ca(2+)-dependent protein kinase from soybean root nodules, suggesting that this is the specific target residue in mung bean sucrose synthase. The apparent affinity for sucrose was increased in this phosphorylated enzyme and also in the S11D and S11E mutant enzymes, although the affinities for UDP-glucose and fructose were similar in the wild-type, phosphorylated wild-type, and mutant enzymes. These results suggest that a monoanionic (1-) side chain at position 11 mimics the Ser11-P2- residue to bind and cleave sucrose for the synthesis of UDP-glucose. Since the S11E mutant enzyme showed the lowest K(m) (sucrose) and the highest catalytic efficiency of the recombinant proteins, the enzymic properties of this S11E mutant were further characterized. The results showed that replacement of Ser11 with Glu11 modestly protected the sucrose synthesis activity against phenolic glycosides and altered the enzyme nucleotide specificity. We postulate that the introduction of negative charge at Ser11 is possibly involved in the enzymatic perturbation of sucrose synthase. PMID:10050318

Nakai, T; Konishi, T; Zhang, X Q; Chollet, R; Tonouchi, N; Tsuchida, T; Yoshinaga, F; Mori, H; Sakai, F; Hayashi, T



A Mitotic Phosphorylation Feedback Network Connects Cdk1, Plk1, 53BP1, and Chk2 to Inactivate the G2/M DNA Damage Checkpoint  

PubMed Central

DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular mechanisms involved in sustaining and ultimately inactivating cell cycle checkpoints are largely unknown. Here, we explored feedback mechanisms that control the maintenance and termination of checkpoint function by computationally identifying an evolutionary conserved mitotic phosphorylation network within the DNA damage response. We demonstrate that the non-enzymatic checkpoint adaptor protein 53BP1 is an in vivo target of the cell cycle kinases Cyclin-dependent kinase-1 and Polo-like kinase-1 (Plk1). We show that Plk1 binds 53BP1 during mitosis and that this interaction is required for proper inactivation of the DNA damage checkpoint. 53BP1 mutants that are unable to bind Plk1 fail to restart the cell cycle after ionizing radiation-mediated cell cycle arrest. Importantly, we show that Plk1 also phosphorylates the 53BP1-binding checkpoint kinase Chk2 to inactivate its FHA domain and inhibit its kinase activity in mammalian cells. Thus, a mitotic kinase-mediated negative feedback loop regulates the ATM-Chk2 branch of the DNA damage signaling network by phosphorylating conserved sites in 53BP1 and Chk2 to inactivate checkpoint signaling and control checkpoint duration. PMID:20126263

van Vugt, Marcel A. T. M.; Reinhardt, H. Christian; Ong, Shao-En; Tan, Chris S.; Miao, Hua; Keezer, Susan M.; Li, Jeijin; Pawson, Tony; Lewis, Timothy A.; Carr, Steven A.; Smerdon, Stephen J.; Brummelkamp, Thijn R.; Yaffe, Michael B.



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

E-print Network

: 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

Blanchette, Robert A.


Linkage disequilibrium between enzymatic loci in natural populations of Drosophila simulans  

E-print Network

Linkage disequilibrium between enzymatic loci in natural populations of Drosophila simulans measured in three Mediterranean populations of Drosophila simulans. Gametic frequencies were estimated are not repeatable from sample to sample. Key words :Drosophila simulans, enzymatic polymorphism, linkage

Boyer, Edmond


Huntingtin-interacting protein 1 phosphorylation by receptor tyrosine kinases.  


Huntingtin-interacting protein 1 (HIP1) binds inositol lipids, clathrin, actin, and receptor tyrosine kinases (RTKs). HIP1 is elevated in many tumors, and its expression is prognostic in prostate cancer. HIP1 overexpression increases levels of the RTK epidermal growth factor receptor (EGFR) and transforms fibroblasts. Here we report that HIP1 is tyrosine phosphorylated in the presence of EGFR and platelet-derived growth factor ? receptor (PDGF?R) as well as the oncogenic derivatives EGFRvIII, HIP1/PDGF?R (H/P), and TEL/PDGF?R (T/P). We identified a four-tyrosine "HIP1 phosphorylation motif" (HPM) in the N-terminal region of HIP1 that is required for phosphorylation mediated by both EGFR and PDGF?R but not by the oncoproteins H/P and T/P. We also identified a tyrosine residue (Y152) within the HPM motif of HIP1 that inhibits HIP1 tyrosine phosphorylation. The HPM tyrosines are conserved in HIP1's only known mammalian relative, HIP1-related protein (HIP1r), and are also required for HIP1r phosphorylation. Tyrosine-to-phenylalanine point mutations in the HPM of HIP1 result in proapoptotic activity, indicating that an intact HPM may be necessary for HIP1's role in cellular survival. These data suggest that phosphorylation of HIP1 by RTKs in an N-terminal region contributes to the promotion of cellular survival. PMID:23836884

Ames, Heather M; Wang, Anmin A; Coughran, Alanna; Evaul, Kristen; Huang, Sha; Graves, Chiron W; Soyombo, Abigail A; Ross, Theodora S



Whose Entropy: A Maximal Entropy Analysis of Phosphorylation Signaling  

NASA Astrophysics Data System (ADS)

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

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



Formation and Dissociation of Phosphorylated Peptide Radical Cations  

NASA Astrophysics Data System (ADS)

In this study, we generated phosphoserine- and phosphothreonine-containing peptide radical cations through low-energy collision-induced dissociation (CID) of the ternary metal-ligand phosphorylated peptide complexes [CuII(terpy) p M]·2+ and [CoIII(salen) p M]·+ [ p M: phosphorylated angiotensin III derivative; terpy: 2,2':6',2''-terpyridine; salen: N, N '-ethylenebis(salicylideneiminato)]. Subsequent CID of the phosphorylated peptide radical cations ( p M·+) revealed fascinating gas-phase radical chemistry, yielding (1) charge-directed b- and y-type product ions, (2) radical-driven product ions through cleavages of peptide backbones and side chains, and (3) different degrees of formation of [M - H3PO4]·+ species through phosphate ester bond cleavage. The CID spectra of the p M·+ species and their non-phosphorylated analogues featured fragment ions of similar sequence, suggesting that the phosphoryl group did not play a significant role in the fragmentation of the peptide backbone or side chain. The extent of neutral H3PO4 loss was influenced by the peptide sequence and the initial sites of the charge and radical. A preliminary density functional theory study, at the B3LYP 6-311++G(d,p) level of theory, of the neutral loss of H3PO4 from a prototypical model— N-acetylphosphorylserine methylamide—revealed several factors governing the elimination of neutral phosphoryl groups through charge- and radical-induced mechanisms.

Kong, Ricky P. W.; Quan, Quan; Hao, Qiang; Lai, Cheuk-Kuen; Siu, Chi-Kit; Chu, Ivan K.



Protein phosphorylation differs significantly among ontogenetic phases in Malus seedlings  

PubMed Central

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



Tyrosine phosphorylation of clathrin heavy chain under oxidative stress.  


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

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



Prediction of Cyclin-Dependent Kinase Phosphorylation Substrates  

PubMed Central

Protein phosphorylation, mediated by a family of enzymes called cyclin-dependent kinases (Cdks), plays a central role in the cell-division cycle of eukaryotes. Phosphorylation by Cdks directs the cell cycle by modifying the function of regulators of key processes such as DNA replication and mitotic progression. Here, we present a novel computational procedure to predict substrates of the cyclin-dependent kinase Cdc28 (Cdk1) in the Saccharomyces cerevisiae. Currently, most computational phosphorylation site prediction procedures focus solely on local sequence characteristics. In the present procedure, we model Cdk substrates based on both local and global characteristics of the substrates. Thus, we define the local sequence motifs that represent the Cdc28 phosphorylation sites and subsequently model clustering of these motifs within the protein sequences. This restraint reflects the observation that many known Cdk substrates contain multiple clustered phosphorylation sites. The present strategy defines a subset of the proteome that is highly enriched for Cdk substrates, as validated by comparing it to a set of bona fide, published, experimentally characterized Cdk substrates which was to our knowledge, comprehensive at the time of writing. To corroborate our model, we compared its predictions with three experimentally independent Cdk proteomic datasets and found significant overlap. Finally, we directly detected in vivo phosphorylation at Cdk motifs for selected putative substrates using mass spectrometry. PMID:17668044

Chang, Emmanuel J.; Begum, Rashida; Chait, Brian T.; Gaasterland, Terry



"Ready, Set, Go": Checkpoint regulation by Cdk1 inhibitory phosphorylation.  


ABSTRACT Cell cycle checkpoints prevent mitosis from occurring before DNA replication and repair are completed during S and G2 phases. The checkpoint mechanism involves inhibitory phosphorylation of Cdk1, a conserved kinase that regulates the onset of mitosis. Metazoans have two distinct Cdk1 inhibitory kinases with specialized developmental functions: Wee1 and Myt1. Ayeni et al used transgenic Cdk1 phospho-acceptor mutants to analyze how the distinct biochemical properties of these kinases affected their functions. They concluded from their results that phosphorylation of Cdk1 on Y15 was necessary and sufficient for G2/M checkpoint arrest in imaginal wing discs, whereas phosphorylation on T14 promoted chromosome stability by a different mechanism. A curious relationship was also noted between Y15 inhibitory phosphorylation and T161 activating phosphorylation. These unexpected complexities in Cdk1 inhibitory phosphorylation demonstrate that the checkpoint mechanism is not a simple binary "off/on" switch, but has at least three distinct states: "Ready", to prevent chromosome damage and apoptosis, "Set", for developmentally regulated G2 phase arrest, and "Go", when Cdc25 phosphatases remove inhibitory phosphates to trigger Cdk1 activation at the G2/M transition. PMID:25483135

Ayeni, Jo; Campbell, Sd



Phosphorylation of Mutant Huntingtin at Serine 116 Modulates Neuronal Toxicity  

PubMed Central

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

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



Growth of calcium phosphate on phosphorylated chitin fibres.  


Calcium phosphate growth on chitin phosphorylated fibres was studied using scanning electron microscopy and energy dispersive X-ray analysis (SEM, EDX), micro-Fourier transform infrared spectroscopy (FTIR), and solid state magic angle spinning nuclear magnetic resonance (MAS NMR) techniques. The C6 chemical shift positions of 13C MAS NMR in the chitin fibres phosphorylated using urea and H3PO4 are obvious indicating that phosphorylation takes place not in the C1 but in the C6 region. Micro-FTIR and 31P MAS NMR suggested that ammonium hydrogen phosphate formed during the phosphorylation procedure. Chitin fibres phosphorylated using urea and H3PO4 and then soaked in saturated Ca(OH)2 solution at ambient temperature, which lead to the formation of thin coatings formed by partial hydrolysis of the PO4 functionalities, were found to stimulate the growth of a calcium phosphate coating on their surfaces after soaking in 1.5xSBF solution for as little as one day. The thin layer after Ca(OH)2 treatment functioned as a nucleation layer for further calcium phosphate deposition after soaking in 1.5xSBF solution. EDX-measured Ca : P ratios of the coatings of Ca(OH)2-treated phosphorylated chitin in 1.5xSBF solution suggested that calcium-deficient apatite was formed. PMID:15348722

Yokogawa, Y; Paz Reyes, J; Mucalo, M R; Toriyama, M; Kawamoto, Y; Suzuki, T; Nishizawa, K; Nagata, F; Kamayama, T



Combination of 31P-NMR magnetization transfer and radioisotope exchange methods for assessment of an enzyme reaction mechanism: rate-determining steps of the creatine kinase reaction.  


