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Sample records for accelerate gtp hydrolysis

  1. Noncanonical Myo9b-RhoGAP Accelerates RhoA GTP Hydrolysis by a Dual-Arginine-Finger Mechanism.

    PubMed

    Yi, Fengshuang; Kong, Ruirui; Ren, Jinqi; Zhu, Li; Lou, Jizhong; Wu, Jane Y; Feng, Wei

    2016-07-31

    The GTP hydrolysis activities of Rho GTPases are stimulated by GTPase-activating proteins (GAPs), which contain a RhoGAP domain equipped with a characteristic arginine finger and an auxiliary asparagine for catalysis. However, the auxiliary asparagine is missing in the RhoGAP domain of Myo9b (Myo9b-RhoGAP), a unique motorized RhoGAP that specifically targets RhoA for controlling cell motility. Here, we determined the structure of Myo9b-RhoGAP in complex with GDP-bound RhoA and magnesium fluoride. Unexpectedly, Myo9b-RhoGAP contains two arginine fingers at its catalytic site. The first arginine finger resembles the one within the canonical RhoGAP domains and inserts into the nucleotide-binding pocket of RhoA, whereas the second arginine finger anchors the Switch I loop of RhoA and interacts with the nucleotide, stabilizing the transition state of GTP hydrolysis and compensating for the lack of the asparagine. Mutating either of the two arginine fingers impaired the catalytic activity of Myo9b-RhoGAP and affected the Myo9b-mediated cell migration. Our data indicate that Myo9b-RhoGAP accelerates RhoA GTP hydrolysis by a previously unknown dual-arginine-finger mechanism, which may be shared by other noncanonical RhoGAP domains lacking the auxiliary asparagine. PMID:27363609

  2. Modeling the mechanisms of biological GTP hydrolysis.

    PubMed

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

    2015-09-15

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

  3. Exploring potassium-dependent GTP hydrolysis in TEES family GTPases.

    PubMed

    Rafay, Abu; Majumdar, Soneya; Prakash, Balaji

    2012-01-01

    GTPases are important regulatory proteins that hydrolyze GTP to GDP. A novel GTP-hydrolysis mechanism is employed by MnmE, YqeH and FeoB, where a potassium ion plays a role analogous to the Arginine finger of the Ras-RasGAP system, to accelerate otherwise slow GTP hydrolysis rates. In these proteins, two conserved asparagines and a 'K-loop' present in switch-I, were suggested as attributes of GTPases employing a K(+)-mediated mechanism. Based on their conservation, a similar mechanism was suggested for TEES family GTPases. Recently, in Dynamin, Fzo1 and RbgA, which also conserve these attributes, a similar mechanism was shown to be operative. Here, we probe K(+)-activated GTP hydrolysis in TEES (TrmE-Era-EngA-YihA-Septin) GTPases - Era, EngB and the two contiguous G-domains, GD1 and GD2 of YphC (EngA homologue) - and also in HflX, another GTPase that also conserves the same attributes. While GD1-YphC and Era exhibit a K(+)-mediated activation of GTP hydrolysis, surprisingly GD2-YphC, EngB and HflX do not. Therefore, the attributes identified thus far, do not necessarily predict a K(+)-mechanism in GTPases and hence warrant extensive structural investigations. PMID:23650596

  4. Direct incorporation of GDP into microtubules without GTP hydrolysis

    SciTech Connect

    Lin, C.M.; Hamel, E.

    1987-05-01

    Tubulin bearing (8-/sup 14/C)GDP in the exchangeable nucleotide binding site was prepared, and its polymerization was examined with microtubule-associated proteins containing minimal nucleoside diphosphate kinase and nonspecific phosphatase contamination. Although microtubule assembly required GTP, significant incorporation of tubulin-bound GDP into microtubules without exchange of the radiolabeled GDP for GTP was observed under reaction conditions which favored an increased proportion of tubulin x GDP relative to tubulin x GTP. These were low GTP concentrations, low Mg/sup 2 +/ concentrations, high tubulin concentrations, and exogenous GDP in the reaction mixture. The minimum tubulin x GTP:tubulin x GDP ratio required for microtubule assembly was determined to establish the relative importance of the two tubulin species in the initiation of assembly and was found to be about 2:1. In addition, the relative efficiency with which tubulin x GTP and tubulin x GDP were incorporated into microtubules was determined. They found that tubulin x GDP was incorporated into polymer about half as efficiently as tubulin x GTP.

  5. Light- and GTP-activated hydrolysis of phosphatidylinositol bisphosphate in squid photoreceptor membranes

    SciTech Connect

    Baer, K.M.; Saibil, H.R.

    1988-01-05

    Light stimulates the hydrolysis of exogenous, (/sup 3/H)inositol-labeled phosphatidylinositol bisphosphate (PtdInsP2) added to squid photoreceptor membranes, releasing inositol trisphosphate (InsP3). At free calcium levels of 0.05 microM or greater, hydrolysis of the labeled lipid is stimulated up to 4-fold by GTP and light together, but not separately. This activity is the biochemical counterpart of observations on intact retina showing that a rhodopsin-activated GTP-binding protein is involved in visual transduction in invertebrates, and that InsP3 release is correlated with visual excitation and adaptation. Using an in vitro assay, we investigated the calcium and GTP dependence of the phospholipase activity. At calcium concentrations between 0.1 and 0.5 microM, some hydrolysis occurs independently of GTP and light, with a light- and GTP-activated component superimposed. At 1 microM calcium there is no background activity, and hydrolysis absolutely requires both GTP and light. Ion exchange chromatography on Dowex 1 (formate form) of the water-soluble products released at 1 microM calcium reveals that the product is almost entirely InsP3. Invertebrate rhodopsin is homologous in sequence and function to vertebrate visual pigment, which modulates the concentration of cyclic GMP through the mediation of the GTP-binding protein transducin. While there is some evidence that light also modulates PtdInsP2 content in vertebrate photoreceptors, the case for its involvement in phototransduction is stronger for the invertebrate systems. The results reported here support the scheme of rhodopsin----GTP-binding protein----phospholipase C activation in invertebrate photoreceptors.

  6. Characterization of GTP binding and hydrolysis in plasma membranes of zucchini

    NASA Technical Reports Server (NTRS)

    Perdue, D. O.; Lomax, T. L.

    1992-01-01

    We have investigated the possibility that G-protein-like entities may be present in the plasma membrane (PM) of zucchini (Cucurbita pepo L.) hypocotyls by examining a number of criteria common to animal and yeast G-proteins. The GTP binding and hydrolysis characteristics of purified zucchini PM are similar to the characteristics of a number of known G-proteins. Our results demonstrate GTP binding to a single PM site having a Kd value between 16-31 nM. This binding has a high specificity for guanine nucleotides, and is stimulated by Mg2+, detergents, and fluoride or aluminium ions. The GTPase activity (Km = 0.49 micromole) of zucchini PM shows a sensitivity to NaF similar to that seen for other G-proteins. Localization of GTP mu 35S binding to nitrocellulose blots of proteins separated by SDS-PAGE indicates a 30-kDa protein as the predominant GTP-binding species in zucchini PM. Taken together, these data indicate that plant PM contains proteins which are biochemically similar to previously characterized G-proteins.

  7. Activation of GTP hydrolysis in mRNA-tRNA translocation by elongation factor G

    PubMed Central

    Li, Wen; Liu, Zheng; Koripella, Ravi Kiran; Langlois, Robert; Sanyal, Suparna; Frank, Joachim

    2015-01-01

    During protein synthesis, elongation of the polypeptide chain by each amino acid is followed by a translocation step in which mRNA and transfer RNA (tRNA) are advanced by one codon. This crucial step is catalyzed by elongation factor G (EF-G), a guanosine triphosphatase (GTPase), and accompanied by a rotation between the two ribosomal subunits. A mutant of EF-G, H91A, renders the factor impaired in guanosine triphosphate (GTP) hydrolysis and thereby stabilizes it on the ribosome. We use cryogenic electron microscopy (cryo-EM) at near-atomic resolution to investigate two complexes formed by EF-G H91A in its GTP state with the ribosome, distinguished by the presence or absence of the intersubunit rotation. Comparison of these two structures argues in favor of a direct role of the conserved histidine in the switch II loop of EF-G in GTPase activation, and explains why GTP hydrolysis cannot proceed with EF-G bound to the unrotated form of the ribosome. PMID:26229983

  8. Protein export from the nucleus requires the GTPase Ran and GTP hydrolysis.

    PubMed Central

    Moroianu, J; Blobel, G

    1995-01-01

    Nuclei of digitonin-permeabilized cells that had been preloaded with a model transport substrate in a cytosol-dependent import reaction were subsequently incubated to investigate which conditions would result in export of transport substrate. We found that up to 80% of the imported substrate was exported when recombinant human Ran and GTP were present in the export reaction. Ran-mediated export was inhibited by nonhydrolyzable GTP analogs and also by wheat germ agglutinin but was unaffected by a nonhydrolyzable ATP analog. Moreover, a recombinant human Ran mutant that was deficient in its GTPase activity inhibited export. These data indicate that export of proteins from the nucleus requires Ran and GTP hydrolysis but not ATP hydrolysis. We also found that digitonin-permeabilized cells were depleted of their endogenous nuclear Ran, thus allowing detection of Ran as a limiting factor for export. In contrast, most endogenous karyopherin alpha was retained in nuclei of digitonin-permeabilized cells. Unexpectedly, exogenously added, fluorescently labeled Ran, although it accessed the nuclear interior, was found to dock at the nuclear rim in a punctate pattern, suggesting the existence of Ran-binding sites at the nuclear pore complex. Images Fig. 3 Fig. 6 Fig. 7 PMID:7753805

  9. The Role of Gln61 in HRas GTP Hydrolysis: A Quantum Mechanics/Molecular Mechanics Study

    PubMed Central

    Martín-García, Fernando; Mendieta-Moreno, Jesús Ignacio; López-Viñas, Eduardo; Gómez-Puertas, Paulino; Mendieta, Jesús

    2012-01-01

    Activation of the water molecule involved in GTP hydrolysis within the HRas⋅RasGAP system is analyzed using a tailored approach based on hybrid quantum mechanics/molecular mechanics (QM/MM) simulation. A new path emerges: transfer of a proton from the attacking water molecule to a second water molecule, then a different proton is transferred from this second water molecule to the GTP. Gln61 will stabilize the transient OH− and H3O+ molecules thus generated. This newly proposed mechanism was generated by using, for the first time to our knowledge, the entire HRas-RasGAP protein complex in a QM/MM simulation context. It also offers a rational explanation for previous experimental results regarding the decrease of GTPase rate found in the HRas Q61A mutant and the increase exhibited by the HRas Q61E mutant. PMID:22225809

  10. Thermodynamic and structural analysis of microtubule assembly: the role of GTP hydrolysis.

    PubMed

    Vulevic, B; Correia, J J

    1997-03-01

    Different models have been proposed that link the tubulin heterodimer nucleotide content and the role of GTP hydrolysis with microtubule assembly and dynamics. Here we compare the thermodynamics of microtubule assembly as a function of nucleotide content by van't Hoff analysis. The thermodynamic parameters of tubulin assembly in 30-100 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM MgSO4, 2 mM EGTA, pH 6.9, in the presence of a weakly hydrolyzable analog, GMPCPP, the dinucleotide analog GMPCP plus 2 M glycerol, and GTP plus 2 M glycerol were obtained together with data for taxol-GTP/GDP tubulin assembly (GMPCPP and GMPCP are the GTP and GDP nucleotide analogs where the alpha beta oxygen has been replaced by a methylene, -CH2-). All of the processes studied are characterized by a positive enthalpy, a positive entropy, and a large, negative heat capacity change. GMPCP-induced assembly has the largest negative heat capacity change and GMPCPP has the second largest, whereas GTP/2 M glycerol- and taxol-induced assembly have more positive values, respectively. A large, negative heat capacity is most consistent with the burial of water-accessible hydrophobic surface area, which gives rise to the release of bound water. The heat capacity changes observed with GTP/2 M glycerol-induced and with taxol-induced assembly are very similar, -790 +/- 190 cal/mol/k, and correspond to the burial of 3330 +/- 820 A2 of nonpolar surface area. This value is shown to be very similar to an estimate of the buried nonpolar surface in a reconstructed microtubule lattice. Polymerization data from GMPCP- and GMPCPP-induced assembly are consistent with buried nonpolar surface areas that are 3 and 6 times larger. A linear enthalpy-entropy and enthalpy-free energy plot for tubulin polymerization reactions verifies that enthalpy-entropy compensation for this system is based upon true biochemical correlation, most likely corresponding to a dominant hydrophobic effect. Entropy analysis suggests

  11. Ras-catalyzed hydrolysis of GTP: a new perspective from model studies.

    PubMed Central

    Maegley, K A; Admiraal, S J; Herschlag, D

    1996-01-01

    Despite the biological and medical importance of signal transduction via Ras proteins and despite considerable kinetic and structural studies of wild-type and mutant Ras proteins, the mechanism of Ras-catalyzed GTP hydrolysis remains controversial. We take a different approach to this problem: the uncatalyzed hydrolysis of GTP is analyzed, and the understanding derived is applied to the Ras-catalyzed reaction. Evaluation of previous mechanistic proposals from this chemical perspective suggests that proton abstraction from the attacking water by a general base and stabilization of charge development on the gamma-phosphoryl oxygen atoms would not be catalytic. Rather, this analysis focuses attention on the GDP leaving group, including the beta-gamma bridge oxygen of GTP, the atom that undergoes the largest change in charge in going from the ground state to the transition state. This leads to a new catalytic proposal in which a hydrogen bond from the backbone amide of Gly-13 to this bridge oxygen is strengthened in the transition state relative to the ground state, within an active site that provides a template complementary to the transition state. Strengthened transition state interactions of the active site lysine, Lys-16, with the beta-nonbridging phosphoryl oxygens and a network of interactions that positions the nucleophilic water molecule and gamma-phosphoryl group with respect to one another may also contribute to catalysis. It is speculated that a significant fraction of the GAP-activated GTPase activity of Ras arises from an additional interaction of the beta-gamma bridge oxygen with an Arg side chain that is provided in trans by GAP. The conclusions for Ras and related G proteins are expected to apply more widely to other enzymes that catalyze phosphoryl (-PO(3)2-) transfer, including kinases and phosphatases. Images Fig. 1 Fig. 2 Fig. 3 PMID:8710841

  12. Exceptionally large entropy contributions enable the high rates of GTP hydrolysis on the ribosome

    PubMed Central

    Åqvist, Johan; Kamerlin, Shina C.L.