The theoretical analysis of a reversible enzyme reaction performed in this work shows that the 31P-NMR magnetization (saturation) transfer technique combined with a radioisotope exchange method may potentially provide information on the position of rate-determining step(s). It depends on chemical shifts of NMR signals of nuclei of interest in free and enzyme-bound forms of substrate(s) and product(s) of the reaction. The creatine kinase reaction (MgATP + creatine----MgADP + P-creatine) has been used as a model. Chemical shifts of 31P in binary, ternary and transitional state substrate-enzyme complexes have been estimated by the variable frequency saturation transfer (VFST) method. This method is based on selective irradiation of numerous points in the spectrum and observation of changes in the intensity of visible line(s) which occur due to chemical exchange between it and lines which are not visible in the routine spectrum. Also, dissociation rate constants of MgADP-containing complexes were determined. Magnetization exchange rates, P-creatine----[gamma-P]MgATP and [beta-P]MgADP----[beta-P]MgATP, were compared with radioisotope exchange rates, [gamma-32P-MgATP----P-creatine and [3H]MgADP----MgATP at different [P-creatine]/[creatine] ratios and at different temperatures. All these exchange rates were close to each other at 30-37 degrees C and [PCr]/[Cr] ratios lower than 2. It is concluded that phosphoryl group transfer is the rate-determining step of the overall creatine kinase reaction under these conditions. However, at lower temperatures (below 25 degrees C) or at high [PCr]/[Cr] ratios ([ADP] less than 20 microM) the rate-determining step seems to be shifted toward dissociation of nucleotide substrates from enzyme-substrate complexes, since exchange rates became significantly different. This approach is useful for analysis of mechanism of enzymatic reactions and also can be applied to non-enzymatic reactions and evaluation of small rapidly exchangeable metabolite pools. PMID:2248962

Kupriyanov, V V; Balaban, R S; Lyulina, N V; Steinschneider AYa; Saks, V A



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...


Lime pretreatment and fermentation of enzymatically hydrolyzed sugarcane bagasse.  


Sugarcane bagasse was subjected to lime (calcium hydroxide) pretreatment and enzymatic hydrolysis for second-generation ethanol production. A central composite factorial design was performed to determine the best combination of pretreatment time, temperature, and lime loading, as well as to evaluate the influence of enzymatic loadings on hydrolysis conversion. The influence of increasing solids loading in the pretreatment and enzymatic hydrolysis stages was also determined. The hydrolysate was fermented using Saccharomyces cerevisiae in batch and continuous mode. In the continuous fermentation, the hydrolysates were concentrated with molasses. Lime pretreatment significantly increased the enzymatic digestibility of sugarcane bagasse without the need for prior particle size reduction. In the optimal pretreatment conditions (90 h, 90 °C, 0.47 glime/g bagasse) and industrially realistic conditions of hydrolysis (12.7 FPU/g of cellulase and 7.3 CBU/g of ?-glucosidase), 139.6 kglignin/ton raw bagasse and 126.0 kg hemicellulose in the pretreatment liquor per ton raw bagasse were obtained. The hydrolysate from lime pretreated sugarcane bagasse presented low amounts of inhibitors, leading to ethanol yield of 164.1 kgethanol/ton raw bagasse. PMID:23334836

Rabelo, Sarita C; Maciel Filho, Rubens; Costa, Aline C




E-print Network

ENZYMATIC HYDROLYSIS OF CLOVER- GRASS MIXTURES FOR ETHANOL PRODUCTION MARTÍN, C.1,2 , THOMSEN, M. H, Risø National Laboratory, P.O.Box 49, DK-4000 Roskilde, Denmark Clover (Trifolium repens L.)-grass.g. organic farming systems. Since clover and grass are rich in carbohydrates, mainly cellulose



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...


Quantitative analysis of enzymatic fractionation of multiple substrate mixtures.  


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

Kaki, Shiva Shanker; Adlercreutz, Patrick



Regioselective Enzymatic Carboxylation of Phenols and Hydroxystyrene Derivatives  

PubMed Central

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



Enzymatic aminoacylation of tRNA with unnatural amino acids  

E-print Network

Enzymatic aminoacylation of tRNA with unnatural amino acids Matthew C. T. Hartman, KristopherRNAs with amino acids. We have developed an AARS assay based on mass spectrometry that can be used to rapidly functional properties. Remarkably, many -amino acids, N-methyl amino acids, and , -disubstituted amino acids

Heller, Eric


The Preparation and Enzymatic Hydrolysis of a Library of Esters  

ERIC Educational Resources Information Center

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…

Sanford, Elizabeth M.; Smith, Traci L.



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 ...


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...


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...



Microsoft Academic Search

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




Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose  

Microsoft Academic Search

Lignocellulose is a potential substrate for ethanol production. However, high cellulose conversion requires high enzyme loading, which makes the process less economically feasible. Addition of surfactants to enzymatic hydrolysis of lignocellulose increases the conversion of cellulose into soluble sugars. The mechanism is not known for the increase of lignocellulose hydrolysis by surfactant addition, therefore, experiments were designed to explore mechanisms

Torny Eriksson; Johan Börjesson; Folke Tjerneld



Structural modifications of lignocellulosics by pretreatments to enhance enzymatic hydrolysis  

Microsoft Academic Search

In this work an evaluation was made of a wide variety of single and multiple pretreatment methods for enhancing the rate of enzymatic hydrolysis of wheat straw. A multiple pretreatment consisted of a physical pretreatment followed by a chemical pretreatment. The structural features of wheat straw, including the specific surface area, crystallinity index, and lignin content, were measured to understand

M. M. Gharpuray; Yong-Hyun Lee; L. T. Fan



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...


Effect of acetyl groups on enzymatic hydrolysis of cellulosic substrates  

Microsoft Academic Search

Evidence showed that acetyl groups introduced during acetic acid delignification was a primary cause of the poor enzymatic digestibility of acetic acid pulp. The inhi- bition by acetyl groups could be removed by saponifi- cation. Acetyl groups might inhibit the enzymes by interfering with the productive binding (hydrogen bonds) between cellulose and the catalytic domain of cellulases, by affecting the

Xuejun Pan; Neil Gilkes; Jack N. Saddler




Technology Transfer Automated Retrieval System (TEKTRAN)

To control the dimensional stability of wool, enzymes can modify the scalar surface structure by proteolysis of the polypeptide network so that the surface properties no longer assist in entanglement. Wool is resistant to enzyme attack. Efficient enzymatic systems generally include a reducing agen...


ORIGINAL PAPER Secreted enzymatic activities of ectomycorrhizal fungi  

E-print Network

ORIGINAL PAPER Secreted enzymatic activities of ectomycorrhizal fungi as a case study of functional fungi (ECMf) are dominant members of the soil microbial community in temperate and boreal forests by the root, the main ECM fungus and other associated organisms as bacteria or other fungi. But, for the sake

Paris-Sud XI, Université de


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

PubMed Central

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

Munnecke, D M



Coupled chemo(enzymatic) reactions in continuous flow  

PubMed Central

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

Yuryev, Ruslan; Strompen, Simon



Enzymatic, immunological and phylogenetic characterization of Brucella suis urease  

Microsoft Academic Search

BACKGROUND: The sequenced genomes of the Brucella spp. have two urease operons, ure-1 and ure-2, but there is evidence that only one is responsible for encoding an active urease. The present work describes the purification and the enzymatic and phylogenomic characterization of urease from Brucella suis strain 1330. Additionally, the urease reactivity of sera from patients diagnosed with brucellosis was

Araceli Contreras-Rodriguez; Jose Quiroz-Limon; Ana M Martins; Humberto Peralta; Eric Avila-Calderon; Nammalwar Sriranganathan; Stephen M Boyle; Ahide Lopez-Merino



Enhancing Enzymatic Digestibility of Wheat Straw by Ethanol Cooking Pretreatment  

Microsoft Academic Search

Ethanol cooking is an important process for the fractionation of biomass components. This work evaluate the effectiveness of sulphate acid catalyzed ethanol cooking pretreatment on enzymatic hydrolysis of wheat straw. The wheat straw samples, which were impregnated with 1.0% sulphate acid for about 12 h at room temperature, were submitted to ethanol cooking pretreatment. The inżuence of both pretreatment temperature

Peng Luo; Zhong Liu



Enzymatic properties and cholesterol content of mitochondrial outer mem-  

E-print Network

Enzymatic properties and cholesterol content of mitochondrial outer mem- branes in liver of obese and microsomes. Simultaneously microsomes were shown to be richer in cholesterol than mitochondrial outer, it was determined that outer membranes are extremely poor in cholesterol, far richer in to- tal fatty acids than

Boyer, Edmond


Development of an oxygen-rich biosensor using enzymatic reaction  

Microsoft Academic Search

This work reports a novel strategy for the development of an O2-rich biosensor. The principle is based on an enzymatic reaction between catalase and H2O2 to release O2, thus to increase the O2 amount in the enzyme matrix. This method improves the determination reliability by alleviating the O2 dependence.

Yue Cui; John P. Barford; Reinhard Renneberg



Analyses of Phosphorylation Events in the Rubella Virus Capsid Protein: Role in Early Replication Events  

Microsoft Academic Search

The Rubella virus capsid protein is phosphorylated prior to virus assembly. Our previous data are consistent with a model in which dynamic phosphorylation of the capsid regulates its RNA binding activity and, in turn, nucleocapsid assembly. In the present study, the process of capsid phosphorylation was examined in further detail. We show that phosphorylation of serine 46 in the RNA

LokMan J. Law; Carolina S. Ilkow; Wen-Pin Tzeng; Matthew Rawluk; David T. Stuart; Teryl K. Frey; Tom C. Hobman



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

E-print Network

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

Allen, John F.


In Vitro Phosphorylation Does not Influence the Aggregation Kinetics of WT ?-Synuclein in Contrast to Its Phosphorylation Mutants  

PubMed Central

The aggregation of alpha-synuclein (?-SYN) into fibrils is characteristic for several neurodegenerative diseases, including Parkinson’s disease (PD). Ninety percent of ?-SYN deposited in Lewy Bodies, a pathological hallmark of PD, is phosphorylated on serine129. ?-SYN can also be phosphorylated on tyrosine125, which is believed to regulate the membrane binding capacity and thus possibly its normal function. A better understanding of the effect of phosphorylation on the aggregation of ?-SYN might shed light on its role in the pathogenesis of PD. In this study we compare the aggregation properties of WT ?-SYN with the phospho-dead and phospho-mimic mutants S129A, S129D, Y125F and Y125E and in vitro phosphorylated ?-SYN using turbidity, thioflavin T and circular dichroism measurements as well as transmission electron microscopy. We show that the mutants S129A and S129D behave similarly compared to wild type (WT) ?-SYN, while the mutants Y125F and Y125E fibrillate significantly slower, although all mutants form fibrillar structures similar to the WT protein. In contrast, in vitro phosphorylation of ?-SYN on either S129 or Y125 does not significantly affect the fibrillization kinetics. Moreover, FK506 binding proteins (FKBPs), enzymes with peptidyl-prolyl cis-trans isomerase activity, still accelerate the aggregation of phosphorylated ?-SYN in vitro, as was shown previously for WT ?-SYN. In conclusion, our results illustrate that phosphorylation mutants can display different aggregation properties compared to the more biologically relevant phosphorylated form of ?-SYN. PMID:24434619

Schreurs, Sarah; Gerard, Melanie; Derua, Rita; Waelkens, Etienne; Taymans, Jean-Marc; Baekelandt, Veerle; Engelborghs, Yves




PubMed Central

Our experiments investigated associations of specific isoforms of protein kinase C (PKC) with individual proteins in the cardiac troponin complex. Troponin I (cTnI) associated with PKC ? and ? and troponin T (cTnT) associated with PKC ?, ?, and ?. Based on its association with cTnI, we hypothesized that PKC? is a major regulator of myofilament protein phosphorylation. To test this, we infected adult cardiac myocytes with adenoviral constructs containing DsRed monomer-tagged wild type (WT) and the following constitutively active forms of PKC?: the pseudo-substrate region (A119E), 3?-phospho-inositide-dependent kinase-1 (T410E), and auto-phosphorylation (T560E). The A119E and T410E mutants displayed increased localization to the Z-discs compared with WT and T560E. Immunoprecipitations were performed in myocytes expressing PKC? using PKC phospho-motif antibodies to determine the phosphophorylation of cTnI, cTnT, tropomyosin, myosin-binding protein C, and desmin. We did not find serine (Ser) phosphorylation of cTnI or cTnT. However, we observed a significant decrease in threonine (Thr) phosphorylation of cTnI and cTnT notably by PKC? T560E. Ser phosphorylation of tropomyosin was increased by all three active mutants of PKC?. Ser/Thr phosphorylation of myosin-binding protein C increased primarily by PKC? A119E. Both PKC? A119E and T410E mutants increased desmin Ser/Thr phosphorylation. To explain the apparent Thr dephosphorylation of cTnI and cTnT, we hypothesized that PKC? exists as a complex with p21-activated kinase-1 (Pak1) and protein phosphatase 2A (PP2A), and this was confirmed by immunoprecipitation Western blot. Our data demonstrate that PKC? is a novel regulator of myofilament protein phosphorylation. PMID:17724026

Wu, Steven C.; Solaro, R. John



Molecular dynamics study of enhanced Man5B enzymatic activity  

PubMed Central

Background Biofuels are a well-known alternative to the largely used fossil-derived fuels, however the competition with food production is an ethical dilemma. Fortunately a solution is offered by second-generation biofuels which can be produced from agricultural waste or, more specifically, from plant cell wall polysaccharides. The conversion process involves typically enzymatic hydrolysis of lignocellulosic biomass and then separation of its constituent sugars that are further fermented to produce ethanol. Over the years several technologies have been developed that allow this conversion process to occur and the objective is now to make this process cost-competitive in today’s markets. Results We observe that reduction of enzymatic efficiency in the presence of gluco-oligosaccharides is associated with a loss of the enzyme’s flexibility, the latter being required to bind new substrate, while the presence of manno-oligosaccharides does not pose this problem. Molecular dynamics simulations identify key contacts between substrates and the enzyme catalytic pocket that might be modified through site-directed mutagenesis to prevent loss of enzymatic efficiency. Conclusions Based on previous experimental studies and the new molecular dynamics data, we suggest that cellohexaose in the active site pocket slows down or even inhibits Man5B enzymatic activity. The assumption of such a mechanism is reasonable since when the gluco-oligosaccharide substrate is attached to the catalytic pocket it takes much longer to leave the pocket and thus prevents other substrates from reaching the active site. The insight is of crucial importance since the inhibition of enzymes by the enzymatic product or by an unsuitable substrate is a major technological problem in reducing the competitiveness of second-generation biofuel production. PMID:24976862



Preparation of icariside II from icariin by enzymatic hydrolysis method.  


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

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



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...


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

ERIC Educational Resources Information Center

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

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



Structural Basis for Inactivation of the Human Pyruvate Dehydrogenase Complex by Phosphorylation: Role of Disordered Phosphorylation Loops  

SciTech Connect

We report the crystal structures of the phosporylated pyruvate dehydrogenase (E1p) component of the human pyruvate dehydrogenase complex (PDC). The complete phosphorylation at Ser264-{alpha} (site 1) of a variant E1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the PDC core. We show that unlike its unmodified counterpart, the presence of a phosphoryl group at Ser264-{alpha} prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three phosphorylation sites. The disordering of these phosphorylation loops is caused by a previously unrecognized steric clash between the phosphoryl group at site 1 and a nearby Ser266-{alpha}, which nullifies a hydrogen-bonding network essential for maintaining the loop conformations. The disordered phosphorylation loops impede the binding of lipoyl domains of the PDC core to E1p, negating the reductive acetylation step. This results in the disruption of the substrate channeling in the PDC, leading to the inactivation of this catalytic machine.

Kato, Masato; Wynn, R. Max; Chuang, Jacinta L.; Tso, Shih-Chia; Machius, Mischa; Li, Jun; Chuang, David T. (UTSMC)



Structural Basis for Inactivation of the Human Pyruvate Dehydrogenase Complex by Phosphorylation: Role of Disordered Phosphorylation Loops  

PubMed Central

SUMMARY We report the crystal structures of the phosporylated pyruvate dehydrogenase (E1p) component of the human pyruvate dehydrogenase complex (PDC). The complete phosphorylation at Ser264-? (site 1) of a variant E1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the PDC core. We show that unlike its unmodified counterpart, the presence of a phosphoryl group at Ser264-? prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three phosphorylation sites. The disordering of these phosphorylation loops is caused by a previously unrecognized steric clash between the phosphoryl group at site 1 and a nearby Ser266-?, which nullifies a hydrogen-bonding network essential for maintaining the loop conformations. The disordered phosphorylation loops impede the binding of lipoyl domains of the PDC core to E1p negating the reductive acetylation step. This results in the disruption of the substrate channeling in the PDC, leading to the inactivation of this catalytic machine. PMID:19081061

Kato, Masato; Max Wynn, R; Chuang, Jacinta L; Tso, Shih-Chia; Machius, Mischa; Li, Jun; Chuang, David T.



"Transfer Shock" or "Transfer Ecstasy?"  

ERIC Educational Resources Information Center

The alleged characteristic drop in grade point average (GPA) of transfer students and the subsequent rise in GPA was investigated in this study. No statistically significant difference was found in first term junior year GPA between junior college transfers and native Florida State University students after the variance accounted for by the…

Nickens, John M.


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

NASA Astrophysics Data System (ADS)

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.

Szczepaniak, Krzysztof; Worch, Remigiusz; Grzyb, Joanna



Phosphorylation of hepatic phenobarbital-inducible cytochrome P-450.  

PubMed Central

The major phenobarbital-inducible cytochrome P-450 purified from rat liver, a member of family II of the cytochrome P-450 gene superfamily, is rapidly phosphorylated by cAMP-dependent protein kinase. The phosphorylation reaches greater than 0.5 mol phosphate/mol P-450 after 5 min and is accompanied by a decrease in enzyme activity. The serine residue in position 128 was shown to be the sole phosphorylation site and a conformational change of the protein was indicated by a shift of the carbon monoxide difference spectrum of the reduced cytochrome from 450 to 420 nm. Comparison of amino acid sequences of various cytochrome P-450 families revealed a highly conserved arginine residue in the immediate vicinity of the phosphorylated serine residue which constitutes the kinase recognition sequence. It also revealed that only the members of the cytochrome P-450 family II carry this kinase recognition sequence. To find out whether this phosphorylation also occurs in vivo, the exchangeable phosphate pool of intact hepatocytes derived from phenobarbital-pretreated rats was labeled with 32Pi followed by an incubation of the cells with the membrane-permeating dibutyryl-cAMP or with the adenylate cyclase stimulator glucagon to activate endogenous kinase. As a result, a microsomal polypeptide with the same electrophoretic mobility as cytochrome P-450 became strongly labeled. Peptide mapping and immunoprecipitation with monospecific antibodies identified this protein as the major phenobarbital-inducible cytochrome P-450. It becomes phosphorylated at the same serine residues as in the cell-free phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2583091

Pyerin, W; Taniguchi, H



Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy.  


The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knock-in mice expressing nonphosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knock-in mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knock-in mice compared with their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild-type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knock-in mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knock-in and wild-type mice, indicating that mTORC1 was still activated in the knock-in mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild-type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knock-in mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth.Kidney International advance online publication, 17 September 2014; doi:10.1038/ki.2014.302. PMID:25229342

Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang



Phosphorylation and glycosylation of the luteinizing hormone receptor.  


Purified testicular and ovarian luteinizing hormone/human chorionic gonadotropin (hCG) receptors are phosphorylated at serine and threonine residues by the catalytic subunit of the cAMP-dependent protein kinase (protein kinase A). Occupancy of the receptors by hCG significantly increased the rate but not the extent of phosphorylation. However, prolonged preincubation of receptors with hCG reduced the subsequent rate of receptor phosphorylation. Identical phosphopeptide maps were obtained for the phosphorylated ovarian and testicular receptors. The phosphorylated receptor, like the native receptor, bound to wheat germ lectin and hCG-Sepharose and migrated as a single band of Mr 90,000 (testis) and Mr 85,000 (ovary) on NaDodSO4/PAGE. Neuraminidase treatment of receptors caused reductions of molecular weight to 82,000 (testis) and 77,000 (ovary), and further treatment with O-Glycanase had minimal effect on molecular size. However, deglycosylation with N-Glycosidase and endoglycosidase F produced a single labeled polypeptide of Mr 59,000 for both gonadal receptors. Treatment of native receptors with neuraminidase caused no apparent change in binding of gonadotropin to blotted receptors, whereas deglycosylated receptors showed a major reduction in hormone binding. These results indicate that luteinizing hormone/hCG receptors are sialoglycoproteins with predominantly N-linked glycosyl residues that account for the size difference between testicular and ovarian receptors and that may participate in the interaction with gonadotropin. Receptor occupancy by agonist leads to a conformational change that facilitates its phosphorylation during initial binding and reduces the rate of phosphorylation after more prolonged exposure to hCG. PMID:2922394

Minegishi, T; Delgado, C; Dufau, M L



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

SciTech Connect

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.