    2015-01-01

    Protein synthesis on the ribosome involves hydrolysis of GTP in several key steps of the mRNA translation cycle. These steps are catalyzed by the translational GTPases of which elongation factor Tu (EF-Tu) is the fastest GTPase known. Here, we use extensive computer simulations to explore the origin of its remarkably high catalytic rate on the ribosome and show that it is made possible by a very large positive activation entropy. This entropy term (TΔS‡) amounts to more than 7 kcal/mol at 25 °C. It is further found to be characteristic of the reaction mechanism utilized by the translational, but not other, GTPases and it enables these enzymes to attain hydrolysis rates exceeding 500 s−1. This entropy driven mechanism likely reflects the very high selection pressure on the speed of protein synthesis, which drives the rate of each individual GTPase towards maximal turnover rate of the whole translation cycle. PMID:26497916

  13. Exceptionally large entropy contributions enable the high rates of GTP hydrolysis on the ribosome.

    PubMed

    Åqvist, Johan; Kamerlin, Shina C L

    2015-10-26

    Protein synthesis on the ribosome involves hydrolysis of GTP in several key steps of the mRNA translation cycle. These steps are catalyzed by the translational GTPases of which elongation factor Tu (EF-Tu) is the fastest GTPase known. Here, we use extensive computer simulations to explore the origin of its remarkably high catalytic rate on the ribosome and show that it is made possible by a very large positive activation entropy. This entropy term (TΔS(‡)) amounts to more than 7 kcal/mol at 25 °C. It is further found to be characteristic of the reaction mechanism utilized by the translational, but not other, GTPases and it enables these enzymes to attain hydrolysis rates exceeding 500 s(-1). This entropy driven mechanism likely reflects the very high selection pressure on the speed of protein synthesis, which drives the rate of each individual GTPase towards maximal turnover rate of the whole translation cycle.

  14. GTP-dependent hydrolysis of phosphatidylinositol-4,5-bisphosphate by a soluble phospholipase C from adult human epidermis

    SciTech Connect

    Fisher, G.J.; Baldassare, J.J.; Voorhees, J.J.

    1987-05-01

    The effects of tumor promoting phorbol esters, which activate protein kinase (C (PK-C), on epidermis suggest that PK-C is important in the regulation of epidermal growth and differentiation. Since in vivo PK-C is activated by the products of phospholipase C (PL-C)-catalyzed hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP/sub 2/), they have investigated the properties of this reaction. Soluble PL-C from adult human epidermis was incubated with sonicated lipid vesicles containing (/sup 3/H-inositol)PIP/sub 2/, for 10 minutes at 37/sup 0/C. Water soluble reaction products were extracted, chromatographed and quantitated. In the presence of physiological concentrations of Ca/sup + +/ magnesium and GTP PIP/sub 2/, but not phosphatidylinositol, was hydrolyzed by PL-C (11.7 nmol/min/mg). Addition of GTP or GTP..gamma..S stimulated activity greater than 15 fold. Half maximal and maximal activity were observed at 10 ..mu..M and 100 ..mu..M GTP..gamma..S, respectively. ATP was unable to substitute for GTP, and GDP/S inhibited PIP/sub 2/ hydrolysis in a dose dependent manner. Activity was sensitive to pH, and exhibited a sharp optimum at pH 6.5. In addition, the PL-C preparation specifically bound (/sup 35/S)GTP S. These data demonstrate that adult human epidermis contains PL-C activity that specifically hydrolyzes PIP/sub 2/ and suggest the involvement of a GTP-binding regulatory protein in this reaction.

  15. Hydrolysis of Guanosine Triphosphate (GTP) by the Ras·GAP Protein Complex: Reaction Mechanism and Kinetic Scheme.

    PubMed

    Khrenova, Maria G; Grigorenko, Bella L; Kolomeisky, Anatoly B; Nemukhin, Alexander V

    2015-10-01

    Molecular mechanisms of the hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (Pi) by the Ras·GAP protein complex are fully investigated by using modern modeling tools. The previously hypothesized stages of the cleavage of the phosphorus-oxygen bond in GTP and the formation of the imide form of catalytic Gln61 from Ras upon creation of Pi are confirmed by using the higher-level quantum-based calculations. The steps of the enzyme regeneration are modeled for the first time, providing a comprehensive description of the catalytic cycle. It is found that for the reaction Ras·GAP·GTP·H2O → Ras·GAP·GDP·Pi, the highest barriers correspond to the process of regeneration of the active site but not to the process of substrate cleavage. The specific shape of the energy profile is responsible for an interesting kinetic mechanism of the GTP hydrolysis. The analysis of the process using the first-passage approach and consideration of kinetic equations suggest that the overall reaction rate is a result of the balance between relatively fast transitions and low probability of states from which these transitions are taking place. Our theoretical predictions are in excellent agreement with available experimental observations on GTP hydrolysis rates.

  16. Mechanism of muscarinic receptor-induced K+ channel activation as revealed by hydrolysis-resistant GTP analogues

    PubMed Central

    1988-01-01

    The role of a guanine nucleotide-binding protein (Gk) in the coupling between muscarinic receptor activation and opening of an inwardly rectifying K+ channel [IK(M)] was examined in cardiac atrial myocytes, using hydrolysis-resistant GTP analogues. In the absence of muscarinic agonist, GTP analogues produced a membrane current characteristic of IK(M). The initial rate of appearance of this receptor-independent IK(M) was measured for the various analogues in order to explore the kinetic properties of IK(M) activation. We found that IK(M) activation is controlled solely by the intracellular analogue/GTP ratio and not by the absolute concentrations of the nucleotides. Analogues competed with GTP for binding to Gk with the following relative affinities: GTP gamma S greater than GTP greater than GppNHp greater than GppCH2p. At sufficiently high intracellular concentrations, however, all GTP analogues produced the same rate of IK(M) activation. This analogue- independent limiting rate is likely to correspond to the rate of GDP release from inactive, GDP-bound Gk. Muscarinic receptor stimulation by nanomolar concentrations of acetylcholine (ACh), which do not elicit IK(M) under control conditions, catalyzed IK(M) activation in the presence of GTP analogues. The rate of Gk activation by ACh (kACh) was found to be described by the simple relationship kACh = 8.4 X 10(8) min- 1 M-1.[ACh] + 0.44 min-1, the first term of which presumably reflects the agonist-catalyzed rate of GDP release from the Gk.GDP complex, while the second term corresponds to the basal rate of receptor- independent GDP release. Combined with the estimated K0.5 of the IK(M)- [ACh] dose-effect relationship, 160 nM, this result also allowed us to estimate the rate of Gk.GTP hydrolysis, kcat, to be near 135 min-1. These results provide, for the first time, a quantitative description of the salient features of G-protein function in vivo. PMID:2455765

  17. Steady-state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis. The role of the allosteric activator GTP incoupling between glutamine hydrolysis and CTP synthesis.

    PubMed

    Willemoës, Martin; Sigurskjold, Bent W

    2002-10-01

    CTP synthase catalyzes the reaction glutamine + UTP + ATP --> glutamate + CTP + ADP + Pi. The rate of the reaction is greatly enhanced by the allosteric activator GTP. We have studied the glutaminase half-reaction of CTP synthase from Lactococcus lactis and its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia coli enzyme, GTP activation of the L. lactis enzyme did not result in similar kcat values for the glutaminase activity and glutamine hydrolysis coupled to CTP synthesis. GTP activation of the glutaminase reaction never reached the levels of GTP-activated CTP synthesis, not even when the active site was saturated with UTP and the nonhydrolyzeable ATP-binding analog adenosine 5'-[gamma-thio]triphosphate. Furthermore, under conditions where the rate of glutamine hydrolysis exceeded that of CTP synthesis, GTP would stimulate CTP synthesis. These results indicate that the L. lactis enzyme differs significantly from the E. coli enzyme. For the E. coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis. An alternative mechanism for the action of GTP on L. lactis CTP synthase is suggested. Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself. PMID:12354108

  18. ATP/GTP hydrolysis is required for oxazole and thiazole biosynthesis in the peptide antibiotic microcin B17.

    PubMed

    Milne, J C; Eliot, A C; Kelleher, N L; Walsh, C T

    1998-09-22

    In the maturation of the Escherichia coli antibiotic Microcin B17, the product of the mcbA gene is modified posttranslationally by the multimeric Microcin synthetase complex (composed of McbB, C, and D) to cyclize four Cys and four Ser residues to four thiazoles and four oxazoles, respectively. The purified synthetase shows an absolute requirement for ATP or GTP in peptide substrate heterocyclization, with GTP one-third as effective as ATP in initial rate studies. The ATPase/GTPase activity of the synthetase complex is conditional in that ADP or GDP formation requires the presence of substrate; noncyclizable versions of McbA bind to synthetase, but do not induce the NTPase activity. The stoichiometry of ATP hydrolysis and heterocycle formation is 5:1 for a substrate that contains two potential sites of modification. However, at high substrate concentrations (>50Km) heterocycle formation is inhibited, while ATPase activity occurs undiminished, consistent with uncoupling of NTP hydrolysis and heterocycle formation at high substrate concentrations. Sequence homology reveals that the McbD subunit has motifs reminiscent of the Walker B box in ATP utilizing enzymes and of motifs found in small G protein GTPases. Mutagenesis of three aspartates to alanine in these motifs (D132, D147, and D199) reduced Microcin B17 production in vivo and heterocycle formation in vitro, suggesting that the 45 kDa McbD has a regulated ATPase/GTPase domain in its N-terminal region necessary for peptide heterocyclization.

  19. Effects of the wasp venom peptide, mastoparan, on GTP hydrolysis in rat brain membranes

    PubMed Central

    Odagaki, Y; Nishi, N; Koyama, T

    1997-01-01

    The effects of mastoparan, a wasp venom toxin, on GTP hydrolyzing activity were examined in rat brain membranes. Mastoparan inhibited the low-affinity GTPase activity, defined as the amount of 32Pi released from 0.3 μM [γ-32P]-GTP in the presence of 100 μM unlabelled GTP, in a concentration-dependent manner. This inhibitory effect of mastoparan on low-affinity GTPase activity was diminished by increasing concentrations of UDP and was completely attenuated at 20 mM, indicating that activation of nucleoside diphosphokinase (NDPK) is inolved in the phenomenon. In the presence of 20 mM UDP, mastoparan stimulated the high-affinity GTPase activity by increasing the Vmax value without affecting the apparent KM for GTP. Mastoparan-stimulated high-affinity GTPase activity was apparent at concentrations higher than 1 μM, in a concentration-dependent manner, but without saturation even at 100 μM. Mastoparan-induced high-affinity GTPase activity showed a characteristic sensitivity to MgCl2, quite different from that seen in L-glutamate-stimulated activity, a representative of receptor-mediated G-protein activation. There appeared to be a simple additive interaction between mastoparan- and L-glutamate-stimulated high-affinity GTPase activities, indicting that distinct pools of G-proteins are involved in receptor-independent and receptor-mediated G-protein activation. These results suggest that G-proteins in brain membranes are functionally altered by mastoparan through multiple mechanisms of action and that the mastoparan-induced, direct G-protein activating process lacks a synergistic or antagonistic interaction with an agonist-induced, receptor-mediated activation of G-proteins. PMID:9257921

  20. A Novel Domain in Translational GTPase BipA Mediates Interaction with the 70S Ribosome and Influences GTP Hydrolysis

    SciTech Connect

    deLivron, M.; Makanji, H; Lane, M; Robinson, V

    2009-01-01

    BipA is a universally conserved prokaryotic GTPase that exhibits differential ribosome association in response to stress-related events. It is a member of the translation factor family of GTPases along with EF-G and LepA. BipA has five domains. The N-terminal region of the protein, consisting of GTPase and {beta}-barrel domains, is common to all translational GTPases. BipA domains III and V have structural counterparts in EF-G and LepA. However, the C-terminal domain (CTD) of the protein is unique to the BipA family. To investigate how the individual domains of BipA contribute to the biological properties of the protein, deletion constructs were designed and their GTP hydrolysis and ribosome binding properties assessed. Data presented show that removal of the CTD abolishes the ability of BipA to bind to the ribosome and that ribosome complex formation requires the surface provided by domains III and V and the CTD. Additional mutational analysis was used to outline the BipA-70S interaction surface extending across these domains. Steady state kinetic analyses revealed that successive truncation of domains from the C-terminus resulted in a significant increase in the intrinsic GTP hydrolysis rate and a loss of ribosome-stimulated GTPase activity. These results indicate that, similar to other translational GTPases, the ribosome binding and GTPase activities of BipA are tightly coupled. Such intermolecular regulation likely plays a role in the differential ribosome binding by the protein.

  1. High resolution microtubule structures reveal the structural transitions in αβ–tubulin upon GTP hydrolysis

    PubMed Central

    Alushin, Gregory M.; Lander, Gabriel C.; Kellogg, Elizabeth H.; Zhang, Rui; Baker, David; Nogales, Eva

    2014-01-01

    Summary Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice, and is inhibited by anticancer agents like Taxol. We provide new insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7–5.6 Å) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α–tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α– and β–tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces. PMID:24855948

  2. The Rap-RapGAP complex: GTP hydrolysis without catalytic glutamine and arginine residues.

    PubMed

    Scrima, Andrea; Thomas, Christoph; Deaconescu, Delia; Wittinghofer, Alfred

    2008-04-01

    The GTP-binding protein Rap1 regulates integrin-mediated and other cell adhesion processes. Unlike most other Ras-related proteins, it contains a threonine in switch II instead of a glutamine (Gln61 in Ras), a residue crucial for the GTPase reaction of most G proteins. Furthermore, unlike most other GTPase-activating proteins (GAPs) for small G proteins, which supply a catalytically important Arg-finger, no arginine residue of RapGAP makes a significant contribution to the GTPase reaction of Rap1. For a detailed understanding of the reaction mechanism, we have solved the structure of Rap1 in complex with Rap1GAP. It shows that the Thr61 of Rap is away from the active site and that an invariant asparagine of RapGAPs, the Asn-thumb, takes over the role of the cis-glutamine of Ras, Rho or Ran. The structure and biochemical data allow to further explain the mechanism and to define the important role of a conserved tyrosine. The structure and biochemical data furthermore show that the RapGAP homologous region of the tumour suppressor Tuberin is sufficient for catalysis on Rheb.

  3. Sliding of a 43S ribosomal complex from the recognized AUG codon triggered by a delay in eIF2-bound GTP hydrolysis

    PubMed Central

    Terenin, Ilya M.; Akulich, Kseniya A.; Andreev, Dmitry E.; Polyanskaya, Sofya A.; Shatsky, Ivan N.; Dmitriev, Sergey E.