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



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

PubMed Central

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

Shi, Feng; Shi, Jikui; Li, Yongfu



Interhelical Interaction and Receptor Phosphorylation Regulate the Activation Kinetics of Different Human ?1-Adrenoceptor Variants.  


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

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



Enzymatically quiescent heparanase augments T cell interactions with VCAM-1 and extracellular matrix components under versatile dynamic contexts.  


During their migration into inflammatory sites, immune cells, such as T cells, secrete extracellular matrix (ECM)-degrading enzymes, such as heparanase, which, under mildly acidic conditions, degrade heparan sulfate proteoglycans (HSPG). We have previously shown that at pH 7.2, human placental heparanase loses its enzymatic activity, while retaining its ability to bind HSPG and promote T cell adhesion to unfractionated ECM. We now demonstrate that the 65-kDa recombinant human heparanase, which is devoid of enzymatic activity, but can still bind HSPG, captures T cells under shear flow conditions and mediates their rolling and arrest, in the absence or presence of stromal cell-derived factor 1 alpha (SDF-1 alpha; CXCL12), in an alpha(4)beta(1)-VCAM-1-dependent manner. Furthermore, heparanase binds to and induces T cell adhesion to key ECM components, like fibronectin and hyaluronic acid, in beta(1) integrin- and CD44-specific manners, respectively, via the activation of the protein kinase C and phosphatidylinositol 3-kinase intracellular signaling machineries. Although the nature of the putative T cell heparanase-binding moiety is unknown, it appears that heparanase exerts its proadhesive activity by interacting with the T cells' surface HSPG, because pretreatment of the cells with heparinase abolished their subsequent response to heparanase. Also, heparanase augmented the SDF-1 alpha-triggered phosphorylation of Pyk-2 and extracellular signal-regulated kinase-2 implicated in integrin functioning. Moreover, heparanase, which had no chemotactic effect on T cells on its own, augmented the SDF-1 alpha-induced T cell chemotaxis across fibronectin. These findings add another dimension to the known versatility of heparanase as a key regulator of T cell activities during inflammation, both in the context of the vasculature and at extravascular sites. PMID:15100255

Sotnikov, Ilya; Hershkoviz, Rami; Grabovsky, Valentin; Ilan, Neta; Cahalon, Liora; Vlodavsky, Israel; Alon, Ronen; Lider, Ofer



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

PubMed Central

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

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



Regulation of yeast central metabolism by enzyme phosphorylation  

PubMed Central

As a frequent post-translational modification, protein phosphorylation regulates many cellular processes. Although several hundred phosphorylation sites have been mapped to metabolic enzymes in Saccharomyces cerevisiae, functionality was demonstrated for few of them. Here, we describe a novel approach to identify in vivo functionality of enzyme phosphorylation by combining flux analysis with proteomics and phosphoproteomics. Focusing on the network of 204 enzymes that constitute the yeast central carbon and amino-acid metabolism, we combined protein and phosphoprotein levels to identify 35 enzymes that change their degree of phosphorylation during growth under five conditions. Correlations between previously determined intracellular fluxes and phosphoprotein abundances provided first functional evidence for five novel phosphoregulated enzymes in this network, adding to nine known phosphoenzymes. For the pyruvate dehydrogenase complex E1 ? subunit Pda1 and the newly identified phosphoregulated glycerol-3-phosphate dehydrogenase Gpd1 and phosphofructose-1-kinase complex ? subunit Pfk2, we then validated functionality of specific phosphosites through absolute peptide quantification by targeted mass spectrometry, metabolomics and physiological flux analysis in mutants with genetically removed phosphosites. These results demonstrate the role of phosphorylation in controlling the metabolic flux realised by these three enzymes. PMID:23149688

Oliveira, Ana Paula; Ludwig, Christina; Picotti, Paola; Kogadeeva, Maria; Aebersold, Ruedi; Sauer, Uwe



Interplay between sumoylation and phosphorylation for protection against ?-synuclein inclusions.  


Parkinson disease is associated with the progressive loss of dopaminergic neurons from the substantia nigra. The pathological hallmark of the disease is the accumulation of intracytoplasmic inclusions known as Lewy bodies that consist mainly of post-translationally modified forms of ?-synuclein. Whereas phosphorylation is one of the major modifications of ?-synuclein in Lewy bodies, sumoylation has recently been described. The interplay between ?-synuclein phosphorylation and sumoylation is poorly understood. Here, we examined the interplay between these modifications as well as their impact on cell growth and inclusion formation in yeast. We found that ?-synuclein is sumoylated in vivo at the same sites in yeast as in human cells. Impaired sumoylation resulted in reduced yeast growth combined with an increased number of cells with inclusions, suggesting that this modification plays a protective role. In addition, inhibition of sumoylation prevented autophagy-mediated aggregate clearance. A defect in ?-synuclein sumoylation could be suppressed by serine 129 phosphorylation by the human G protein-coupled receptor kinase 5 (GRK5) in yeast. Phosphorylation reduced foci formation, alleviated yeast growth inhibition, and partially rescued autophagic ?-synuclein degradation along with the promotion of proteasomal degradation, resulting in aggregate clearance in the absence of a small ubiquitin-like modifier. These findings suggest a complex interplay between sumoylation and phosphorylation in ?-synuclein aggregate clearance, which may open new horizons for the development of therapeutic strategies for Parkinson disease. PMID:25231978

Shahpasandzadeh, Hedieh; Popova, Blagovesta; Kleinknecht, Alexandra; Fraser, Paul E; Outeiro, Tiago F; Braus, Gerhard H



Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication  

PubMed Central

Merkel Cell Polyomavirus (MCPyV) was recently discovered as a novel human polyomavirus that is associated with ~80% of Merkel Cell Carcinomas. The Large Tumor antigen (LT) is an early viral protein which has a variety of functions, including manipulation of the cell cycle and initiating viral DNA replication. Phosphorylation plays a critical regulatory role for polyomavirus LT proteins, but no investigation of MCPyV LT phosphorylation has been performed to date. In this report mass spectrometry analysis reveals three unique phosphorylation sites: T271, T297 and T299. In vivo replication assays confirm that phosphorylation of T271 does not play a role in viral replication, while modification at T297 and T299 have dramatic and opposing effects on LT’s ability to initiate replication from the viral origin. We test these mutants for their ability to bind, unwind, and act as a functional helicase at the viral origin. These studies provide a framework for understanding how phosphorylation of LT may dynamically regulate viral replication. Although the natural host cell of MCPyV has not yet been established, this work provides a foundation for understanding how LT activity is regulated and provides tools for better exploring this regulation in both natural host cells and Merkel cells. PMID:25006834

Diaz, Jason; Wang, Xin; Tsang, Sabrina H.; Jiao, Jing; You, Jianxin



?-Synuclein phosphorylation as a therapeutic target in Parkinson's disease.  


Phosphorylation is a key post-translational modification necessary for normal cellular signaling and, therefore, lies at the heart of cellular function. In neurodegenerative disorders, abnormal hyperphosphorylation of pathogenic proteins is a common phenomenon that contributes in important ways to the disease process. A prototypical protein that is hyperphosphorylated in the brain is ?-synuclein (?-syn) - found in Lewy bodies and Lewy neurites - the pathological hallmarks of Parkinson's disease (PD) and other ?-synucleinopathies. The genetic linkage of ?-syn to PD as well as its pathological association in both genetic and sporadic cases have made it the primary protein of interest. In understanding how ?-syn dysfunction occurs, increasing focus is being placed on its abnormal aggregation and the contribution of phosphorylation to this process. Studies of both the kinases and phosphatases that regulate ?-syn phosphorylation are beginning to reveal the roles of this post-translational modification in disease pathogenesis. Modulation of ?-syn phosphorylation may ultimately prove to be a viable strategy for disease-modifying therapeutic interventions. In this review, we explore mechanisms related to ?-syn phosphorylation, its biophysical and functional consequences, and its role in neurodegeneration. PMID:22499677

Braithwaite, Steven P; Stock, Jeffry B; Mouradian, M Maral



Phosphorylation and desensitization of alpha1d-adrenergic receptors.  

PubMed Central

In rat-1 fibroblasts stably expressing rat alpha(1d)-adrenoceptors, noradrenaline and PMA markedly decreased alpha(1d)-adrenoceptor function (noradrenaline-elicited increases in calcium in whole cells and [(35)S]guanosine 5'-[gamma-thio]triphosphate binding in membranes), suggesting homologous and heterologous desensitizations. Photoaffinity labelling, Western blotting and immunoprecipitation identified alpha(1d)-adrenoceptors as a broad band of 70-80 kDa. alpha(1d)-Adrenoceptors were phosphorylated in the basal state and noradrenaline and PMA increased it. The effect of noradrenaline was concentration-dependent (EC(50) 75 nM), rapid (maximum at 1 min) and transient. Phorbol ester-induced phosphorylation was concentration-dependent (EC(50) 25 nM), slightly slower (maximum at 5 min) and stable for at least 60 min. Inhibitors of protein kinase C decreased the effect of phorbol esters but not that of noradrenaline. Evidence of cross-talk of alpha(1d)-adrenoceptors with receptors endogenously expressed in rat-1 fibroblasts was given by the ability of endothelin, lysophosphatidic acid and bradykinin to induce alpha(1d)-adrenoceptor phosphorylation. In summary, it is shown for the first time here that alpha(1d)-adrenoceptors are phosphoproteins and that receptor phosphorylation is increased by the natural ligand, noradrenaline, by direct activation of protein kinase C and via cross-talk with other receptors endogenously expressed in rat-1 fibroblasts. Receptor phosphorylation has functional repercussions. PMID:11171057

García-Sáinz, J A; Vázquez-Cuevas, F G; Romero-Avila, M T



Phosphorylation Barcoding as a Mechanism of Directing GPCR Signaling  

NSDL National Science Digital Library

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

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



Rock-dependent calponin 3 phosphorylation regulates myoblast fusion.  


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

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



Cellular Functions Regulated by Phosphorylation of EGFR on Tyr845  

PubMed Central

The Src gene product (Src) and the epidermal growth factor receptor (EGFR) are prototypes of oncogene products and function primarily as a cytoplasmic non-receptor tyrosine kinase and a transmembrane receptor tyrosine kinase, respectively. The identification of Src and EGFR, and the subsequent extensive investigations of these proteins have long provided cutting edge research in cancer and other molecular and cellular biological studies. In 1995, we reported that the human epidermoid carcinoma cells, A431, contain a small fraction of Src and EGFR in which these two kinase were in physical association with each other, and that Src phosphorylates EGFR on tyrosine 845 (Y845) in the Src-EGFR complex. Y845 of EGFR is located in the activation segment of the kinase domain, where many protein kinases contain kinase-activating autophosphorylation sites (e.g., cAMP-dependent protein kinase, Src family kinases, transmembrane receptor type tyrosine kinases) or trans-phosphorylation sites (e.g., cyclin-dependent protein kinase, mitogen-activated protein kinase, Akt protein kinase). A number of studies have demonstrated that Y845 phosphorylation serves an important role in cancer as well as normal cells. Here we compile the experimental facts involving Src phosphorylation of EGFR on Y845, by which cell proliferation, cell cycle control, mitochondrial regulation of cell metabolism, gamete activation and other cellular functions are regulated. We also discuss the physiological relevance, as well as structural insights of the Y845 phosphorylation. PMID:23702846

Sato, Ken-ichi



Queuine mediated inhibition in phosphorylation of tyrosine phosphoproteins in cancer.  