    2016-01-01

    During eukaryotic translation initiation, 43S ribosomal complex scans mRNA leader unless an AUG codon in an appropriate context is found. Establishing the stable codon–anticodon base-pairing traps the ribosome on the initiator codon and triggers structural rearrangements, which lead to Pi release from the eIF2-bound GTP. It is generally accepted that AUG recognition by the scanning 43S complex sets the final point in the process of start codon selection, while latter stages do not contribute to this process. Here we use translation reconstitution approach and kinetic toe-printing assay to show that after the 48S complex is formed on an AUG codon, in case GTP hydrolysis is impaired, the ribosomal subunit is capable to resume scanning and slides downstream to the next AUG. In contrast to leaky scanning, this sliding is not limited to AUGs in poor nucleotide contexts and occurs after a relatively long pause at the recognized AUG. Thus, recognition of an AUG per se does not inevitably lead to this codon being selected for initiation of protein synthesis. Instead, it is eIF5-induced GTP hydrolysis and Pi release that irreversibly trap the 48S complex, and this complex is further stabilized by eIF5B and 60S joining. PMID:26717981

  4. The hinge region of Escherichia coli ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis.

    PubMed

    Dey, D; Oleinikov, A V; Traut, R R

    1995-01-01

    A variant form of Escherichia coli ribosomal protein L7/L12 that lacked residues 42 to 52 (L7/L12: delta 42-52) in the hinge region was shown previously to be completely inactive in supporting polyphenylalanine synthesis although it bound to L7/L12 deficient core particles with the normal stoichiometry of four copies per particle (Oleinikov AV, Perroud B, Wang B, Traut RR (1993) J Biol Chem, 268, 917-922). The result suggested that the hinge confers flexibility that is required for activity because the resulting bent conformation allows the distal C-terminal domain to occupy a location on the body of the large ribosomal subunit proximal to the base of the L7/L12 stalk where elongation factors bind. Factor binding to the hinge-truncated variant was tested. As an alternative strategy to deleting residues from the hinge, seven amino acid residues within the putative hinge region were replaced by seven consecutive proline residues in an attempt to confer increased rigidity that might reduce or eliminate the bending of the molecule inferred to be functionally important. This variant, L7/L12:(Pro)7, remained fully active in protein synthesis. Whereas the binding of both factors in ribosomes containing L7/L12:delta 42-52 was decreased by about 50%, there was no loss of factor binding in ribosomes containing L7/L12:(Pro)7, as predicted from the retention of protein synthesis activity. The factor:ribosome complexes that contained L7/L12:delta 42-52 had the same low level of GTP hydrolysis as the core particles completely lacking L7/L12 and EF-G did not support translocation measured by the reaction of phe-tRNA bound in the A site with puromycin. It is concluded that the hinge region is required for the functionally productive binding of elongation factors, and the defect in protein synthesis reported previously is due to this defect. The variant produced by the introduction of the putative rigid Pro7 sequence retains sufficient flexibility for full activity.

  5. The arginine finger of RasGAP helps Gln-61 align the nucleophilic water in GAP-stimulated hydrolysis of GTP.

    PubMed

    Resat, H; Straatsma, T P; Dixon, D A; Miller, J H

    2001-05-22

    The Ras family of GTPases is a collection of molecular switches that link receptors on the plasma membrane to signaling pathways that regulate cell proliferation and differentiation. The accessory GTPase-activating proteins (GAPs) negatively regulate the cell signaling by increasing the slow intrinsic GTP to GDP hydrolysis rate of Ras. Mutants of Ras are found in 25-30% of human tumors. The most dramatic property of these mutants is their insensitivity to the negative regulatory action of GAPs. All known oncogenic mutants of Ras map to a small subset of amino acids. Gln-61 is particularly important because virtually all mutations of this residue eliminate sensitivity to GAPs. Despite its obvious importance for carcinogenesis, the role of Gln-61 in the GAP-stimulated GTPase activity of Ras has remained a mystery. Our molecular dynamics simulations of the p21ras-p120GAP-GTP complex suggest that the local structure around the catalytic region can be different from that revealed by the x-ray crystal structure. We find that the carbonyl oxygen on the backbone of the arginine finger supplied in trans by p120GAP (Arg-789) interacts with a water molecule in the active site that is forming a bridge between the NH(2) group of the Gln-61 and the gamma-phosphate of GTP. Thus, Arg-789 may play a dual role in generating the nucleophile as well as stabilizing the transition state for PO bond cleavage. PMID:11371635

  6. Inhibition of Escherichia coli CTP synthase by glutamate gamma-semialdehyde and the role of the allosteric effector GTP in glutamine hydrolysis.

    PubMed

    Bearne, S L; Hekmat, O; Macdonnell, J E

    2001-05-15

    Cytidine 5'-triphosphate synthase catalyses the ATP-dependent formation of CTP from UTP with either ammonia or glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Escherichia coli CTP synthase, overexpressed as a hexahistidine-tagged form, was purified to high specific activity with the use of metal-ion-affinity chromatography. Unfused CTP synthase, generated by the enzymic removal of the hexahistidine tag, displayed an activity identical with that of the purified native enzyme and was used to study the effect of GTP on the inhibition of enzymic activity by glutamate gamma-semialdehyde. Glutamate gamma-semialdehyde is expected to inhibit CTP synthase by reacting reversibly with the active-site Cys-379 to form an analogue of a tetrahedral intermediate in glutamine hydrolysis. Indeed, glutamate gamma-semialdehyde is a potent linear mixed-type inhibitor of CTP synthase with respect to glutamine (K(is) 0.16+/-0.03 mM; K(ii) 0.4+/-0.1 mM) and a competitive inhibitor with respect to ammonia (K(i) 0.39+/-0.06 mM) in the presence of GTP at pH 8.0. The mutant enzyme (C379A), which is fully active with ammonia but has no glutamine-dependent activity, is not inhibited by glutamate gamma-semialdehyde. Although glutamate gamma-semialdehyde exists in solution primarily in its cyclic form, Delta(1)-pyrroline-5-carboxylate, the variation of inhibition with pH, and the weak inhibition by cyclic analogues of Delta(1)-pyrroline-5-carboxylate (L-proline, L-2-pyrrolidone and pyrrole-2-carboxylate) confirm that the rare open-chain aldehyde species causes the inhibition. When ammonia is employed as the substrate in the absence of GTP, the enzyme's affinity for glutamate gamma-semialdehyde is decreased approx. 10-fold, indicating that the allosteric effector, GTP, functions by stabilizing the protein conformation that binds the tetrahedral intermediate(s) formed during glutamine hydrolysis. PMID:11336655

  7. GTP hydrolysis of TC10 promotes neurite outgrowth through exocytic fusion of Rab11- and L1-containing vesicles by releasing exocyst component Exo70.

    PubMed

    Fujita, Akane; Koinuma, Shingo; Yasuda, Sayaka; Nagai, Hiroyuki; Kamiguchi, Hiroyuki; Wada, Naoyuki; Nakamura, Takeshi

    2013-01-01

    The use of exocytosis for membrane expansion at nerve growth cones is critical for neurite outgrowth. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking to the plasma membrane. Recent studies have shown that TC10 and its effector Exo70, a component of the exocyst tethering complex, contribute to neurite outgrowth. However, the molecular mechanisms of the neuritogenesis-promoting functions of TC10 remain to be established. Here, we propose that GTP hydrolysis of vesicular TC10 near the plasma membrane promotes neurite outgrowth by accelerating vesicle fusion by releasing Exo70. Using Förster resonance energy transfer (FRET)-based biosensors, we show that TC10 activity at the plasma membrane decreased at extending growth cones in hippocampal neurons and nerve growth factor (NGF)-treated PC12 cells. In neuronal cells, TC10 activity at vesicles was higher than its activity at the plasma membrane, and TC10-positive vesicles were found to fuse to the plasma membrane in NGF-treated PC12 cells. Therefore, activity of TC10 at vesicles is presumed to be inactivated near the plasma membrane during neuronal exocytosis. Our model is supported by functional evidence that constitutively active TC10 could not rescue decrease in NGF-induced neurite outgrowth induced by TC10 depletion. Furthermore, TC10 knockdown experiments and colocalization analyses confirmed the involvement of Exo70 in TC10-mediated trafficking in neuronal cells. TC10 frequently resided on vesicles containing Rab11, which is a key regulator of recycling pathways and implicated in neurite outgrowth. In growth cones, most of the vesicles containing the cell adhesion molecule L1 had TC10. Exocytosis of Rab11- and L1-positive vesicles may play a central role in TC10-mediated neurite outgrowth. The combination of this study and our previous work on the role of TC10 in EGF-induced exocytosis in HeLa cells suggests that the signaling machinery containing TC10 proposed here may be

  8. Polarization of Diploid Daughter Cells Directed by Spatial Cues and GTP Hydrolysis of Cdc42 in Budding Yeast

    PubMed Central

    Narayan, Monisha; Chou, Ching-Shan; Park, Hay-Oak

    2013-01-01

    Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast exhibit a characteristic pattern of budding, which depends on cell-type-specific cortical markers, reflecting a genetic programming for the site of cell polarization. The Cdc42 GTPase plays a key role in cell polarization in various cell types. Although previous studies in budding yeast suggested positive feedback loops whereby Cdc42 becomes polarized, these mechanisms do not include spatial cues, neglecting the normal patterns of budding. Here we combine live-cell imaging and mathematical modeling to understand how diploid daughter cells establish polarity preferentially at the pole distal to the previous division site. Live-cell imaging shows that daughter cells of diploids exhibit dynamic polarization of Cdc42-GTP, which localizes to the bud tip until the M phase, to the division site at cytokinesis, and then to the distal pole in the next G1 phase. The strong bias toward distal budding of daughter cells requires the distal-pole tag Bud8 and Rga1, a GTPase activating protein for Cdc42, which inhibits budding at the cytokinesis site. Unexpectedly, we also find that over 50% of daughter cells lacking Rga1 exhibit persistent Cdc42-GTP polarization at the bud tip and the distal pole, revealing an additional role of Rga1 in spatiotemporal regulation of Cdc42 and thus in the pattern of polarized growth. Mathematical modeling indeed reveals robust Cdc42-GTP clustering at the distal pole in diploid daughter cells despite random perturbation of the landmark cues. Moreover, modeling predicts different dynamics of Cdc42-GTP polarization when the landmark level and the initial level of Cdc42-GTP at the division site are perturbed by noise added in the model. PMID:23437206

  9. Accelerated Hydrolysis of Aspirin Using Alternating Magnetic Fields

    NASA Astrophysics Data System (ADS)

    Reinscheid, Uwe M.

    2009-08-01

    The major problem of current drug-based therapy is selectivity. As in other areas of science, a combined approach might improve the situation decisively. The idea is to use the pro-drug principle together with an alternating magnetic field as physical stimulus, which can be applied in a spatially and temporarily controlled manner. As a proof of principle, the neutral hydrolysis of aspirin in physiological phosphate buffer of pH 7.5 at 40 °C was chosen. The sensor and actuator system is a commercially available gold nanoparticle (NP) suspension which is approved for animal usage, stable in high concentrations and reproducibly available. Applying the alternating magnetic field of a conventional NMR magnet system accelerated the hydrolysis of aspirin in solution.

  10. Hydrolysis of GTP by p21NRAS, the NRAS protooncogene product, is accompanied by a conformational change in the wild-type protein: use of a single fluorescent probe at the catalytic site.

    PubMed Central

    Neal, S E; Eccleston, J F; Webb, M R

    1990-01-01

    2'(3')-O-(N-Methyl)anthraniloylguanosine 5'-triphosphate (mantGTP) is a fluorescent analogue of GTP that has similar properties to the physiological substrate in terms of its binding constant and the kinetics of its interactions with p21NRAS, the NRAS protooncogene product. There is a 3-fold increase in fluorescence intensity when mantGTP binds to p21NRAS. The rate constant for the cleavage of mantGTP complexed with the protein is similar to that of GTP, and cleavage is accompanied by a fluorescence intensity change in the wild-type protein complex. A two-phase fluorescence change also occurs when the nonhydrolyzable analogue 2'(3')-O-(N-methyl)anthraniloylguanosine 5'-[beta, gamma-imido]triphosphate (mantp[NH]ppG) binds to wild-type p21NRAS. The second phase occurs at the same rate as the second phase observed after mantGTP binding. Thus this second phase is probably a conformation change of the p21NRAS nucleotiside triphosphate complex and that the change controls the rate of GTP hydrolysis on the protein. With a transforming mutant, [Asp12]-p21NRAS, there is no second phase of the fluorescence change after mantGTP or mantp[NH]ppG binding, even though mantGTP is hydrolyzed. This shows that an equivalent conformational change does not occur and thus the mutant may stay in a "GTP-like" conformation throughout the GTPase cycle. These results are discussed in terms of the proposed role of p21NRAS in signal transduction and the transforming properties of the mutant. PMID:2185475

  11. The guanine cap of human guanylate-binding protein 1 is responsible for dimerization and self-activation of GTP hydrolysis.

    PubMed

    Wehner, Mark; Kunzelmann, Simone; Herrmann, Christian

    2012-01-01

    Human guanylate-binding protein 1 (hGBP1) belongs to the superfamily of large, dynamin-related GTPases. The expression of hGBP1 is induced by stimulation with interferons (mainly interferon-γ), and it plays a role in different cellular responses to inflammatory cytokines, e.g. pathogen defence, control of proliferation, and angiogenesis. Although other members of the dynamin superfamily show a diversity of cellular functions, they share a common GTPase mechanism that relies on nucleotide-controlled oligomerization and self-activation of the GTPase. Previous structural studies on hGBP1 have suggested a mechanism of GTPase and GDPase activity that, as a critical step, involves dimerization of the large GTP-binding domains. In this study, we show that the guanine cap of hGBP1 is the key structural element responsible for dimerization, and is thereby essential for self-activation of the GTPase activity. Studies of concentration-dependent GTP hydrolysis showed that mutations of residues in the guanine cap, in particular Arg240 and Arg244, resulted in higher dissociation constants of the dimer, whereas the maximum hydrolytic activity was largely unaffected. Additionally, we identified an intramolecular polar contact (Lys62-Asp255) whose mutation leads to a loss of self-activation capability and controlled oligomer formation. We suggest that this contact structurally couples the guanine cap to the switch regions of the GTPase, translating the structural changes that occur upon nucleotide binding to a change in oligomerization and self-activation. PMID:22059445

  12. Magnesium activation of GTP hydrolysis or incubation in S-adenosyl-l-methionine reverses iron-56-particle-induced decrements in oxotremorine enhancement of K+-evoked striatal release of dopamine.

    PubMed

    Joseph, J A; Shukitt-Hale, B; McEwen, J; Rabin, B

    1999-12-01

    Previous research has determined that the deficits in motor behavior seen in aged animals irradiated with (56)Fe particles involved alterations in muscarinic receptor sensitivity. In the present experiments, we determined whether increasing either membrane fluidity by exposure of striatal slices from irradiated ((56)Fe particles) animals to S-adenosyl-l-methionine (SAM) or GTP hydrolysis with Mg(2+) would reverse this (56)Fe-particle-induced loss of muscarinic receptor sensitivity, as has been observed in aged animals. Results indicated that, while increasing Mg(2+) concentrations in the incubation medium was effective in reducing the radiation effects, SAM was able to effect some reversal of the radiation effects only at the lower concentration (200 microM). These results suggest that similar mechanisms may be involved in the deficits in signal transduction seen after (56)Fe-particle irradiation to those seen in aging, and that these may include changes in the membrane structure or composition that could alter subsequent responsiveness of transduction pathways. The results further suggest that, as has been reported previously, (56)Fe-particle irradiation may accelerate brain aging, and that since these HZE particles contribute at least 1% of the dose that astronauts would receive from cosmic rays, long-term exposure on extended space flights (e.g. to Mars) may produce similar deficits that could have immediate or delayed effects on behavior. PMID:10581534

  13. Integration of Fourier Transform Infrared Spectroscopy, Fluorescence Spectroscopy, Steady-state Kinetics and Molecular Dynamics Simulations of Gαi1 Distinguishes between the GTP Hydrolysis and GDP Release Mechanism.

    PubMed

    Schröter, Grit; Mann, Daniel; Kötting, Carsten; Gerwert, Klaus

    2015-07-10

    Gα subunits are central molecular switches in cells. They are activated by G protein-coupled receptors that exchange GDP for GTP, similar to small GTPase activation mechanisms. Gα subunits are turned off by GTP hydrolysis. For the first time we employed time-resolved FTIR difference spectroscopy to investigate the molecular reaction mechanisms of Gαi1. FTIR spectroscopy is a powerful tool that monitors reactions label free with high spatio-temporal resolution. In contrast to common multiple turnover assays, FTIR spectroscopy depicts the single turnover GTPase reaction without nucleotide exchange/Mg(2+) binding bias. Global fit analysis resulted in one apparent rate constant of 0.02 s(-1) at 15 °C. Isotopic labeling was applied to assign the individual phosphate vibrations for α-, β-, and γ-GTP (1243, 1224, and 1156 cm(-1), respectively), α- and β-GDP (1214 and 1134/1103 cm(-1), respectively), and free phosphate (1078/991 cm(-1)). In contrast to Ras · GAP catalysis, the bond breakage of the β-γ-phosphate but not the Pi release is rate-limiting in the GTPase reaction. Complementary common GTPase assays were used. Reversed phase HPLC provided multiple turnover rates and tryptophan fluorescence provided nucleotide exchange rates. Experiments were complemented by molecular dynamics simulations. This broad approach provided detailed insights at atomic resolution and allows now to identify key residues of Gαi1 in GTP hydrolysis and nucleotide exchange. Mutants of the intrinsic arginine finger (Gαi1-R178S) affected exclusively the hydrolysis reaction. The effect of nucleotide binding (Gαi1-D272N) and Ras-like/all-α interface coordination (Gαi1-D229N/Gαi1-D231N) on the nucleotide exchange reaction was furthermore elucidated.

  14. Low Intensity Uniform Ultrasound Accelerates 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 an improvement of rates in the hydrolysis of cellulosic materials to sugars, ...

  15. Rejection of tmRNA·SmpB after GTP hydrolysis by EF-Tu on ribosomes stalled on intact mRNA.

    PubMed

    Kurita, Daisuke; Miller, Mickey R; Muto, Akira; Buskirk, Allen R; Himeno, Hyouta

    2014-11-01

    Messenger RNAs lacking a stop codon trap ribosomes at their 3' ends, depleting the pool of ribosomes available for protein synthesis. In bacteria, a remarkable quality control system rescues and recycles stalled ribosomes in a process known as trans-translation. Acting as a tRNA, transfer-messenger RNA (tmRNA) is aminoacylated, delivered by EF-Tu to the ribosomal A site, and accepts the nascent polypeptide. Translation then resumes on a reading frame within tmRNA, encoding a short peptide tag that targets the nascent peptide for degradation by proteases. One unsolved issue in trans-translation is how tmRNA and its protein partner SmpB preferentially recognize stalled ribosomes and not actively translating ones. Here, we examine the effect of the length of the 3' extension of mRNA on each step of trans-translation by pre-steady-state kinetic methods and fluorescence polarization binding assays. Unexpectedly, EF-Tu activation and GTP hydrolysis occur rapidly regardless of the length of the mRNA, although the peptidyl transfer to tmRNA decreases as the mRNA 3' extension increases and the tmRNA·SmpB binds less tightly to the ribosome with an mRNA having a long 3' extension. From these results, we conclude that the tmRNA·SmpB complex dissociates during accommodation due to competition between the downstream mRNA and the C-terminal tail for the mRNA channel. Rejection of the tmRNA·SmpB complex during accommodation is reminiscent of the rejection of near-cognate tRNA from the ribosome in canonical translation.

  16. [Magnetic Magnesium Isotope Accelerates ATP Hydrolysis Catalyzed by Myosin].

    PubMed

    Koltover, V K; Labyntseva, R D; Karandashev, V K; Kosterin, S O

    2016-01-01

    In this paper, we present the results of experimental studies on the influence of different magnesium isotopes, the magnetic 25Mg and nonmagnetic 24Mg and 26Mg on ATP activity of the isolated myosin subfragment-1. The reaction rate in the presence of magetic 25Mg isotope turned out to be 2.0-2.5 times higher than that using nonmagnetic 24Mg and 2 Mg isotopes. No magnetic isotope effect was observed in the absence of the enzyme as in spontaneous ATP hydrolysis in aqueous solution. Hence, a significant catalytic effect of the magnetic 25Mg isotope (nuclear spin catalysis) was observed in the enzymatic hydrolysis of ATP.

  17. Regenerating cellulose from ionic liquids for an accelerated enzymatic hydrolysis

    SciTech Connect

    Zhao, Hua; Jones, Cecil L; Baker, Gary A; Xia, Shuqian; Olubajo, Olarongbe; Person, Vernecia

    2009-01-01

    The efficient conversion of lignocellulosic materials into fuel ethanol has become a research priority in producing affordable and renewable energy. The pretreatment of lignocelluloses is known to be key to the fast enzymatic hydrolysis of cellulose. Recently, certain ionic liquids (ILs)were found capable of dissolving more than 10 wt% cellulose. Preliminary investigations [Dadi, A.P., Varanasi, S., Schall, C.A., 2006. Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step. Biotechnol. Bioeng. 95, 904 910; Liu, L., Chen, H., 2006. Enzymatic hydrolysis of cellulose materials treated with ionic liquid [BMIM]Cl. Chin. Sci. Bull. 51, 2432 2436; Dadi, A.P., Schall, C.A., Varanasi, S., 2007. Mitigation of cellulose recalcitrance to enzymatic hydrolysis by ionic liquid pretreatment. Appl. Biochem. Biotechnol. 137 140, 407 421] suggest that celluloses regenerated from IL solutions are subject to faster saccharification than untreated substrates. These encouraging results offer the possibility of using ILs as alternative and nonvolatile solvents for cellulose pretreatment. However, these studies are limited to two chloride-based ILs: (a) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), which is a corrosive, toxic and extremely hygroscopic solid (m.p. 70 C), and (b) 1-allyl-3-methylimidazolium chloride ([AMIM]Cl), which is viscous and has a reactive side-chain. Therefore, more in-depth research involving other ILs is much needed to explore this promising pretreatment route. For this reason, we studied a number of chloride- and acetate-based ILs for cellulose regeneration, including several ILs newly developed in our laboratory. This will enable us to select inexpensive, efficient and environmentally benign solvents for processing cellulosic biomass. Our data confirm that all regenerated celluloses are less crystalline (58 75% lower) and more accessible to cellulase (>2 times) than untreated substrates. As a result, regenerated Avicel

  18. GTP-specific fab fragment-based GTPase activity assay.

    PubMed

    Kopra, Kari; Rozwandowicz-Jansen, Anita; Syrjänpää, Markku; Blaževitš, Olga; Ligabue, Alessio; Veltel, Stefan; Lamminmäki, Urpo; Abankwa, Daniel; Härmä, Harri

    2015-03-17

    GTPases are central cellular signaling proteins, which cycle between a GDP-bound inactive and a GTP-bound active conformation in a controlled manner. Ras GTPases are frequently mutated in cancer and so far only few experimental inhibitors exist. The most common methods for monitoring GTP hydrolysis rely on luminescent GDP- or GTP-analogs. In this study, the first GTP-specific Fab fragment and its application are described. We selected Fab fragments using the phage display technology. Six Fab fragments were found against 2'/3'-GTP-biotin and 8-GTP-biotin. Selected antibody fragments allowed specific detection of endogenous, free GTP. The most potent Fab fragment (2A4(GTP)) showed over 100-fold GTP-specificity over GDP, ATP, or CTP and was used to develop a heterogeneous time-resolved luminescence based assay for the monitoring of GTP concentration. The method allows studying the GEF dependent H-Ras activation (GTP binding) and GAP-catalyzed H-Ras deactivation (GTP hydrolysis) at nanomolar protein concentrations.

  19. Nylon 6.6 accelerated aging studies : thermal-oxidative degradation and its interaction with hydrolysis.

    SciTech Connect

    Bernstein, Robert; Derzon, Dora Kay; Gillen, Kenneth T.

    2004-06-01

    Accelerated aging of Nylon 6.6 fibers used in parachutes has been conducted by following the tensile strength loss under both thermal-oxidative and 100% relative humidity conditions. Thermal-oxidative studies (air circulating ovens) were performed for time periods of weeks to years at temperatures ranging from 37 C to 138 C. Accelerated aging humidity experiments (100% RH) were performed under both an argon atmosphere to examine the 'pure' hydrolysis pathway, and under an oxygen atmosphere (oxygen partial pressure close to that occurring in air) to mimic true aging conditions. As expected the results indicated that degradation caused by humidity is much more important than thermal-oxidative degradation. Surprisingly when both oxygen and humidity were present the rate of degradation was dramatically enhanced relative to humidity aging in the absence of oxygen. This significant and previously unknown phenomena underscores the importance of careful accelerated aging that truly mimics real world storage conditions.

  20. How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis

    PubMed Central

    2011-01-01

    Background In order to generate biofuels, insoluble cellulosic substrates are pretreated and subsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safe and environmentally friendly manner is to apply, under mild conditions, non-hydrolyzing proteins such as swollenin - naturally produced in low yields by the fungus Trichoderma reesei. To yield sufficient swollenin for industrial applications, the first aim of this study is to present a new way of producing recombinant swollenin. The main objective is to show how swollenin quantitatively affects relevant physical properties of cellulosic substrates and how it affects subsequent hydrolysis. Results After expression in the yeast Kluyveromyces lactis, the resulting swollenin was purified. The adsorption parameters of the recombinant swollenin onto cellulose were quantified for the first time and were comparable to those of individual cellulases from T. reesei. Four different insoluble cellulosic substrates were then pretreated with swollenin. At first, it could be qualitatively shown by macroscopic evaluation and microscopy that swollenin caused deagglomeration of bigger cellulose agglomerates as well as dispersion of cellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin on cellulose particle size, maximum cellulase adsorption and cellulose crystallinity were quantified. The pretreatment with swollenin resulted in a significant decrease in particle size of the cellulosic substrates as well as in their crystallinity, thereby substantially increasing maximum cellulase adsorption onto these substrates. Subsequently, the pretreated cellulosic substrates were hydrolyzed with cellulases. Here, pretreatment of cellulosic substrates with swollenin, even in non-saturating concentrations, significantly accelerated the hydrolysis. By correlating particle size and crystallinity of the cellulosic substrates with initial hydrolysis rates, it could be shown that the swollenin

  1. A tRNA splicing operon: Archease endows RtcB with dual GTP/ATP cofactor specificity and accelerates RNA ligation

    PubMed Central

    Desai, Kevin K.; Cheng, Chin L.; Bingman, Craig A.; Phillips, George N.; Raines, Ronald T.

    2014-01-01

    Archease is a 16-kDa protein that is conserved in all three domains of life. In diverse bacteria and archaea, the genes encoding Archease and the tRNA ligase RtcB are localized into an operon. Here we provide a rationale for this operon organization by showing that Archease and RtcB from Pyrococcus horikoshii function in tandem, with Archease altering the catalytic properties of the RNA ligase. RtcB catalyzes the GTP and Mn(II)-dependent joining of either 2′,3′-cyclic phosphate or 3′-phosphate termini to 5′-hydroxyl termini. We find that catalytic concentrations of Archease are sufficient to activate RtcB, and that Archease accelerates both the RNA 3′-P guanylylation and ligation steps. In addition, we show that Archease can alter the NTP specificity of RtcB such that ATP, dGTP or ITP is used efficiently. Moreover, RtcB variants that have inactivating substitutions in the guanine-binding pocket can be rescued by the addition of Archease. We also present a 1.4 Å-resolution crystal structure of P. horikoshii Archease that reveals a metal-binding site consisting of conserved carboxylates located at the protein tip. Substitution of the Archease metal-binding residues drastically reduced Archease-dependent activation of RtcB. Thus, evolution has sought to co-express archease and rtcB by creating a tRNA splicing operon. PMID:24435797

  2. Bacterial tubulin TubZ-Bt transitions between a two-stranded intermediate and a four-stranded filament upon GTP hydrolysis

    PubMed Central

    Montabana, Elizabeth A.; Agard, David A.

    2014-01-01

    Cytoskeletal filaments form diverse superstructures that are highly adapted for specific functions. The recently discovered TubZ subfamily of tubulins is involved in type III plasmid partitioning systems, facilitating faithful segregation of low copy-number plasmids during bacterial cell division. One such protein, TubZ-Bt, is found on the large pBtoxis plasmid in Bacillus thuringiensis, and interacts via its extended C terminus with a DNA adaptor protein TubR. Here, we use cryo-electron microscopy to determine the structure of TubZ-Bt filaments and light scattering to explore their mechanism of polymerization. Surprisingly, we find that the helical filament architecture is remarkably sensitive to nucleotide state, changing from two-stranded to four-stranded depending on the ability of TubZ-Bt to hydrolyze GTP. We present pseudoatomic models of both the two- and four-protofilament forms based on cryo-electron microscopy reconstructions (10.8 Å and 6.9 Å, respectively) of filaments formed under different nucleotide states. These data lead to a model in which the two-stranded filament is a necessary intermediate along the pathway to formation of the four-stranded filament. Such nucleotide-directed structural polymorphism is to our knowledge an unprecedented mechanism for the formation of polar filaments. PMID:24550513

  3. Ras and GTPase-activating protein (GAP) drive GTP into a precatalytic state as revealed by combining FTIR and biomolecular simulations.