Protein phosphorylation or dephosphorylation is the most important regulatory switch of signal transduction contributing to control of cell proliferation. The reversibility of phosphorylation and dephosphorylation is due to the activities of kinases and phosphatase, which determine protein phosphorylation level of cell under different physiological and pathological conditions. Receptor tyrosine kinase (RTK) mediated cellular signaling is precisely coordinated and tightly controlled in normal cells which ensures regulated mitosis. Deregulation of RTK signaling resulting in aberrant activation in RTKs leads to malignant transformation. Queuine is one of the modified base of tRNA which participates in down regulation of tyrosine kinase activity. The guanine analogue queuine is a nutrient factor to eukaryotes and occurs as free base or modified nucleoside queuosine into the first anticodon position of specific tRNAs. The tRNAs are often queuine deficient in cancer and fast proliferating tissues. The present study is aimed to investigate queuine mediated inhibition in phosphorylation of tyrosine phosphorylated proteins in lymphoma bearing mouse. The result shows high level of cytosolic and membrane associated tyrosine phosphoprotein in DLAT cancerous mouse liver compared to normal. Queuine treatments down regulate the level of tyrosine phosphoproteins, which suggests that queuine is involved in regulation of mitotic signaling pathways. PMID:17514433

Pathak, Chandramani; Jaiswal, Yogesh K; Vinayak, Manjula



A Phosphorylated Pseudokinase Complex Controls Cell Wall Synthesis in Mycobacteria  

PubMed Central

Prokaryotic cell wall biosynthesis is coordinated with cell growth and division, but the mechanisms regulating this dynamic process remain obscure. Here, we describe a phosphorylation-dependent regulatory complex that controls peptidoglycan (PG) biosynthesis in Mycobacterium tuberculosis. We found that PknB, a PG-responsive Ser-Thr protein kinase (STPK), initiates complex assembly by phosphorylating a kinase-like domain in the essential PG biosynthetic protein, MviN. This domain was structurally diverged from active kinases and did not mediate phosphotransfer. Threonine phosphorylation of the pseudokinase domain recruited the FhaA protein through its forkhead-associated (FHA) domain. The crystal structure of this phosphorylated pseudokinase–FHA domain complex revealed the basis of FHA domain recognition, which included unexpected contacts distal to the phosphorylated threonine. Conditional degradation of these proteins in mycobacteria demonstrated that MviN was essential for growth and PG biosynthesis and that FhaA regulated these processes at the cell poles and septum. Controlling this spatially localized PG regulatory complex is only one of several cellular roles ascribed to PknB, suggesting that the capacity to coordinate signaling across multiple processes is an important feature conserved between eukaryotic and prokaryotic STPK networks. PMID:22275220

Gee, Christine L.; Papavinasasundaram, Kadamba G.; Blair, Sloane R.; Baer, Christina E.; Falick, Arnold M.; King, David S.; Griffin, Jennifer E.; Venghatakrishnan, Harene; Zukauskas, Andrew; Wei, Jun-Rong; Dhiman, Rakesh K.; Crick, Dean C.; Rubin, Eric J.; Sassetti, Christopher M.; Alber, Tom



Cardiac troponin I tyrosine 26 phosphorylation decreases myofilament Ca(2+) sensitivity and accelerates deactivation.  


Troponin I (TnI), the inhibitory subunit of the troponin complex, can be phosphorylated as a key regulatory mechanism to alter the calcium regulation of contraction. Recent work has identified phosphorylation of TnI Tyr-26 in the human heart with unknown functional effects. We hypothesized that TnI Tyr-26N-terminal phosphorylation decreases calcium sensitivity of the thin filament, similar to the desensitizing effects of TnI Ser-23/24 phosphorylation. Our results demonstrate that Tyr-26 phosphorylation and pseudo-phosphorylation decrease calcium binding to troponin C (TnC) on the thin filament and calcium sensitivity of force development to a similar magnitude as TnI Ser-23/24 pseudo-phosphorylation. To investigate the effects of TnI Tyr-26 phosphorylation on myofilament deactivation, we measured the rate of calcium dissociation from TnC. Results demonstrate that filaments containing Tyr-26 pseudo-phosphorylated TnI accelerate the rate of calcium dissociation from TnC similar to that of TnI Ser-23/24. Finally, to assess functional integration of TnI Tyr-26 with Ser-23/24 phosphorylation, we generated recombinant TnI phospho-mimetic substitutions at all three residues. Our biochemical analyses demonstrated no additive effect on calcium sensitivity or calcium-sensitive force development imposed by Tyr-26 and Ser-23/24 phosphorylation integration. However, integration of Tyr-26 phosphorylation with pseudo-phosphorylated Ser-23/24 further accelerated thin filament deactivation. Our findings suggest that TnI Tyr-26 phosphorylation functions similarly to Ser-23/24N-terminal phosphorylation to decrease myofilament calcium sensitivity and accelerate myofilament relaxation. Furthermore, Tyr-26 phosphorylation can buffer the desensitization of Ser-23/24 phosphorylation while further accelerating thin filament deactivation. Therefore, the functional integration of TnI phosphorylation may be a common mechanism to modulate Ser-23/24 phosphorylation function. PMID:25252176

Salhi, Hussam E; Walton, Shane D; Hassel, Nathan C; Brundage, Elizabeth A; de Tombe, Pieter P; Janssen, Paul M L; Davis, Jonathan P; Biesiadecki, Brandon J



The dynamical nature of enzymatic catalysis.  


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 Ĺ 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

Callender, Robert; Dyer, R Brian



The Dynamical Nature of Enzymatic Catalysis  

PubMed Central

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 Ĺ 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

Callender, Robert; Dyer, R. Brian



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

PubMed Central

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



An enzymatically stable kyotorphin analog induces pain in subattomol doses.  


Intraplantar injection of the enzymatically stable, N-methylated kyotorphin analog Tyr(NMe)-Arg-OH produced marked and sharp nociceptive flexor responses in a dose-dependent manner. A significant response was observed with this compound at a dose of 0. 01 amol (6000 molecules). Tyr(NMe)-Arg-OH-nociception was completely blocked by the kyotorphin antagonist leucyl-arginine and its enzymatically stable, N-methylated analog, as well as by CP-99994, a specific neurokinin 1 antagonist. These findings suggest that the nociceptive effect produced by Tyr(NMe)-Arg-OH in subattomol doses occurs via specific interaction with the kyotorphin receptor and that the extraordinary potency observed may result from amplification through local substance P release. PMID:10876055

Ueda, H; Inoue, M; Weltrowska, G; Schiller, P W



Cyclic enzymatic solid phase synthesis of isotopically labeled DNA oligonucleotides.  


Isotopic labeling of DNA using standard solid phase synthesis requires expensive phosphoramidites that are used in large excess. We have developed a protocol where enzymatic, cyclic, solid phase synthesis of DNA facilitates a more economical use of the less expensive labeled DNA triphosphates (dNTP). In this approach, the DNA template is immobilized on an epoxy-activated solid support. Both the support and the linkage between DNA and resin are inert to high pH conditions which are required for product release in this scheme. Efficient covalent attachment of the DNA to the resin was achieved when the reaction was carried out in MgCl2/CAPS. The enzymatic fill in reaction as well as product release and recycling conditions were optimized for efficient reuse of dNTPs without any purification. The developed protocol was used to generate a selectively [(13)C, (15)N] G labeled 10-mer duplex. PMID:20183571

Khan, Ahmed M; Mishra, Subrata H; Germann, Markus W



Alginate oligosaccharides: enzymatic preparation and antioxidant property evaluation.  


Alginate oligosaccharides (AOs) prepared from alginate, by alginate lyase-mediated depolymerization, were structurally characterized by mass spectrometry, infrared spectrometry and thin layer chromatography. Studies of their antioxidant activities revealed that AOs were able to completely (100%) inhibit lipid oxidation in emulsions, superiorly to ascorbic acid (89% inhibition). AOs showed radical scavenging activity towards ABTS?, hydroxyl, and superoxide radicals, which might explain their excellent antioxidant activity. The radical scavenging activity is suggested to originate mainly from the presence of the conjugated alkene acid structure formed during enzymatic depolymerization. According to the resonance hybrid theory, the parent radicals of AOs are delocalized through allylic rearrangement, and as a consequence, the reactive intermediates are stabilized. AOs were weak ferrous ion chelators. This work demonstrated that AOs obtained from a facile enzymatic treatment of abundant alginate is an excellent natural antioxidant, which may find applications in the food industry. PMID:24996323

Falkeborg, Mia; Cheong, Ling-Zhi; Gianfico, Carlo; Sztukiel, Katarzyna Magdalena; Kristensen, Kasper; Glasius, Marianne; Xu, Xuebing; Guo, Zheng



Rational design of functional and tunable oscillating enzymatic networks  

NASA Astrophysics Data System (ADS)

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.

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.



Rational design of functional and tunable oscillating enzymatic networks.  


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. PMID:25615670

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



Enzymatic methods in food analysis: determination of ascorbic acid.  


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

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



Loosening xyloglucan accelerates the enzymatic degradation of cellulose in wood.  


In order to create trees in which cellulose, the most abundant component in biomass, can be enzymatically hydrolyzed highly for the production of bioethanol, we examined the saccharification of xylem from several transgenic poplars, each overexpressing either xyloglucanase, cellulase, xylanase, or galactanase. The level of cellulose degradation achieved by a cellulase preparation was markedly greater in the xylem overexpressing xyloglucanase and much greater in the xylems overexpressing xylanase and cellulase than in the xylem of the wild-type plant. Although a high degree of degradation occurred in all xylems at all loci, the crystalline region of the cellulose microfibrils was highly degraded in the xylem overexpressing xyloglucanase. Since the complex between microfibrils and xyloglucans could be one region that is particularly resistant to cellulose degradation, loosening xyloglucan could facilitate the enzymatic hydrolysis of cellulose in wood. PMID:19825667

Kaida, Rumi; Kaku, Tomomi; Baba, Kei'ichi; Oyadomari, Masafumi; Watanabe, Takashi; Nishida, Koji; Kanaya, Toshiji; Shani, Ziv; Shoseyov, Oded; Hayashi, Takahisa



Enzymatic hydrolysis of steryl glycosides for their analysis in foods.  