    PubMed

    Rudack, Till; Xia, Fei; Schlitter, Jürgen; Kötting, Carsten; Gerwert, Klaus

    2012-09-18

    Members of the Ras superfamily regulate many cellular processes. They are down-regulated by a GTPase reaction in which GTP is cleaved into GDP and P(i) by nucleophilic attack of a water molecule. Ras proteins accelerate GTP hydrolysis by a factor of 10(5) compared to GTP in water. GTPase-activating proteins (GAPs) accelerate hydrolysis by another factor of 10(5) compared to Ras alone. Oncogenic mutations in Ras and GAPs slow GTP hydrolysis and are a factor in many cancers. Here, we elucidate in detail how this remarkable catalysis is brought about. We refined the protein-bound GTP structure and protein-induced charge shifts within GTP beyond the current resolution of X-ray structural models by combining quantum mechanics and molecular mechanics simulations with time-resolved Fourier-transform infrared spectroscopy. The simulations were validated by comparing experimental and theoretical IR difference spectra. The reactant structure of GTP is destabilized by Ras via a conformational change from a staggered to an eclipsed position of the nonbridging oxygen atoms of the γ- relative to the β-phosphates and the further rotation of the nonbridging oxygen atoms of α- relative to the β- and γ-phosphates by GAP. Further, the γ-phosphate becomes more positive although two of its oxygen atoms remain negative. This facilitates the nucleophilic attack by the water oxygen at the phosphate and proton transfer to the oxygen. Detailed changes in geometry and charge distribution in the ligand below the resolution of X-ray structure analysis are important for catalysis. Such high resolution appears crucial for the understanding of enzyme catalysis.

  4. In vitro mutation analysis of Arabidopsis thaliana small GTP-binding proteins and detection of GAP-like activities in plant cells.

    PubMed

    Anai, T; Matsui, M; Nomura, N; Ishizaki, R; Uchimiya, H

    1994-06-13

    Previously, we have reported the molecular cloning of ara genes encoding a small GTP-binding protein from Arabidopsis thaliana. The criterion based on amino acid sequences suggest that such an ara gene family can be classified to be of the YPT/rab type. To examine the biochemical properties of ARA proteins, several deletions and point mutations were introduced into ara cDNAs. Mutant proteins were expressed in E. coli as GST-chimeric molecules and analyzed in terms of their GTP-binding or GTP-hydrolysing ability in vitro. The results indicate that four conserved amino acid sequence regions of ARA proteins are necessary for GTP-binding. A point mutation of Asn at position 72 for ARA-2, or 71 for ARA-4, to Ile decreased GTP-binding and a point mutation of Gln at position 126 for ARA-2, or 125 for ARA-4, to Leu suppressed GTP-hydrolysis activity. Furthermore, certain factors associated with the membrane fraction accelerated GTPase activities of ARA proteins, suggesting the presence of GTPase activating protein(s) (GAP(s)) in the vesicular transport system of higher plant cells.

  5. Analysis of Arf GTP-binding Protein Function in Cells

    PubMed Central

    Cohen, Lee Ann; Donaldson, Julie G.

    2010-01-01

    This unit describes techniques and approaches that can be used to study the functions of the ADP-ribosylation factor (Arf) GTP-binding proteins in cells. There are 6 mammalian Arfs and many more Arf-like proteins (Arls) and these proteins are conserved in eukaryotes from yeast to man. Like all GTPases, Arfs cycle between GDP-bound, inactive and GTP-bound active conformations, facilitated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that catalyze GTP binding and hydrolysis respectively. Here we describe approaches that can be taken to examine the localization and function of Arf and Arl proteins in cells (Protocol 1). We also provide a simple protocol for measuring activation (GTP-binding) of specific Arf proteins in cells using a pull-down assay (Protocol 2). We then discuss approaches that can be taken to assess function of GEFs and GAPs in cells (Protocol 3). PMID:20853342

  6. Acceleration of the Enzymatic Hydrolysis of Cotton Waste Celluloses by Low Intensity Uniform Ultrasound Field

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cost-competitive production of bio-ethanol and other biofuels is currently impeded, mostly by high cost and low efficiency of enzymatic hydrolysis of feedstock biomass and especially plant celluloses. Despite substantial reduction in the cost of production of cellulolytic enzymes in recent times...

  7. Apolipoprotein AV accelerates plasma hydrolysis of triglyceride-rich lipoproteins by interaction with proteoglycan-bound lipoprotein lipase.

    PubMed

    Merkel, Martin; Loeffler, Britta; Kluger, Malte; Fabig, Nathalie; Geppert, Gesa; Pennacchio, Len A; Laatsch, Alexander; Heeren, Joerg

    2005-06-01

    Apolipoprotein A5 (APOA5) is associated with differences in triglyceride levels and familial combined hyperlipidemia. In genetically engineered mice, apoAV plasma levels are inversely correlated with plasma triglycerides. To elucidate the mechanism by which apoAV influences plasma triglycerides, metabolic studies and in vitro assays resembling physiological conditions were performed. In human APOA5 transgenic mice (hAPOA5tr), catabolism of chylomicrons and very low density lipoprotein (VLDL) was accelerated due to a faster plasma hydrolysis of triglycerides by lipoprotein lipase (LPL). Hepatic VLDL and intestinal chylomicron production were not affected. The functional interplay between apoAV and LPL was further investigated by cross-breeding a human LPL transgene with the apoa5 knock-out and the hAPOA5tr to an lpl-deficient background. Increased LPL activity completely normalized hypertriglyceridemia of apoa5-deficient mice; however, overexpression of human apoAV modulated triglyceride levels only slightly when LPL was reduced. To reflect the physiological situation in which LPL is bound to cell surface proteoglycans, we examined hydrolysis in the presence or absence of proteoglycans. Without proteoglycans, apoAV derived either from triglyceride-rich lipoproteins, hAPOA5tr high density lipoprotein, or a recombinant source did not alter the LPL hydrolysis rate. In the presence of proteoglycans, however, apoAV led to a significant and dose-dependent increase in LPL-mediated hydrolysis of VLDL triglycerides. These results were confirmed in cell culture using a proteoglycan-deficient cell line. A direct interaction between LPL and apoAV was found by ligand blotting. It is proposed, that apoAV reduces triglyceride levels by guiding VLDL and chylomicrons to proteoglycan-bound LPL for lipolysis.

  8. Apolipoprotein AV Accelerates Plasma Hydrolysis OfTriglyceride-Rich Lipoproteins By Interaction With Proteoglycan BoundLipoprotein Lipase

    SciTech Connect

    Merkel, Martin; Loeffler, Britta; Kluger, Malte; Fabig, Nathalie; Geppert, Gesa; Pennacchio, Len A.; Laatsch, Alexander; Heeren, Joerg

    2005-02-22

    Apolipoprotein A5 (APOA5) is associated with differences intriglyceride levels and familial combined hyperlipidemia. In genetically engineered mice, apoAV plasma levels are inversely correlated with plasmatriglycerides. To elucidate the mechanism by which apoAV influences plasma triglycerides, metabolic studies and in vitro assays resembling physiological conditions were performed. In hAPOA5 transgenic mice(hAPOA5tr), catabolism of chylomicrons and VLDL was accelerated due to a faster plasma hydrolysis of triglycerides by lipoprotein lipase (LPL).Hepatic VLDL and intestinal chylomicron production were not affected. The functional interplay between apoAV and LPL was further investigated by crossbreeding a human LPL transgene with the apoa5 knockout, and the hAPOA5tr to an LPL deficient background. Increased LPL activity completely normalized hypertriglyceridemia of apoa5 deficient mice,however, over expression of human apoAV modulated triglyceride levels only slightly when LPL was reduced. To reflect the physiological situation in which LPL is bound to cell surface proteoglycans, we examined hydrolysis in the presence or absence of proteoglycans. Without proteoglycans, apoAV derived either from triglyceride-rich lipoproteins, hAPOA5tr HDL, or a recombinant source did not alter the LPL hydrolysis rate. In the presence of proteoglycans, however, apoAV led to a significant and dose-dependent increase in LPL mediated hydrolysis of VLDL triglycerides. These results were confirmed in cell culture using a proteoglycan-deficient cell line.A direct interaction between LPL and apoAV was found by ligand blotting.It is proposed, that apoAV reduces triglyceride levels by guiding VLDL and chylomicrons to proteoglycans bound LPL for lipolysis.

  9. Focused ultrasound-assisted acceleration of enzymatic hydrolysis of alkylphenols and 17β-oestradiol glucuronide in fish bile.

    PubMed

    Vallejo, Asier; Usobiaga, Aresatz; Ortiz-Zarragoitia, Maren; Cajaraville, Miren P; Fernández, Luis A; Zuloaga, Olatz

    2010-11-01

    According to the European Water Framework Directive (WFD), alkylphenols, such as octylphenols and nonylphenols, and 17β-oestradiol are considered as priority or emerging pollutants, respectively, mainly due to their possible properties as endocrine-disrupting compounds (EDCs). EDCs are accumulated in liver, fat, kidney and bile in the glucuronide form. In order to determine the concentration of these compounds in bile, an enzymatic hydrolysis step is necessary. This step is usually long (~16 h), and in this sense, ultrasound probes were studied as a possible alternative energy source to accelerate this process. Enzymatic hydrolysis was reduced to 20 min using an ultrasound probe at one cycle and 10% of amplitude. For validation of analytical procedure, nonylphenol glucuronide (4NP-G), 4-tert-octylphenol glucuronide (4tOP-G) and 4-n-octylphenol glucuronide (4nOP-G) were synthesised while 17β-oestradiol glucuronide (E2-G) was commercially available. Bile from thick-lip grey mullets (Chelon labrosus) was spiked with known amounts of 4NP-G, 4tOP-G, 4nOP-G and E2-G and submitted to the optimised procedure. Good recoveries (77-122%), precision in the 5% to 12% range and limits of detection, ranging from the low nanogramme per gramme level for 4tOP, 4nOP and E2 to the low microgramme per gramme level for nonylphenols, were obtained. The optimised method was applied for the determination of alkylphenol in the bile of thick-lip grey mullets fish bile from the Urdaibai estuary (UNESCO reserve of the Biosphere, Bay of Biscay), and high concentrations (2.3-14.2 μg/g), such as those obtained in polluted areas, were measured. E2 was determined in the bile of thick-lip grey mullets, intraperitoneally injected with E2.

  10. Evidence for a vasopressin receptor-GTP binding protein complex

    SciTech Connect

    Fitzgerald, T.J.; Uhing, R.J.; Exton, J.H.

    1986-05-01

    Plasma membranes from the livers of rats were able to hydrolyze the ..gamma..-phosphate from guanosine-5'-triphosphate (GTP). The rate of GTP hydrolysis could be decreased to 10% of its initial rate by the addition of adenosine-5'-triphosphate with a concomitant decrease in the K/sub m/ for GTP from approx. 10/sup -3/ M to 10/sup -6/ M. The low K/sub m/ GTPase activity was inhibited by the addition of nonhydrolyzable analogs of GTP. In addition, the GTPase activity was stimulated from 10 to 30% over basal by the addition of vasopressin. A dose dependency curve showed that the maximum stimulation was obtained with 10/sup -8/ M vasopressin. Identical results were obtained from plasma membranes that had been solubilized with 1% digitonin. When membranes that had been solubilized in the presence of (Phenylalanyl-3,4,5-/sup 3/H(N))vasopressin were subjected to sucrose gradient centrifugation, the majority of bound (/sup 3/H)vasopressin migrated with an approximate molecular weight of 300,000. Moreover, there was a GTPase activity that migrated with the bound (/sup 3/H)vasopressin. This peak of bound (/sup 3/H)vasopressin was decreased by 90% when the sucrose gradient centrifugation was run in the presence of 10/sup -5/ M guanosine-5'-O-(3-thiotriphosphate). These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.

  11. Inhibition of E. coli CTP synthase by the "positive" allosteric effector GTP.

    PubMed

    MacDonnell, Jennifer E; Lunn, Faylene A; Bearne, Stephen L

    2004-06-01

    Cytidine 5'-triphosphate (CTP) synthase catalyzes the ATP-dependent formation of CTP from UTP using either ammonia or l-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as a positive allosteric effector to promote catalysis of glutamine hydrolysis. We show that at concentrations exceeding approximately 0.15 mM, GTP actually behaves as a negative allosteric effector of E. coli CTP synthase, inhibiting glutamine-dependent CTP formation. In addition, GTP inhibits NH(3)-dependent CTP formation in a concentration-dependent manner. However, GTP does not inhibit the enzyme's intrinsic glutaminase activity. Although the activation of CTP synthase by GTP does not display cooperative behavior, inhibition of both CTP synthase-catalyzed ammonia- and glutamine-dependent CTP synthesis by GTP do exhibit positive cooperativity. These results suggest that GTP binding affects CTP synthase catalysis in two ways: it activates enzyme-catalyzed glutamine hydrolysis and it inhibits the utilization of NH(3) as a substrate by the synthase domain. PMID:15158730

  12. Identification of a second GTP-bound magnesium ion in archaeal initiation factor 2.

    PubMed

    Dubiez, Etienne; Aleksandrov, Alexey; Lazennec-Schurdevin, Christine; Mechulam, Yves; Schmitt, Emmanuelle

    2015-03-11

    Eukaryotic and archaeal translation initiation processes involve a heterotrimeric GTPase e/aIF2 crucial for accuracy of start codon selection. In eukaryotes, the GTPase activity of eIF2 is assisted by a GTPase-activating protein (GAP), eIF5. In archaea, orthologs of eIF5 are not found and aIF2 GTPase activity is thought to be non-assisted. However, no in vitro GTPase activity of the archaeal factor has been reported to date. Here, we show that aIF2 significantly hydrolyses GTP in vitro. Within aIF2γ, H97, corresponding to the catalytic histidine found in other translational GTPases, and D19, from the GKT loop, both participate in this activity. Several high-resolution crystal structures were determined to get insight into GTP hydrolysis by aIF2γ. In particular, a crystal structure of the H97A mutant was obtained in the presence of non-hydrolyzed GTP. This structure reveals the presence of a second magnesium ion bound to GTP and D19. Quantum chemical/molecular mechanical simulations support the idea that the second magnesium ion may assist GTP hydrolysis by helping to neutralize the developing negative charge in the transition state. These results are discussed in light of the absence of an identified GAP in archaea to assist GTP hydrolysis on aIF2.

  13. Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

    SciTech Connect

    Simonetti, Angelita; Fabbretti, Attilio; Hazemann, Isabelle; Jenner, Lasse; Gualerzi, Claudio O.; Klaholz, Bruno P.

    2013-06-01

    The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue.

  14. Interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly

    SciTech Connect

    Lin, C.M.; Hamel, E.

    1987-11-03

    The author previously reported that direct incorporation of GDP (i.e., without an initial hydrolysis of GTP) into microtubules occurs throughout an assembly cycle in a constant proportion. The exact proportion varied with reaction conditions, becoming greater under all conditions in which tubulin-GDP increased relative to tubulin-GTP (low Mg/sup 2 +/ and GTP concentrations, high tubulin concentrations, and in the presence of exogeneous GDP). These findings led the authors to explore further interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly. They have now determined the minimum amount of tubulin-GTP required for the initiation of microtubule assembly and the relative efficiency with which tubulin-GDP participates in microtubule elongation. When (8-/sup 14/C)GTP, (8-/sup 14/C)GDP, and tubulin concentrations were varied at a constant Mg/sup 2 +/ concentration (0.2 mM), initiation of assembly required that 35% of the nucleotide-bearing tubulin be in the form of tubulin-GTP, and incorporation of tubulin-GDP into microtubules during elongation was only 60% as efficient as would be predicted on the basis of its proportional concentration in the reaction mixtures. Very different results were obtained when the Mg/sup 2 +/ concentration was varied. In the absence of exogenous Mg/sup 2 +/, only 20% tubulin-GTP was required for initiation, and tubulin-GDP was directly incorporated into microtubules half as efficiently as would be predicted on the basis of its concentration in the reaction mixture. At the highest Mg/sup 2 +/ concentration examined (4 mM), 80% tubulin-GTP was required for initiation of assembly, and tubulin-GDP was incorporated into microtubules as efficiently as tubulin-GTP.

  15. Acceleration of the Enzymatic Hydrolysis of Corn Stover and Sugar Cane Bagasse Celluloses by Low Intensity Uniform Ultrasound

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cost-competitive production of bio-ethanol and other biofuels is currently impeded, mostly by high cost and low efficiency of enzymatic hydrolysis of feedstock biomass and especially plant celluloses. Despite substantial reduction in the cost of production of cellulolytic enzymes in recent times...

  16. Phospholipases as GTPase activity accelerating proteins (GAPs) in plants.

    PubMed

    Pandey, Sona

    2016-05-01

    GTPase activity accelerating proteins (GAPs) are key regulators of the G-protein signaling cycle. By facilitating effective hydrolysis of the GTP bound on Gα proteins, GAPs control the timing and amplitude of the signaling cycle and ascertain the availability of the inactive heterotrimer for the next round of activation. Until very recently, the studies of GAPs in plants were focused exclusively on the regulator of G-protein signaling (RGS) protein. We now show that phospholipase Dα1 (PLDα1) is also a bona fide GAP in plants and together with the RGS protein controls the level of active Gα protein. PMID:27124090

  17. Lid L11 of the glutamine amidotransferase domain of CTP synthase mediates allosteric GTP activation of glutaminase activity.

    PubMed

    Willemoës, Martin; Mølgaard, Anne; Johansson, Eva; Martinussen, Jan

    2005-02-01

    GTP is an allosteric activator of CTP synthase and acts to increase the k(cat) for the glutamine-dependent CTP synthesis reaction. GTP is suggested, in part, to optimally orient the oxy-anion hole for hydrolysis of glutamine that takes place in the glutamine amidotransferase class I (GATase) domain of CTP synthase. In the GATase domain of the recently published structures of the Escherichia coli and Thermus thermophilus CTP synthases a loop region immediately proceeding amino acid residues forming the oxy-anion hole and named lid L11 is shown for the latter enzyme to be flexible and change position depending on the presence or absence of glutamine in the glutamine binding site. Displacement or rearrangement of this loop may provide a means for the suggested role of allosteric activation by GTP to optimize the oxy-anion hole for glutamine hydrolysis. Arg359, Gly360 and Glu362 of the Lactococcus lactis enzyme are highly conserved residues in lid L11 and we have analyzed their possible role in GTP activation. Characterization of the mutant enzymes R359M, R359P, G360A and G360P indicated that both Arg359 and Gly360 are involved in the allosteric response to GTP binding whereas the E362Q enzyme behaved like wild-type enzyme. Apart from the G360A enzyme, the results from kinetic analysis of the enzymes altered at position 359 and 360 showed a 10- to 50-fold decrease in GTP activation of glutamine dependent CTP synthesis and concomitant four- to 10-fold increases in K(A) for GTP. The R359M, R359P and G360P also showed no GTP activation of the uncoupled glutaminase reaction whereas the G360A enzyme was about twofold more active than wild-type enzyme. The elevated K(A) for GTP and reduced GTP activation of CTP synthesis of the mutant enzymes are in agreement with a predicted interaction of bound GTP with lid L11 and indicate that the GTP activation of glutamine dependent CTP synthesis may be explained by structural rearrangements around the oxy-anion hole of the GATase

  18. Identification of different roles for RanGDP and RanGTP in nuclear protein import.

    PubMed Central

    Görlich, D; Panté, N; Kutay, U; Aebi, U; Bischoff, F R

    1996-01-01

    The importin-alpha/beta heterodimer and the GTPase Ran play key roles in nuclear protein import. Importin binds the nuclear localization signal (NLS). Translocation of the resulting import ligand complex through the nuclear pore complex (NPC) requires Ran and is terminated at the nucleoplasmic side by its disassembly. The principal GTP exchange factor for Ran is the nuclear protein RCC1, whereas the major RanGAP is cytoplasmic, predicting that nuclear Ran is mainly in the GTP form and cytoplasmic Ran is in the GDP-bound form. Here, we show that nuclear import depends on cytoplasmic RanGDP and free GTP, and that RanGDP binds to the NPC. Therefore, import might involve nucleotide exchange and GTP hydrolysis on NPC-bound Ran. RanGDP binding to the NPC is not mediated by the Ran binding sites of importin-beta, suggesting that translocation is not driven from these sites. Consistently, a mutant importin-beta deficient in Ran binding can deliver its cargo up to the nucleoplasmic side of the NPC. However, the mutant is unable to release the import substrate into the nucleoplasm. Thus, binding of nucleoplasmic RanGTP to importin-beta probably triggers termination, i.e. the dissociation of importin-alpha from importin-beta and the subsequent release of the import substrate into the nucleoplasm. Images PMID:8896452

  19. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions

    NASA Astrophysics Data System (ADS)

    Ostrem, Jonathan M.; Peters, Ulf; Sos, Martin L.; Wells, James A.; Shokat, Kevan M.

    2013-11-01

    Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner.

  20. Human septin isoforms and the GDP-GTP cycle.

    PubMed

    Zent, Eldar; Wittinghofer, Alfred

    2014-02-01

    Septins form oligomeric complexes consisting of septins from different subgroups, which form filaments that are involved in a number of biological processes. They are GTP-binding proteins that contain all the necessary elements to perform the general GDP-to-GTP conformational switch. It is however unclear whether or not such a switch is important for the dynamics of septin filaments. Here we investigate the complex GTPase reaction of members of each of the four human septin groups, which is dominated by the stability of dimer formation via the nucleotide binding or so-called G-interface. The results also show that the actual hydrolysis reaction is very similar for three septin groups in the monomeric state while the Sept6 has no GTPase activity. Sept7, the only member of the Sept7 subgroup, forms a very tight G-interface dimer in the GDP-bound state. Here we show that the stability of the interface is dramatically decreased by exchanging GDP with a nucleoside triphosphate, which is believed to influence filament formation and dynamics via Sept7.

  1. Synthesis of hierarchical NiCo2O4 hollow nanorods via sacrificial-template accelerate hydrolysis for electrochemical glucose oxidation.

    PubMed

    Yang, Jiao; Cho, Misuk; Lee, Youngkwan

    2016-01-15

    Hierarchical NiCo2O4 hollow nanorods (HR) were directly grown on stainless steel via a sacrificial template accelerated hydrolysis and post calcination using ZnO nanorod as a template. The composition of the NiCo2O4 HR electrode was determined using X-ray diffraction and X-ray photoelectron spectroscopy. The morphology of the NiCo2O4 HR is comprised of nanoflakes that were characterized with scanning electron microscopy and transmission electron microscopy. The mixed-valence metal oxide and hollow structure provided high chemical reactivity and a large surface area for glucose oxidation in an alkaline solution. Under an optimal applied potential of +0.6 V, the developed NiCo2O4 HR electrode showed a broad detection range of 0.0003–1.0 mM, a sensitivity of 1685.1 μA mM−1 cm−2, and a low detection limit of 0.16 μM. These results represent a significant improvement over both NiO and Co3O4 HR. The developed NiCo2O4 HR electrode not only demonstrated excellent selectivity in the presence of several electro-active species, but also exhibited high stability following a 200 cycles voltammetry test. PMID:26281005

  2. Structural requirements for the activation of Escherichia coli CTP synthase by the allosteric effector GTP are stringent, but requirements for inhibition are lax.

    PubMed

    Lunn, Faylene A; MacDonnell, Jennifer E; Bearne, Stephen L

    2008-01-25

    Cytidine 5'-triphosphate synthase catalyzes the ATP-dependent formation of CTP from UTP using either NH(3) or l-glutamine (Gln) as the source of nitrogen. GTP acts as an allosteric effector promoting Gln hydrolysis but inhibiting Gln-dependent CTP formation at concentrations of >0.15 mM and NH(3)-dependent CTP formation at all concentrations. A structure-activity study using a variety of GTP and guanosine analogues revealed that only a few GTP analogues were capable of activating Gln-dependent CTP formation to varying degrees: GTP approximately 6-thio-GTP > ITP approximately guanosine 5'-tetraphosphate > O(6)-methyl-GTP > 2'-deoxy-GTP. No activation was observed with guanosine, GMP, GDP, 2',3'-dideoxy-GTP, acycloguanosine, and acycloguanosine monophosphate, indicating that the 5'-triphosphate, 2'-OH, and 3'-OH are required for full activation. The 2-NH(2) group plays an important role in binding recognition, whereas substituents at the 6-position play an important role in activation. The presence of a 6-NH(2) group obviates activation, consistent with the inability of ATP to substitute for GTP. Nucleotide and nucleoside analogues of GTP and guanosine, respectively, all inhibited NH(3)- and Gln-dependent CTP formation (often in a cooperative manner) to a similar extent (IC(50) approximately 0.2-0.5 mM). This inhibition appeared to be due solely to the purine base and was relatively insensitive to the identity of the purine with the exception of inosine, ITP, and adenosine (IC(50) approximately 4-12 mM). 8-Oxoguanosine was the best inhibitor identified (IC(50) = 80 microM). Our findings suggest that modifying 2-aminopurine or 2-aminopurine riboside may serve as an effective strategy for developing cytidine 5'-triphosphate synthase inhibitors. PMID:18003612

  3. A GTP-binding protein regulates the activity of (1-->3)-beta-glucan synthase, an enzyme directly involved in yeast cell wall morphogenesis.

    PubMed

    Mol, P C; Park, H M; Mullins, J T; Cabib, E

    1994-12-01

    Synthesis of (1-->3)-beta-D-glucan, the major structural component of the yeast cell wall, is synchronized with the budding cycle. Membrane-bound, GTP-stimulated (1-->3)-beta-glucan synthase was dissociated by stepwise treatment with salt and detergents into two soluble fractions, A and B, both required for activity. Fraction A was purified about 800-fold by chromatography on Mono Q and Sephacryl S-300 columns. During purification, GTP binding to protein correlated with synthase complementing activity. A 20-kDa GTP-binding protein was identified by photolabeling in the purified preparation. This preparation no longer required GTP for activity, but incubation with another fraction from the Mono Q column (A1) led to hydrolysis of bound GTP to GDP with a concomitant return of the GTP requirement. Thus, fraction A1 appears to contain a GTPase-activating protein. These results show that the GTP-binding protein not only regulates glucan synthase activity but can be regulated in turn, constituting a potential link between cell cycle controls and wall morphogenesis. PMID:7983071

  4. Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes.

    PubMed

    Crepin, Thibaut; Shalak, Vyacheslav F; Yaremchuk, Anna D; Vlasenko, Dmytro O; McCarthy, Andrew; Negrutskii, Boris S; Tukalo, Michail A; El'skaya, Anna V

    2014-11-10

    Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon-anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the 'GTP'- and 'GDP'-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis.

  5. Microtubule +TIP protein EB1 binds to GTP and undergoes dissociation from dimer to monomer on binding GTP.

    PubMed

    Gireesh, K K; Sreeja, Jamuna S; Chakraborti, Soumyananda; Singh, Puja; Thomas, Geethu Emily; Gupta, Hindol; Manna, Tapas

    2014-09-01

    The +TIP protein EB1 autonomously tracks the growing plus end of microtubules and regulates plus-end dynamics. Previous studies have indicated that EB1 can recognize GTP-bound tubulin structures at the plus end, and it localizes on the microtubule surface at a site close to the exchangeable GTP-binding site of tubulin. Although the GTP-dependent structural change in tubulin has been demonstrated to be a critical determinant for recognition of plus ends by EB1, the effect of GTP on the structure of EB1 has remained unclear. Here, we have used spectroscopic, calorimetric, and biochemical methods to analyze the effect of GTP on EB1 in vitro. Isothermal titration calorimetry and tryptophan fluorescence quenching experiments demonstrated that EB1 binds to GTP with a dissociation constant ~30 μM. Circular dichroism measurements showed that EB1 undergoes changes in its secondary structure on binding GTP. Size-exclusion chromatography and urea-induced unfolding analyses revealed that GTP binding induces dissociation of the EB1 dimer to monomers. Size-exclusion chromatography followed by biochemical analysis further determined that EB1-GTP binding involves association of approximately one molecule of GTP per EB1 monomer. The results reveal a hitherto unknown GTP-dependent mechanism of dimer-to-monomer transition in EB1 and further implicate its possible role in regulating the stability of the EB1 dimer vs monomer as well as plus-end regulation in cells. PMID:25111064

  6. Real-time NMR Study of Three Small GTPases Reveals That Fluorescent 2′(3′)-O-(N-Methylanthraniloyl)-tagged Nucleotides Alter Hydrolysis and Exchange Kinetics*

    PubMed Central

    Mazhab-Jafari, Mohammad T.; Marshall, Christopher B.; Smith, Matthew; Gasmi-Seabrook, Geneviève M. C.; Stambolic, Vuk; Rottapel, Robert; Neel, Benjamin G.; Ikura, Mitsuhiko

    2010-01-01

    The Ras family of small GTPases control diverse signaling pathways through a conserved “switch” mechanism, which is turned on by binding of GTP and turned off by GTP hydrolysis to GDP. Full understanding of GTPase switch functions requires reliable, quantitative assays for nucleotide binding and hydrolysis. Fluorescently labeled guanine nucleotides, such as 2′(3′)-O-(N-methylanthraniloyl) (mant)-substituted GTP and GDP analogs, have been widely used to investigate the molecular properties of small GTPases, including Ras and Rho. Using a recently developed NMR method, we show that the kinetics of nucleotide hydrolysis and exchange by three small GTPases, alone and in the presence of their cognate GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors, are affected by the presence of the fluorescent mant moiety. Intrinsic hydrolysis of mantGTP by Ras homolog enriched in brain (Rheb) is ∼10 times faster than that of GTP, whereas it is 3.4 times slower with RhoA. On the other hand, the mant tag inhibits TSC2GAP-catalyzed GTP hydrolysis by Rheb but promotes p120 RasGAP-catalyzed GTP hydrolysis by H-Ras. Guanine nucleotide exchange factor-catalyzed nucleotide exchange for both H-Ras and RhoA was inhibited by mant-substituted nucleotides, and the degree of inhibition depends highly on the GTPase and whether the assay measures association of mantGTP with, or dissociation of mantGDP from the GTPase. These results indicate that the mant moiety has significant and unpredictable effects on GTPase reaction kinetics and underscore the importance of validating its use in each assay. PMID:20018863

  7. GTP-binding proteins in rat liver nuclear envelopes.

    PubMed Central

    Rubins, J B; Benditt, J O; Dickey, B F; Riedel, N

    1990-01-01

    Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of [alpha-32P]GTP binding to Western-blotted proteins and UV crosslinking of [alpha-32P]GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and [alpha-32P]GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with [alpha-32P]GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all [alpha-32P]GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with [alpha-32P]ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membrane vesicles required for reassembly of the nucleus after mitosis. Images PMID:2119502

  8. Modulation of cardiac L-type Ca2+ channels by GTP gamma S in response to isoprenaline, forskolin and photoreleased nucleotides.