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

Münger, Linda H; Nyström, Laura



Biomimetic Materials for Medical Application Through Enzymatic Modification  

Microsoft Academic Search

\\u000a \\u000a Abstract  Living organisms synthesize functional materials, based on proteins and polysaccharides, using enzyme-catalyzed reactions.\\u000a According to the biomimetic approach, biomaterial matrices for tissue engineering are designed to be able to mimic the properties\\u000a and the functions of the extracellular matrix (ECM). In this chapter, the most significant research efforts dedicated to the\\u000a study and the preparation of biomimetic materials through enzymatic

Piergiorgio Gentile; Valeria Chiono; Chiara Tonda-Turo; Susanna Sartori; Gianluca Ciardelli


Hydroxypropylation of cellulose as a pretreatment for enzymatic hydrolysis  

E-print Network

Dr. E. J. Soltes Cellulose derivatives such as carboxymethylcellulose and water soluble cellulose acetate have shown increased ability to be converted to reducing sugars, including glucose, compared to pure cellulose. Hydroxypropylcellulose (HPC... of substituted AHG. Fermentation of enzymatic hydrolyzates, obtained from HPC samples, confirmed that the presence of hydroxypropyl derivatives does not inhibit the production of ethyl alcohol. DEDICATION This thesis is dedicated to Kim, Ryan, and Holly...

Brix, Scott Tyson



Enzymatic browning reactions in apple and apple products.  


This review examines the parameters of enzymatic browning in apple and apple products that is, phenolic compounds, polyphenoloxidases, and other factors (ascorbic acid and peroxidases), both qualitatively and quantitatively. Then the relationships between intensity of browning and the browning parameters are discussed, including a paragraph on the methods used for browning evaluation. Finally, the different methods for the control of browning are presented. PMID:8011143

Nicolas, J J; Richard-Forget, F C; Goupy, P M; Amiot, M J; Aubert, S Y



Enzymatic activity preservation and protection through entrapment within degradable hydrogels.  


This work aims to develop a repeatable enzyme entrapment method that preserves activity within an amicable environment while resisting activity reduction in the presence of environmental challenges. Advances in such methods have wide potential use in biosensor applications. In this work ?-galactosidase (lactase) enzyme was entrapped within hydrogel matrices of acrylamide (ACR) crosslinked with N,N'-methylenebisacrylamide (BIS, non-degradable) or poly(ethylene glycol) diacrylate (PEGDA, degradable) to create "biogels." Diffusivity studies of control, enzyme free, hydrogel constructs showed near-Fickian swelling behavior in PBS regardless of crosslinker type or density. As expected, the swelling rate, Ks , decreased when increasing the crosslink density from 78.6 to 14.7?min?ą over a range of 1-20?mol% PEGDA indicating that diffusivity into the matrix is dependent on crosslink density. Fabricated biogels were evaluated for maintained enzyme activity in the 7 and 8 pH range. PEGDA crosslinked gels consistently showed improved enzymatic activity retention as compared to BIS crosslinked gels. As PEGDA crosslink density increased from 5 to 10?mol%, enzymatic activity retention post-initial entrapment increased. Higher PEGDA crosslink densities between 15% and 40% decreased enzymatic activity due to assumed steric hindrance of the entrapped enzyme and also decreased substrate and product diffusion. Increased enzymatic stability was observed in 40?mol% PEGDA crosslinked gels. The biogels were pH challenged to 8.0 and stability, measured as retention of activity, was observed to be 91%. Free, non-entrapped, solution based enzyme conversion only retained 23% activity under the same pH challenge conditions. No significant loss of active enzyme was determined to elute out of the biogels during storage in PBS or during biogel wash and recycling. This entrapment method illustrates the potential to sterically hinder and diffusively impede enzymes from performing their function. Degradation of the network crosslinks can then potentially enable the reactivation of the enzyme at a site and time dictated by the user. PMID:23744741

Mariani, Angela M; Natoli, Mary E; Kofinas, Peter



Hemoglobin–Oxygen Equilibrium Curves Measured during Enzymatic Oxygen Consumption  

Microsoft Academic Search

A rapid, new method to measure hemoglobin–oxygen equilibrium curves is described using the protocatechuic acid\\/protocatechuic acid 3,4-dioxygenase system [C. Bull and D. P. Ballou (1981)J. Biol. Chem.256, 12673–12680] to deoxygenate hemoglobin solutions enzymatically. The reaction is followed by simultaneous measurements of hemoglobin spectra using a diode array spectrophotometer and oxygen tensions using a polarographic O2microelectrode. Multicomponent analysis allows the determination

Kim D. Vandegriff; Ronald J. Rohlfs; Michael D. Magde; Robert M. Winslow



Enzymatic hydrolysis of cellulose and various pretreated wood fractions  

Microsoft Academic Search

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

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



Enzymatic activity of allergenic house dust and storage mite extracts.  


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

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



Enzymatic synthesis of neopentylpolyol esters in organic media  

Microsoft Academic Search

Utilization of lipases for synthesis of esters of hydrophilic polyols has been investigated. The choice of a suitable solvent\\u000a is crucial in this type of reaction. An interesting case is fatty acid esters from neopentylpolyols, such as trimethylolpropane,\\u000a which are of great interest as high temperature lubricants. Enzymatic synthesis of trimethylolpropane tricaprylate was studied\\u000a as an alternative to chemical manufacturing.

Frédéric Monot; Yves Benoit; Daniel Ballerini; Jean-Paul Vandecasteele



Composite aromatic boxes for enzymatic transformations of quaternary ammonium substrates.  


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

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



Sensing core histone phosphorylation — A matter of perfect timing?  

PubMed Central

Systematic analysis of histone modifications has revealed a plethora of posttranslational modifications that mediate changes in chromatin structure and gene expression. Histone phosphorylation is a transient histone modification that becomes induced by extracellular signals, DNA damage or entry into mitosis. Importantly, phosphorylation of histone proteins does lead not only to the binding of specific reader proteins but also to changes in the affinity for readers or writers of other histone modifications. This induces a cross-talk between different chromatin modifications that allows the spatio-temporal control of chromatin-associated events. In this review we will summarize the progress in our current knowledge of factors sensing reversible histone phosphorylation in different biological scenarios. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function. PMID:24747175

Sawicka, Anna; Seiser, Christian



Regulation of GABA Receptor Activity by Neurosteroids and Phosphorylation  

NSDL National Science Digital Library

These two animations show two models for how neurosteroids regulate the flow of chloride ions (Cl-) through ionotropic gamma-aminobutyric acid (GABA) receptors. In the first model, binding of the neurosteroid allows a protein kinase C (PKC) phosphorylation site to become accessible. Phosphorylation of the channel increases flux through the channel. In the second model, phosphorylation by PKC allows the neurosteroid to bind and increase flux through the channel. The animations have two parts: (i) a diagrammatic representation of the sequence of events at the channel in the membrane and (ii) a representative current trace of data obtained using electrophysiological techniques. These animations would be useful in teaching how allosteric modulators (neurosteroids) and covalent modulators (kinases) can work together as regulators of protein activity.

Jeffrey Tasker (Tulane University;Department of Cell and Molecular Biology REV)




PubMed Central

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

Solan, Joell L.; Lampe, Paul D.



Crystal Structure of a Phosphorylation-coupled Saccharide Transporter  

SciTech Connect

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.

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



Phosphorylation factors control neurotransmitter and neuromodulator actions at the gamma-aminobutyric acid type A receptor.  


Whole-cell and patch-voltage clamp experiments were carried out on cultured chick spinal cord neurons to investigate the dependence of gamma-aminobutyric acid (GABA)A receptor function on intracellular phosphorylation factors. Without ATP in the intracellular solution, repeated application of 30 microM GABA results in a progressive decline (run-down) of the currents evoked by GABA in standard whole-cell recordings but not when the nystatin-perforated patch method is used. Run-down is also observed in outside-out excised patch recordings, indicating that any enzymatic factors required for run-down must be closely associated with the plasma membrane. Run-down is associated with decreases in both the maximum GABA-induced current and the GABA EC50. Inclusion of magnesium adenosine-5'-O-(3-thio)triphosphate in the intracellular buffer prevents the decline in the maximum GABA response but the GABA EC50 still decreases, resulting in a "run-up" of the response at low (3 microM) GABA concentrations. Run-down is use dependent, requiring repeated activation of the GABAA receptor by high (30 microM) GABA concentrations. However, use-independent run-down can be induced by the inclusion of alkaline phosphatase in the intracellular buffer. The response to 3 microM GABA does not normally run down, but run-down is observed when the response to 3 microM GABA is potentiated with pentobarbital or allopregnanolone, suggesting that run-down is consequence of GABA receptor activation and/or desensitization. Run-down of the potentiated GABA response can be prevented by addition of magnesium adenosine-5'-O-(3-thio)triphosphate to the intracellular solution. Strikingly, run-down results in a significant decrease in the potentiating effects of positive modulators, whereas the inhibitory effects of negative modulators such as pregnenolone sulfate and ZnCl2 are unchanged. The results demonstrate that phosphorylation factors have the capacity to control GABAA receptor pharmacology, affecting the potency and efficacy of GABA, the kinetics of GABAA receptor desensitization, and the sensitivity of the receptor to modulators such as steroids, benzodiazepines, and barbiturates. PMID:7935336

Gyenes, M; Wang, Q; Gibbs, T T; Farb, D H



Creating Advanced Multifunctional Biosensors with Surface Enzymatic Transformations  

PubMed Central

This article summarizes our recent work on the coupling of surface enzyme chemistry and bioaffinity interactions on biopolymer microarrays for the creation of multiplexed biosensors with enhanced selectivity and sensitivity. The surface sensitive techniques of surface plasmon resonance imaging (SPRI) and surface plasmon fluorescence spectroscopy (SPFS) are used to detect the surface enzymatic transformations in real time. Three specific examples of novel coupled surface bioaffinity/surface enzymatic processes are demonstrated: (i) a surface enzymatic amplification method utilizing the enzyme ribonuclease H (RNase H) in conjunction with RNA microarrays that permits the ultrasensitive direct detection of genomic DNA at a concentration of 1 fM without labeling or PCR amplification, (ii) the use of RNA-DNA ligation chemistry to create renewable RNA microarrays from single stranded DNA microarrays, and (iii) the application of T7 RNA polymerase for the on-chip replication of RNA from double stranded DNA microarray elements. In addition, a simple yet powerful theoretical framework that includes the contributions of both enzyme adsorption and surface enzyme kinetics is used to quantitate surface enzyme reactivity. This model is successfully applied to SPRI and SPFS measurements of surface hydrolysis reactions of RNase H and Exonuclease III (Exo III) on oligonucleotide microarrays. PMID:16732647

Lee, Hye Jin; Wark, Alastair W.; Corn, Robert M.