    PubMed Central

    Kozlowski, R. Z.; Goodstadt, L. J.; Twist, V. W.; Powell, T.

    1994-01-01

    1. Using the patch-clamp recording technique, we have investigated the effects of chronic intracellular application of guanosine thiotriphosphate (GTP gamma S) by cell dialysis, on the potentiation of L-type Ca2+ currents (ICa) by isoprenaline and forskolin and also by GTP gamma S and cyclic AMP released intracellularly by flash-photolysis of their caged derivatives. 2. GTP gamma S prevented enhancement of ICa by isoprenaline with an IC50 of approximately 10 microM and considerably reduced the ability of forskolin to increase ICa. In addition GTP gamma S also reduced the time-to-peak response for potentiation of ICa by forskolin. Responses to forskolin were abolished by co-dialysis of cells with the cyclic AMP antagonist, Rp-adenosine-3'-5'-mono-thionophosphate (Rp-cAMPS). 3. Photoreleased GTP gamma S (PR-GTP gamma S; approximately 23 microM) generally induced a biphasic increase in ICa. This response was also inhibited by chronic intracellular dialysis with GTP gamma S with an IC50 of approximately 1 microM. 4. Pretreatment of cells with pertussis toxin (PTX) reversed the inhibitory effect of 100 microM GTP gamma S on isoprenaline-induced stimulation of ICa. However, PTX pretreatment did not restore the activating action of PR-GTP gamma S inhibited by chronic application of GTP gamma S. 5. Photoreleased cyclic AMP (approximately 5 microM; PR-cyclic AMP) increased peak ICa. This effect was inhibited by dialysis of cells with Rp-cAMPS and by stimulation of ICa by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. Co-dialysis of cells with uncaged GTP gamma S reduced the time-to-peak for PR-cyclic AMP mediated activation of ICa but did not affect the magnitude of the response. 6. It is concluded that chronically applied GTP gamma S can (i) inhibit activation of ICa by isoprenaline by interacting with a PTX-sensitive guanosine nucleotide binding (G-) protein located upstream of adenylate cyclase (possibly Gi) and (ii) accelerate the response to cyclic AMP

  9. Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat

    PubMed Central

    Kung, Leslie F; Pagant, Silvere; Futai, Eugene; D'Arcangelo, Jennifer G; Buchanan, Roy; Dittmar, John C; Reid, Robert J D; Rothstein, Rodney; Hamamoto, Susan; Snapp, Erik L; Schekman, Randy; Miller, Elizabeth A

    2012-01-01

    Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly. PMID:22157747

  10. Crystal structure of transglutaminase 2 with GTP complex and amino acid sequence evidence of evolution of GTP binding site.

    PubMed

    Jang, Tae-Ho; Lee, Dong-Sup; Choi, Kihang; Jeong, Eui Man; Kim, In-Gyu; Kim, Young Whan; Chun, Jung Nyeo; Jeon, Ju-Hong; Park, Hyun Ho

    2014-01-01

    Transglutaminase2 (TG2) is a multi-functional protein involved in various cellular processes, including apoptosis, differentiation, wound healing, and angiogenesis. The malfunction of TG2 causes many human disease including inflammatory disease, celiac disease, neurodegenerative diseases, tissue fibrosis, and cancers. Protein cross-linking activity, which is representative of TG2, is activated by calcium ions and suppressed by GTP. Here, we elucidated the structure of TG2 in complex with its endogenous inhibitor, GTP. Our structure showed why GTP is the optimal nucleotide for interacting with and inhibiting TG2. In addition, sequence comparison provided information describing the evolutionary scenario of GTP usage for controlling the activity of TG2.

  11. Subcellular distribution of small GTP binding proteins in pancreas: Identification of small GTP binding proteins in the rough endoplasmic reticulum

    SciTech Connect

    Nigam, S.K. )

    1990-02-01

    Subfractionation of a canine pancreatic homogenate was performed by several differential centrifugation steps, which gave rise to fractions with distinct marker profiles. Specific binding of guanosine 5{prime}-({gamma}-({sup 35}S)thio)triphosphate (GTP({gamma}-{sup 35}S)) was assayed in each fraction. Enrichment of GTP({gamma}-{sup 35}S) binding was greatest in the interfacial smooth microsomal fraction, expected to contain Golgi and other smooth vesicles. There was also marked enrichment in the rough microsomal fraction. Electron microscopy and marker protein analysis revealed the rough microsomes (RMs) to be highly purified rough endoplasmic reticulum (RER). The distribution of small (low molecular weight) GTP binding proteins was examined by a ({alpha}-{sup 32}P)GTP blot-overlay assay. Several apparent GTP binding proteins of molecular masses 22-25 kDa were detected in various subcellular fractions. In particular, at least two such proteins were found in the Golgi-enriched and RM fractions, suggesting that these small GTP binding proteins were localized to the Golgi and RER. To more precisely localize these proteins to the RER, native RMs and RMs stripped of ribosomes by puromycin/high salt were subjected to isopycnic centrifugation. The total GTP({gamma}-{sup 35}S) binding, as well as the small GTP binding proteins detected by the ({alpha}-{sup 32}P)GTP blot overlay, distributed into fractions of high sucrose density, as did the RER marker ribophorin I. Consistent with a RER localization, when the RMS were stripped of ribosomes and subjected to isopycnic centrifugation, the total GTP({gamma}-{sup 35}S) binding and the small GTP binding proteins detected in the blot-overlay assay shifted to fractions of lighter sucrose density along with the RER marker.

  12. Synthesis and application of a novel GTP phosphonamide photoaffinity reagent: Study of exchangeable GTP-binding domain on tubulin

    SciTech Connect

    Chavan, A.J.

    1989-01-01

    The regulatory role played by nucleotides in various biochemical processes has been the topic of study for several years. This has led to the identification of several regulatory proteins which require guanosine triphosphate (GTP), a nucleotide, for their action. The G-protein family utilizes GTP and displays many common features in the mechanism of their action. Chapter 1 is an overview of several GTP-binding proteins including G-proteins. Tubulin, a structural protein also requires GTP for its assembly/disassembly process. The nature of interaction of GTP with tubulin, particularly at the exchangeable GTP-binding site has been under investigation for several years. Chapter 2 presents an overview of the structure of tubulin, and the studies leading up to the present understanding of the GTP-tubulin interaction. To study the exchangeable GTP-binding domain on tubulin which would complement the other techniques, and reagents used, the author synthesized a novel GTP phosphonamide photoaffinity reagent. Chapter 3 describes the synthesis of {sup 125}I-APTG (36). He utilized {sup 125}I-APTG to establish that it could act as a mimic to GTP. Using the combination of anion-exclusion chromatography, gel-filtration and reverse-phase HPLC techniques he isolated residues 65-79 on the {beta}-chain as the major peptide in the GTP-binding domain. He also isolate residues 65-79 and 353-370 on the {alpha}-chain as minor peptides supporting the hypothesis that the phosphate region is in close proximity of the {alpha}-subunit.

  13. Structural model of FeoB, the iron transporter from Pseudomonas aeruginosa, predicts a cysteine lined, GTP-gated pore.

    PubMed

    Seyedmohammad, Saeed; Fuentealba, Natalia Alveal; Marriott, Robert A J; Goetze, Tom A; Edwardson, J Michael; Barrera, Nelson P; Venter, Henrietta

    2016-01-01

    Iron is essential for the survival and virulence of pathogenic bacteria. The FeoB transporter allows the bacterial cell to acquire ferrous iron from its environment, making it an excellent drug target in intractable pathogens. The protein consists of an N-terminal GTP-binding domain and a C-terminal membrane domain. Despite the availability of X-ray crystal structures of the N-terminal domain, many aspects of the structure and function of FeoB remain unclear, such as the structure of the membrane domain, the oligomeric state of the protein, the molecular mechanism of iron transport, and how this is coupled to GTP hydrolysis at the N-terminal domain. In the present study, we describe the first homology model of FeoB. Due to the lack of sequence homology between FeoB and other transporters, the structures of four different proteins were used as templates to generate the homology model of full-length FeoB, which predicts a trimeric structure. We confirmed this trimeric structure by both blue-native-PAGE (BN-PAGE) and AFM. According to our model, the membrane domain of the trimeric protein forms a central pore lined by highly conserved cysteine residues. This pore aligns with a central pore in the N-terminal GTPase domain (G-domain) lined by aspartate residues. Biochemical analysis of FeoB from Pseudomonas aeruginosa further reveals a putative iron sensor domain that could connect GTP binding/hydrolysis to the opening of the pore. These results indicate that FeoB might not act as a transporter, but rather as a GTP-gated channel.

  14. Structural model of FeoB, the iron transporter from Pseudomonas aeruginosa, predicts a cysteine lined, GTP-gated pore

    PubMed Central

    Seyedmohammad, Saeed; Fuentealba, Natalia Alveal; Marriott, Robert A.J.; Goetze, Tom A.; Edwardson, J. Michael; Barrera, Nelson P.; Venter, Henrietta

    2016-01-01

    Iron is essential for the survival and virulence of pathogenic bacteria. The FeoB transporter allows the bacterial cell to acquire ferrous iron from its environment, making it an excellent drug target in intractable pathogens. The protein consists of an N-terminal GTP-binding domain and a C-terminal membrane domain. Despite the availability of X-ray crystal structures of the N-terminal domain, many aspects of the structure and function of FeoB remain unclear, such as the structure of the membrane domain, the oligomeric state of the protein, the molecular mechanism of iron transport, and how this is coupled to GTP hydrolysis at the N-terminal domain. In the present study, we describe the first homology model of FeoB. Due to the lack of sequence homology between FeoB and other transporters, the structures of four different proteins were used as templates to generate the homology model of full-length FeoB, which predicts a trimeric structure. We confirmed this trimeric structure by both blue-native-PAGE (BN-PAGE) and AFM. According to our model, the membrane domain of the trimeric protein forms a central pore lined by highly conserved cysteine residues. This pore aligns with a central pore in the N-terminal GTPase domain (G-domain) lined by aspartate residues. Biochemical analysis of FeoB from Pseudomonas aeruginosa further reveals a putative iron sensor domain that could connect GTP binding/hydrolysis to the opening of the pore. These results indicate that FeoB might not act as a transporter, but rather as a GTP-gated channel. PMID:26934982

  15. Dynamic Role of the GTP Energy Metabolism in Cancers.

    PubMed

    Sasaki, Atsuo T

    2016-01-01

    Rapid growing cells like tumor cells need a vast amount of energy to match their high metabolic demand. Guanine triphosphate (GTP) is one of major cellular metabolites and served as a building block for RNA and DNA as well as an energy source to drive cellular activities such as intracellular trafficking, the cell migration and translation. However, how cancer cells regulate GTP energy levels to adapt for their high demand remain largely unknown yet. In addition, how cells detect GTP levels remains unknown. In this seminar, I will introduce our recent findings that uncover dramatic change of GTP metabolism in cancer cells and a GTP sensing kinase that regulate metabolism for tumorigenesis.(Presented at the 1918th Meeting, March 3, 2016). PMID:27040886

  16. A green fluorescent protein solubility screen in E. coli reveals domain boundaries of the GTP-binding domain in the P element transposase

    PubMed Central

    Sabogal, Alex; Rio, Donald C

    2010-01-01

    Guanosine triphosphate (GTP) binding and hydrolysis events often act as molecular switches in proteins, modulating conformational changes between active and inactive states in many signaling molecules and transport systems. The P element transposase of Drosophila melanogaster requires GTP binding to proceed along its reaction pathway, following initial site-specific DNA binding. GTP binding is unique to P elements and may represent a novel form of transpositional regulation, allowing the bound transposase to find a second site, looping the transposon DNA for strand cleavage and excision. The GTP-binding activity has been previously mapped to the central portion of the transposase protein; however, the P element transposase contains little sequence identity with known GTP-binding folds. To identify soluble, active transposase domains, a GFP solubility screen was used testing the solubility of random P element gene fragments in E. coli. The screen produced a single clone spanning known GTP-binding residues in the central portion of the transposase coding region. This clone, amino acids 275–409 in the P element transposase, was soluble, highly expressed in E.coli and active for GTP-binding activity, therefore is a candidate for future biochemical and structural studies. In addition, the chimeric screen revealed a minimal N-terminal THAP DNA-binding domain attached to an extended leucine zipper coiled-coil dimerization domain in the P element transposase, precisely delineating the DNA-binding and dimerization activities on the primary sequence. This study highlights the use of a GFP-based solubility screen on a large multidomain protein to identify highly expressed, soluble truncated domain subregions. PMID:20842711

  17. X-ray and Cryo-EM structures reveal mutual conformational changes of Kinesin and GTP-state microtubules upon binding

    PubMed Central

    Morikawa, Manatsu; Yajima, Hiroaki; Nitta, Ryo; Inoue, Shigeyuki; Ogura, Toshihiko; Sato, Chikara; Hirokawa, Nobutaka

    2015-01-01

    The molecular motor kinesin moves along microtubules using energy from ATP hydrolysis in an initial step coupled with ADP release. In neurons, kinesin-1/KIF5C preferentially binds to the GTP-state microtubules over GDP-state microtubules to selectively enter an axon among many processes; however, because the atomic structure of nucleotide-free KIF5C is unavailable, its molecular mechanism remains unresolved. Here, the crystal structure of nucleotide-free KIF5C and the cryo-electron microscopic structure of nucleotide-free KIF5C complexed with the GTP-state microtubule are presented. The structures illustrate mutual conformational changes induced by interaction between the GTP-state microtubule and KIF5C. KIF5C acquires the ‘rigor conformation’, where mobile switches I and II are stabilized through L11 and the initial portion of the neck-linker, facilitating effective ADP release and the weak-to-strong transition of KIF5C microtubule affinity. Conformational changes to tubulin strengthen the longitudinal contacts of the GTP-state microtubule in a similar manner to GDP-taxol microtubules. These results and functional analyses provide the molecular mechanism of the preferential binding of KIF5C to GTP-state microtubules. PMID:25777528

  18. Part I: RNA hydrolysis catalyzed by imidazole compounds. Part II. Hydrophobic acceleration of reactions and mimics of thiamin-dependent enzymes

    SciTech Connect

    Kool, E.T.