A Model for Single-Substrate Trimolecular Enzymatic Kinetics  

PubMed Central

Abstract We developed a kinetic model for a single-substrate trimolecular enzymatic system, where a receptor binds and stretches a substrate to expose its cleavage site, allowing an enzyme to bind and cleave it into product. We demonstrated that the general kinetics of the trimolecular enzymatic system is more complex than the Michaelis-Menten kinetics. Under a limiting condition when the enzyme-substrate binding is in fast equilibrium, the enzymatic kinetics of the trimolecular system reduces to the Michaelis-Menten kinetics. In another limiting case when the receptor dissociates negligibly slowly from the substrate, the trimolecular system is simplified to a bimolecular system, which follows the Michaelis-Menten equation if and only if there is no enzyme-substrate complex initially. We applied this model to a particular trimolecular system important to hemostasis and thrombosis, consisting of von Willebrand factor (substrate), platelet glycoprotein Ib? (receptor), and ADAMTS13 (enzyme). Using parameters from independent experiments, our model successfully predicted published data from two single-molecule experiments and fitted/predicted published data from an ensemble experiment. PMID:20441760

Chen, Wei; Zhu, Cheng



Elucidation of Factors Effecting Enzymatic Saccharification using Transgenic Hardwoods  

NASA Astrophysics Data System (ADS)

Three groups of transgenic wood samples were used as starting materials to elucidate the recalcitrance of enzymatic saccharification with/without pretreatments. The first group of transgenic wood samples is low lignin P. trichocarpa. The second group is low xylan P. trichocarpa. The third one is 12 hybrid poplars which have different levels of S/V ratio and lignin content. Four pretreatments were carried out in this research including dilute sulfuric acid, green liquor, auto hydrolysis and ozone delignification. The behavior among pretreatments as a function of removal of lignin appears to be different. Lignin is the major factor of recalcitrance of the lignocellulosic material to ethanol conversion process. Xylan also plays key role in this process. In addition, the crude milled wood lignin was isolated from these three groups of transgenic samples. Lignin carbohydrate complexes was characterized by 1H-13C HMQC and 13C NMR. Thus the effect of LCCs on enzymatic saccharification was elucidated. High S/V ratio propels the lignin removal during pretreatments however; high S/V ratio retards the enzymatic saccharification on the lignocellulosic material without pretreatments. The level of LCCs linkages accounts for additional recalcitrance of the lignocellulosic material to ethanol conversion process. The amount of LCCs linkages is affected by xylan content, lignin content and S/V ratio.

Min, Douyong


Architecture and regulation of negative-strand viral enzymatic machinery  

PubMed Central

Negative-strand (NS) RNA viruses initiate infection with a unique polymerase complex that mediates both mRNA transcription and subsequent genomic RNA replication. For nearly all NS RNA viruses, distinct enzymatic domains catalyzing RNA polymerization and multiple steps of 5? mRNA cap formation are contained within a single large polymerase protein (L). While NS RNA viruses include a variety of emerging human and agricultural pathogens, the enzymatic machinery driving viral replication and gene expression remains poorly understood. Recent insights with Machupo virus and vesicular stomatitis virus have provided the first structural information of viral L proteins, and revealed how the various enzymatic domains are arranged into a conserved architecture shared by both segmented and nonsegmented NS RNA viruses. In vitro systems reconstituting RNA synthesis from purified components provide new tools to understand the viral replicative machinery, and demonstrate the arenavirus matrix protein regulates RNA synthesis by locking a polymerase–template complex. Inhibition of gene expression by the viral matrix protein is a distinctive feature also shared with influenza A virus and nonsegmented NS RNA viruses, possibly illuminating a conserved mechanism for coordination of viral transcription and polymerase packaging PMID:22767259

Kranzusch, Philip J.; Whelan, Sean P.J.



Bio-nanogate controlled enzymatic reaction for virus sensing.  


The objective of this study was to develop an aptamer-based bifunctional bio-nanogate, which could selectively respond to target molecules, and control enzymatic reaction for electrochemical measurements. It was successfully applied for sensitive, selective, rapid, quantitative, and label-free detection of avian influenza viruses (AIV) H5N1. A nanoporous gold film with pore size of ~20nm was prepared by a metallic corrosion method, and the purity was checked by energy-dispersive X-ray spectroscopy (EDS) study. To improve the performance of the bio-nanogate biosensor, its main analytical parameters were studied and optimized. We demonstrated that the developed bio-nanogate was capable of controlling enzymatic reaction for AIV H5N1 sensing within 1h with a detection limit of 2(-9)HAU (hemagglutination units). The enzymatic reaction was able to cause significant current change due to the presence of target AIV. A linear relationship was found in the virus titer range of 2(-10)-2(2)HAU. No interference was observed from non-target AIV subtypes such as H1N1, H2N2, H4N8 and H7N2. The developed approach could be adopted for sensing of other viruses. PMID:25212377

Wang, Ronghui; Xu, Lizhou; Li, Yanbin



Decreasing Tropomyosin Phosphorylation Rescues Tropomyosin-induced Familial Hypertrophic Cardiomyopathy*  

PubMed Central

Studies indicate that tropomyosin (Tm) phosphorylation status varies in different mouse models of cardiac disease. Investigation of basal and acute cardiac function utilizing a mouse model expressing an ?-Tm protein that cannot be phosphorylated (S283A) shows a compensated hypertrophic phenotype with significant increases in SERCA2a expression and phosphorylation of phospholamban Ser-16 (Schulz, E. M., Correll, R. N., Sheikh, H. N., Lofrano-Alves, M. S., Engel, P. L., Newman, G., Schultz Jel, J., Molkentin, J. D., Wolska, B. M., Solaro, R. J., and Wieczorek, D. F. (2012) J. Biol. Chem. 287, 44478–44489). With these results, we hypothesized that decreasing ?-Tm phosphorylation may be beneficial in the context of a chronic, intrinsic stressor. To test this hypothesis, we utilized the familial hypertrophic cardiomyopathy (FHC) ?-Tm E180G model (Prabhakar, R., Boivin, G. P., Grupp, I. L., Hoit, B., Arteaga, G., Solaro, R. J., and Wieczorek, D. F. (2001) J. Mol. Cell. Cardiol. 33, 1815–1828). These FHC hearts are characterized by increased heart:body weight ratios, fibrosis, increased myofilament Ca2+ sensitivity, and contractile defects. The FHC mice die by 6–8 months of age. We generated mice expressing both the E180G and S283A mutations and found that the hypertrophic phenotype was rescued in the ?-Tm E180G/S283A double mutant transgenic animals; these mice exhibited no signs of cardiac hypertrophy and displayed improved cardiac function. These double mutant transgenic hearts showed increased phosphorylation of phospholamban Ser-16 and Thr-17 compared with the ?-Tm E180G mice. This is the first study to demonstrate that decreasing phosphorylation of tropomyosin can rescue a hypertrophic cardiomyopathic phenotype. PMID:23960072

Schulz, Emily M.; Wilder, Tanganyika; Chowdhury, Shamim A. K.; Sheikh, Hajer N.; Wolska, Beata M.; Solaro, R. John; Wieczorek, David F.



Liver mTOR controls IGF-I bioavailability by regulation of protein kinase CK2 and IGFBP-1 phosphorylation in fetal growth restriction.  


Fetal growth restriction (FGR) increases the risk for perinatal complications and predisposes the infant to diabetes and cardiovascular disease later in life. No treatment for FGR is available, and the underlying pathophysiology remains poorly understood. Increased IGFBP-1 phosphorylation has been implicated as an important mechanism by which fetal growth is reduced. However, to what extent circulating IGFBP-1 is phosphorylated in FGR is unknown, and the molecular mechanisms linking FGR to IGFBP-1 phosphorylation have not been established. We used umbilical cord plasma of appropriate for gestational age (AGA) and growth-restricted human fetuses and determined IGFBP-1 and IGF-I concentrations (ELISA) and site-specific IGFBP-1 phosphorylation (Western blotting using IGFBP-1 phospho-site specific antibodies). In addition, we used a baboon model of FGR produced by 30% maternal nutrient restriction and determined mammalian target of rapamycin (mTOR)C1 activity, CK2 expression/activity, IGFBP-1 expression and phosphorylation, and IGF-I levels in baboon fetal liver by Western blot, enzymatic assay, and ELISA. HepG2 cells and primary fetal baboon hepatocytes were used to explore mechanistic links between mTORC1 signaling and IGFBP-1 phosphorylation. IGFBP-1 was hyperphosphorylated at Ser101, Ser119, and Ser169 in umbilical plasma of human FGR fetuses. IGFBP-1 was also hyperphosphorylated at Ser101, Ser119, and Ser169 in the liver of growth-restricted baboon fetus. mTOR signaling was markedly inhibited, whereas expression and activity of CK2 was increased in growth-restricted baboon fetal liver in vivo. Using HepG2 cells and primary fetal baboon hepatocytes, we established a mechanistic link between mTOR inhibition, CK2 activation, IGFBP-1 hyperphosphorylation, and decreased IGF-I-induced IGF-I receptor autophosphorylation. We provide clear evidence for IGFBP-1 hyperphosphorylation in FGR and identified an mTOR and CK2-mediated mechanism for regulation of IGF-I bioavailability. Our findings are consistent with the model that inhibition of mTOR in the fetal liver, resulting in increased CK2 activity and IGFBP-1 hyperphosphorylation, constitutes a novel mechanistic link between nutrient deprivation and restricted fetal growth. PMID:24437487

Abu Shehab, Majida; Damerill, Ian; Shen, Tong; Rosario, Fredrick J; Nijland, Mark; Nathanielsz, Peter W; Kamat, Amrita; Jansson, Thomas; Gupta, Madhulika B



Bioinformatics study of cancer-related mutations within p53 phosphorylation site motifs.  


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

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



Atg1 kinase organizes autophagosome formation by phosphorylating Atg9.  


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

Papinski, Daniel; Kraft, Claudine



Phosphoryl guanidines: a new type of nucleic Acid analogues.  


A new type of nucleic acid analogues with a phosphoryl guanidine group is described. Oxidation of polymer-supported dinucleoside 2-cyanoethyl phosphite by iodine in the presence of 1,1,3,3-tetramethyl guanidine yields a dinucleotide with an internucleoside tetramethyl phosphoryl guanidine (Tmg) group as the main product. The Tmg group is stable under conditions of solid-phase DNA synthesis and subsequent cleavage and deprotection with ammonia. Oligonucleotides with one or more Tmg groups bind their complementary DNA or RNA with affinity similar to that of natural oligodeoxyribonucleotides. PMID:25558402

Kupryushkin, M S; Pyshnyi, D V; Stetsenko, D A



Phosphoryl Guanidines: A New Type of Nucleic Acid Analogues  

PubMed Central

A new type of nucleic acid analogues with a phosphoryl guanidine group is described. Oxidation of polymer-supported dinucleoside 2-cyanoethyl phosphite by iodine in the presence of 1,1,3,3-tetramethyl guanidine yields a dinucleotide with an internucleoside tetramethyl phosphoryl guanidine (Tmg) group as the main product. The Tmg group is stable under conditions of solid-phase DNA synthesis and subsequent cleavage and deprotection with ammonia. Oligonucleotides with one or more Tmg groups bind their complementary DNA or RNA with affinity similar to that of natural oligodeoxyribonucleotides. PMID:25558402

Kupryushkin, M. S.; Pyshnyi, D. V.; Stetsenko, D. A.



The role of hydrogen bonding in the enzymatic reaction catalyzed by HIV-1 protease  

PubMed Central

The hydrogen-bond network in various stages of the enzymatic reaction catalyzed by HIV-1 protease was studied through quantum-classical molecular dynamics simulations. The approximate valence bond method was applied to the active site atoms participating directly in the rearrangement of chemical bonds. The rest of the protein with explicit solvent was treated with a classical molecular mechanics model. Two possible mechanisms were studied, general-acid/general-base (GA/GB) with Asp 25 protonated at the inner oxygen, and a direct nucleophilic attack by Asp 25. Strong hydrogen bonds leading to spontaneous proton transfers were observed in both reaction paths. A single-well hydrogen bond was formed between the peptide nitrogen and outer oxygen of Asp 125. The proton was diffusely distributed with an average central position and transferred back and forth on a picosecond scale. In both mechanisms, this interaction helped change the peptide-bond hybridization, increased the partial charge on peptidyl carbon, and in the GA/GB mechanism, helped deprotonate the water molecule. The inner oxygens of the aspartic dyad formed a low-barrier, but asymmetric hydrogen bond; the proton was not positioned midway and made a slightly elongated covalent bond, transferring from one to the other aspartate. In the GA/GB mechanism both aspartates may help deprotonate the water molecule. We observed the breakage of the peptide bond and found that the protonation of the peptidyl amine group was essential for the peptide-bond cleavage. In studies of the direct nucleophilic mechanism, the peptide carbon of the substrate and oxygen of Asp 25 approached as close as 2.3 ?. PMID:14739332

Trylska, Joanna; Grochowski, Pawe?; McCammon, J. Andrew



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

SciTech Connect

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.

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



Encephalomyocarditis Virus Leader Is Phosphorylated by CK2 and Syk as a Requirement for Subsequent Phosphorylation of Cellular Nucleoporins  

PubMed Central

ABSTRACT Encephalomyocarditis virus and Theilovirus are species in the Cardiovirus genus of the Picornaviridae family. For all cardioviruses, the viral polyprotein is initiated with a short Leader (L) protein unique to this genus. The nuclear magnetic resonance (NMR) structure of LE from encephalomyocarditis virus (EMCV) has been determined. The protein has an NH2-proximal CHCC zinc finger, a central linker, and a contiguous, highly acidic motif. The theiloviruses encode the same domains, with one or two additional, COOH-proximal domains, characteristic of the human Saffold viruses (SafV) and Theiler's murine encephalomyelitis viruses (TMEV), respectively. The expression of a cardiovirus L, in recombinant form, or during infection/transfection, triggers an extensive, cell-dependent, antihost phosphorylation cascade, targeting nucleoporins (Nups) that form the hydrophobic core of nuclear pore complexes (NPC). The consequent inhibition of active nucleocytoplasmic trafficking is potent and prevents the host from mounting an effective antiviral response. For this inhibition, the L proteins themselves must be phosphorylated. In cells (extracts or recombinant form), LE was shown to be phosphorylated at Thr47 and Tyr41. The first reaction (Thr47), catalyzed by casein kinase 2 (CK2), is an obligatory precedent to the second event (Tyr41), catalyzed by spleen tyrosine kinase (Syk). Site mutations in LE, or kinase-specific inhibitors, prevented LE phosphorylation and subsequent Nup phosphorylation. Parallel experiments with LS (SafV-2) and LT (TMEV BeAn) proteins confirmed the general cardiovirus requirement for L phosphorylation, but CK2 was not the culpable kinase. It is likely that LS and LT are both activated by alternative kinases in different cell types, probably reactive within the Theilo-specific domains. IMPORTANCE PMID:24335301

Basta, Holly A.; Bacot-Davis, Valjean R.; Ciomperlik, Jessica J.



Ob/ob Mouse Livers Show Decreased Oxidative Phosphorylation Efficiencies and Anaerobic Capacities after Cold Ischemia  

PubMed Central

Background Hepatic steatosis is a major risk factor for graft failure in liver transplantation. Hepatic steatosis shows a greater negative influence on graft function following prolonged cold ischaemia. As the impact of steatosis on hepatocyte metabolism during extended cold ischaemia is not well-described, we compared markers of metabolic capacity and mitochondrial function in steatotic and lean livers following clinically relevant durations of cold preservation. Methods Livers from 10-week old leptin-deficient obese (ob/ob, n?=?9) and lean C57 mice (n?=?9) were preserved in ice-cold University of Wisconsin solution. Liver mitochondrial function was then assessed using high resolution respirometry after 1.5, 3, 5, 8, 12, 16 and 24 hours of storage. Metabolic marker enzymes for anaerobiosis and mitochondrial mass were also measured in conjunction with non-bicarbonate tissue pH buffering capacity. Results Ob/ob and lean mice livers showed severe (>60%) macrovesicular and mild (<30%) microvesicular steatosis on Oil Red O staining, respectively. Ob/ob livers had lower baseline enzymatic complex I activity but similar adenosine triphosphate (ATP) levels compared to lean livers. During cold storage, the respiratory control ratio and complex I-fueled phosphorylation deteriorated approximately twice as fast in ob/ob livers compared to lean livers. Ob/ob livers also demonstrated decreased ATP production capacities at all time-points analyzed compared to lean livers. Ob/ob liver baseline lactate dehydrogenase activities and intrinsic non-bicarbonate buffering capacities were depressed by 60% and 40%, respectively compared to lean livers. Conclusions Steatotic livers have impaired baseline aerobic and anaerobic capacities compared to lean livers, and mitochondrial function indices decrease particularly from after 5 hours of cold preservation. These data provide a mechanistic basis for the clinical recommendation of shorter cold storage durations in steatotic donor livers. PMID:24956382

Tagaloa, Sherry; Zhang, Linda; Dare, Anna J.; MacDonald, Julia R.; Yeong, Mee-Ling; Bartlett, Adam S. J. R.; Phillips, Anthony R. J.



In vivo gene transfer via intravenous administration of cationic lipid–protamine–DNA (LPD) complexes  

Microsoft Academic Search

A novel LPD formulation has been developed for in vivo gene transfer. It involves the interaction of plasmid DNA with protamine sulfate, a cationic polypeptide, followed by the addition of DOTAP cationic liposomes. Compared with DOTAP\\/DNA complexes, LPD offers better protection of plasmid DNA against enzymatic digestion and gives consistently higher gene expression in mice via tail vein injection. When

S Li; L Huang



Photo-Induced Electron Transfer Between Photosystem 2 via Cross-linked Redox Hydrogels  

E-print Network

Full Paper Photo-Induced Electron Transfer Between Photosystem 2 via Cross-linked Redox Hydrogels was wired to electrode surfaces via osmium-containing redox polymers based on poly(vinyl)imidazol. The redox illumination, the enzymatic reaction could be switched on and a catalytic current was observed at the electrode

Roegner, Matthias


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

PubMed Central

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

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



CGGBP1 phosphorylation constitutes a telomere-protection signal  

PubMed Central

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

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



Quantitation, networking, and function of protein phosphorylation in plant cell.  


Protein phosphorylation is one of the most important post-translational modifications (PTMs) as it participates in regulating various cellular processes and biological functions. It is therefore crucial to identify phosphorylated proteins to construct a phosphor-relay network, and eventually to understand the underlying molecular regulatory mechanism in response to both internal and external stimuli. The changes in phosphorylation status at these novel phosphosites can be accurately measured using a (15)N-stable isotopic labeling in Arabidopsis (SILIA) quantitative proteomic approach in a high-throughput manner. One of the unique characteristics of the SILIA quantitative phosphoproteomic approach is the preservation of native PTM status on protein during the entire peptide preparation procedure. Evolved from SILIA is another quantitative PTM proteomic approach, AQUIP (absolute quantitation of isoforms of post-translationally modified proteins), which was developed by combining the advantages of targeted proteomics with SILIA. Bioinformatics-based phosphorylation site prediction coupled with an MS-based in vitro kinase assay is an additional way to extend the capability of phosphosite identification from the total cellular protein. The combined use of SILIA and AQUIP provides a novel strategy for molecular systems biological study and for investigation of in vivo biological functions of these phosphoprotein isoforms and combinatorial codes of PTMs. PMID:23316209

Zhu, Lin; Li, Ning



Structural determination of the phosphorylation domain of the ryanodine receptor  

E-print Network

Cancer Research Institute, Ontario Cancer Institute, Princess Margaret Cancer Center, University Health; malignant hyperthermia; phosphorylation site; RyR; X-ray crystallography Correspondence A. Gramolini or S mutations in RyR1 are associated with severe conditions including malignant hyperthermia (MH) and central

Ikura, Mitsuhiko


Coregulation of Ion Channels by Neurosteroids and Phosphorylation  

NSDL National Science Digital Library

This Perspective by Tasker is one of two discussing the regulation of ion channels by neurosteroids. Neurosteroids are steroids synthesized in the brain that act as allosteric modulators of ion-channel activity. The dual regulation of γ-aminobutyric acid type A (GABAA) receptors and voltage-gated calcium channels by phosphorylation and neurosteroids is the focus of this Perspective.

Jeffrey Tasker (Tulane University;Department of Cell and Molecular Biology REV)



Regulation of mitochondrial oxidative phosphorylation through cell signaling  

Microsoft Academic Search

The mitochondrial oxidative phosphorylation (OxPhos) system plays a key role in energy production, the generation of free radicals, and apoptosis. A lack of cellular energy, excessive radical production, and dysregulated apoptosis are found alone or in combination in most human diseases, including neurodegenerative diseases, stroke, cardiovascular disorders, ischemia\\/reperfusion, and cancer. In the context of its relevance to human disease, this

Maik Hüttemann; Icksoo Lee; Lobelia Samavati; Hong Yu; Jeffrey W. Doan



Tyrosine phosphorylation allows integration of multiple signaling inputs by IKK?.  


Signaling regulated by NF?B and related transcription factors is centrally important to many inflammatory and autoimmune diseases, cancer, and stress responses. The kinase that directly regulates the canonical NF?B transcriptional pathway, Inhibitor of ?B kinase ? (IKK?), undergoes activation by Ser phosphorylation mediated by NIK or TAK1 in response to inflammatory signals. Using titanium dioxide-based phosphopeptide enrichment (TiO2)-liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS), we analyzed IKK? phosphorylation in human HEK293 cells expressing IKK? and FGFR2, a Receptor tyrosine kinase (RTK) essential for embryonic differentiation and dysregulated in several cancers. We attained unusually high coverage of IKK?, identifying an abundant site of Tyr phosphorylation at Tyr169 within the Activation Loop. The phosphomimic at this site confers a level of kinase activation and NF?B nuclear localization exceeding the iconic mutant S177E/S181E, demonstrating that RTK-mediated Tyr phosphorylation of IKK? has the potential to directly regulate NF?B transcriptional activation. PMID:24386391

Meyer, April N; Drafahl, Kristine A;