    1988-01-01

    Catalysts modeled after the active site groups of the enzyme Ribonuclease A were synthesized and tested for catalysis of the hydrolysis of poly(rU), using a quantitative assay. The most effective of all the catalysts is N,N{prime}-bis-imidazolylmethane, which gave a four-fold rate enhancement as compared to N-methyl-imidazole. The structure/activity relationships are discussed in light of the ribonuclease mechanism. Also examined were reactions catalyzed by the coenzyme thiamine. In an investigation of the effects of restricting conformational freedom, a thiazolium salt was attached in two positions to {beta}-cyclodextrin. Since the catalyst gave about the same rate for tritium exchange from benzaldehyde as singly-attached catalysts, we surmise that any rate enhancement due to the restriction of bond rotations has been lost by forcing the structure into less productive conformations. The benzoin condensation catalyzed by cyanide was also investigated. The reaction was shown to be faster in water than in most organic solvents. Kinetic salt effects and the effects of added {beta}- and {gamma}-cyclodextrin were measured in water; salting-out ions and {gamma}-cyclodextrin increase the rate, while salting-in ions and {beta}-cyclodextrin decrease it. Negative salt effects were observed in formamide, ethylene glycol, and DMSO. All these media effects are discussed in relation to the compact, hydrophobic transition state for the reaction.

  19. GTP gamma S increases Nav1.8 current in small-diameter dorsal root ganglia neurons.

    PubMed

    Saab, Carl Y; Cummins, Theodore R; Waxman, Stephen G

    2003-10-01

    Tetrodotoxin-resistant (TTX-R) sodium current in small-size dorsal root ganglia (DRG) neurons is upregulated by prostaglandin E(2) and serotonin through a protein kinase A (PKA)/protein kinase (PKC) pathway, suggesting G protein modulation of one or more TTX-R channels in these neurons. Recently, GTP(gammaS), a hydrolysis-resistant analogue of GTP, was shown to increase the persistent current produced by the TTX-R Na(v)1.9. In this study, we investigated the modulation of another TTX-R channel, Na(v)1.8, by GTP(gammaS) in small-diameter DRG neurons from rats using whole-cell voltage clamp recordings. Because it has been suggested that fluoride, often used in intracellular recording solutions, may bind to trace amounts of aluminum and activate G proteins, we recorded Na(v)1.8 currents with and without intracellular fluoride, and with the addition of deferoxamine, an aluminum chelator, to prevent fluoride-aluminum binding. Our results show that GTP(gammaS) (100 micro M) caused a significant increase in Na(v)1.8 current (67%) with a chloride-based intracellular solution. Although the inclusion of fluoride instead of chloride in the pipette solution increased the Na(v)1.8 current by 177%, GTP(gammaS) further increased Na(v)1.8 current by 67% under these conditions. While the effect of GTP(gammaS) was prevented by pretreatment with H7 (100 micro M), a non-selective PKA/PKC inhibitor, the fluoride-induced increase in Na(v)1.8 current was not sensitive to H7 (100 micro M), or to inclusion of deferoxamine (1 mM) in the intracellular solution. We conclude that G protein activation by GTP(gammaS) increases Na(v)1.8 current through a PKA/PKC mechanism and that addition of fluoride to the pipette solution further enhances the current, but is not a confounding variable in the study of Na(v)1.8 channel modulation by G proteins independent of a PKA/PKC pathway or binding to aluminum. PMID:12898089

  20. Affinity labeling of GTP-binding proteins in cellular extracts.

    PubMed

    Löw, A; Faulhammer, H G; Sprinzl, M

    1992-05-25

    GTP-binding proteins in cellular extracts from Escherichia coli, Thermus thermophilus, yeast, wheat germ or calf thymus were identified using in situ periodate-oxidized [alpha-32P]GTP as affinity label. Site-specific reaction of individual GTP-binding proteins was achieved by cross-linking the protein-bound 2',3'-dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiff's base formation and subsequent cyanoborohydride reduction. Labeled GTP-binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. In addition cross-linking of [alpha-32P]GTP with GTP-binding proteins was demonstrated in model systems using different purified GTPases, human c-H-ras p21, transducin from bovine retina, polypeptide elongation factor Tu (EF-Tu) from T. thermophilus and initiation factor 2 (IF2) from T. thermophilus. The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras-proteins, elongation and initiation factors, and heterotrimeric signal transducing G-proteins. PMID:1592117

  1. Differential dynamics of RAS isoforms in GDP- and GTP-bound states.

    PubMed

    Kapoor, Abhijeet; Travesset, Alex

    2015-06-01

    RAS subfamily proteins regulates cell growth promoting signaling processes by cycling between active (GTP-bound) and inactive (GDP-bound) states. Different RAS isoforms, though structurally similar, exhibit functional specificity and are associated with different types of cancers and developmental disorders. Understanding the dynamical differences between the isoforms is crucial for the design of inhibitors that can selectively target a particular malfunctioning isoform. In this study, we provide a comprehensive comparison of the dynamics of all the three RAS isoforms (HRAS, KRAS, and NRAS) using extensive molecular dynamics simulations in both the GDP- (total of 3.06 μs) and GTP-bound (total of 2.4 μs) states. We observed significant differences in the dynamics of the isoforms, which rather interestingly, varied depending on the type of the nucleotide bound and the simulation temperature. Both SwitchI (Residues 25-40) and SwitchII (Residues 59-75) differ significantly in their flexibility in the three isoforms. Furthermore, Principal Component Analysis showed that there are differences in the conformational space sampled by the GTP-bound RAS isoforms. We also identified a previously unreported pocket, which opens transiently during MD simulations, and can be targeted to regulate nucleotide exchange reaction or possibly interfere with membrane localization. Further, we present the first simulation study showing GDP destabilization in the wild-type RAS protein. The destabilization of GDP/GTP occurred only in 1/50 simulations, emphasizing the need of guanine nucleotide exchange factors (GEFs) to accelerate such an extremely unfavorable process. This observation along with the other results presented in this article further support our previously hypothesized mechanism of GEF-assisted nucleotide exchange.

  2. Enhanced enzymatic saccharification of pretreated biomass using glycerol thermal processing (GTP).

    PubMed

    Zhang, Wei; Sathitsuksanoh, Noppadon; Barone, Justin R; Renneckar, Scott

    2016-01-01

    Biomass was heated (200-240°C) in the presence of glycerol, for 4-12 min, under shear to disrupt the native cell wall architecture. The impact of this method, named glycerol thermal processing (GTP), on saccharification efficiency of the hardwood Liquidambar styraciflua, and a control cellulose sample was studied as a function of treatment severity. Furthermore, the enzymatic conversion of samples with varying compositions was studied after extraction of the structural polymers. Interestingly, the sweet gum processed materials crystallinity index increased by 10% of the initial value. The experiments revealed that the residual lignin was not a barrier to limiting the digestibility of cellulose after pretreatment yielding up to 70% glucose based on the starting wood material. Further xylan removal greatly improved the cellulose hydrolysis rate, converting nearly 70% of the cellulose into glucose within 24h, and reaching 78% of ultimate glucan digestibility after 72 h.

  3. Theoretical vibrational spectroscopy of intermediates and the reaction mechanism of the guanosine triphosphate hydrolysis by the protein complex Ras-GAP

    NASA Astrophysics Data System (ADS)

    Khrenova, Maria G.; Grigorenko, Bella L.; Nemukhin, Alexander V.

    2016-09-01

    The structures and vibrational spectra of the reacting species upon guanosine triphosphate (GTP) hydrolysis to guanosine diphosphate and inorganic phosphate (Pi) trapped inside the protein complex Ras-GAP were analyzed following the results of QM/MM simulations. The frequencies of the phosphate vibrations referring to the reactants and to Pi were compared to those observed in the experimental FTIR studies. A good correlation between the theoretical and experimental vibrational data provides a strong support to the reaction mechanism of GTP hydrolysis by the Ras-GAP enzyme system revealed by the recent QM/MM modeling. Evolution of the vibrational bands associated with the inorganic phosphate Pi during the elementary stages of GTP hydrolysis is predicted.

  4. Higher-Order Septin Assembly Is Driven by GTP-Promoted Conformational Changes: Evidence From Unbiased Mutational Analysis in Saccharomyces cerevisiae

    PubMed Central

    Weems, Andrew D.; Johnson, Courtney R.; Argueso, Juan Lucas; McMurray, Michael A.

    2014-01-01

    Septin proteins bind GTP and heterooligomerize into filaments with conserved functions across a wide range of eukaryotes. Most septins hydrolyze GTP, altering the oligomerization interfaces; yet mutations designed to abolish nucleotide binding or hydrolysis by yeast septins perturb function only at high temperatures. Here, we apply an unbiased mutational approach to this problem. Mutations causing defects at high temperature mapped exclusively to the oligomerization interface encompassing the GTP-binding pocket, or to the pocket itself. Strikingly, cold-sensitive defects arise when certain of these same mutations are coexpressed with a wild-type allele, suggestive of a novel mode of dominance involving incompatibility between mutant and wild-type molecules at the septin–septin interfaces that mediate filament polymerization. A different cold-sensitive mutant harbors a substitution in an unstudied but highly conserved region of the septin Cdc12. A homologous domain in the small GTPase Ran allosterically regulates GTP-binding domain conformations, pointing to a possible new functional domain in some septins. Finally, we identify a mutation in septin Cdc3 that restores the high-temperature assembly competence of a mutant allele of septin Cdc10, likely by adopting a conformation more compatible with nucleotide-free Cdc10. Taken together, our findings demonstrate that GTP binding and hydrolysis promote, but are not required for, one-time events—presumably oligomerization-associated conformational changes—during assembly of the building blocks of septin filaments. Restrictive temperatures impose conformational constraints on mutant septin proteins, preventing new assembly and in certain cases destabilizing existing assemblies. These insights from yeast relate directly to disease-causing mutations in human septins. PMID:24398420

  5. Intracellular GTP level determines cell's fate toward differentiation and apoptosis

    SciTech Connect

    Meshkini, Azadeh; Yazdanparast, Razieh Nouri, Kazem

    2011-06-15

    Since the adequate supply of guanine nucleotides is vital for cellular activities, limitation of their syntheses would certainly result in modulation of cellular fate toward differentiation and apoptosis. The aim of this study was to set a correlation between the intracellular level of GTP and the induction of relevant signaling pathways involved in the cell's fate toward life or death. In that regard, we measured the GTP level among human leukemia K562 cells exposed to mycophenolic acid (MPA) or 3-hydrogenkwadaphnin (3-HK) as two potent inosine monophosphate dehydrogenase inhibitors. Our results supported the maturation of the cells when the intracellular GTP level was reduced by almost 30-40%. Under these conditions, 3-HK and/or MPA caused up-regulation of PKC{alpha} and PI3K/AKT pathways. Furthermore, co-treatment of cells with hypoxanthine plus 3-HK or MPA, which caused a reduction of about 60% in the intracellular GTP levels, led to apoptosis and activation of mitochondrial pathways through inverse regulation of Bcl-2/Bax expression and activation of caspase-3. Moreover, our results demonstrated that attenuation of GTP by almost 60% augmented the intracellular ROS and nuclear localization of p21 and subsequently led to cell death. These results suggest that two different threshold levels of GTP are needed for induction of differentiation and/or ROS-associated apoptosis. - Graphical abstract: Display Omitted

  6. Detection of GTP-binding proteins in barley aleurone protoplasts.

    PubMed

    Wang, M; Sedee, N J; Heidekamp, F; Snaar-Jagalska, B E

    1993-08-30

    We report the existence of several families of GTP-binding proteins in barley aleurone protoplasts. Partial purified plasma membrane proteins were separated by SDS-PAGE, transferred to a nitrocellulose filter and incubated with either antisera raised against a highly conserved animal G protein alpha subunit peptide/or Ras protein, or with [alpha-32P]GTP. Two sets of proteins of M(r) = 32-36 kDa and 22-24 kDa were strongly recognized by the antisera. Binding of [alpha-32P]GTP was detected on Western blots with proteins of M(r) = 22-24 kDa and 16 kDa. Binding was inhibited by 10(-7)-10(-6) M GTP gamma S, GTP or GDP; binding was not affected by 10(-6)-10(-5) M ATP gamma S or ADP. The kinetics, specificity and the effects of phytohormones in a [35S]GTP gamma S binding assay were also studied in isolated plasma membranes of barley aleurone protoplasts.

  7. Crystal structure of transglutaminase 2 with GTP complex and amino acid sequence evidence of evolution of GTP binding site.

    PubMed

    Jang, Tae-Ho; Lee, Dong-Sup; Choi, Kihang; Jeong, Eui Man; Kim, In-Gyu; Kim, Young Whan; Chun, Jung Nyeo; Jeon, Ju-Hong; Park, Hyun Ho

    2014-01-01

    Transglutaminase2 (TG2) is a multi-functional protein involved in various cellular processes, including apoptosis, differentiation, wound healing, and angiogenesis. The malfunction of TG2 causes many human disease including inflammatory disease, celiac disease, neurodegenerative diseases, tissue fibrosis, and cancers. Protein cross-linking activity, which is representative of TG2, is activated by calcium ions and suppressed by GTP. Here, we elucidated the structure of TG2 in complex with its endogenous inhibitor, GTP. Our structure showed why GTP is the optimal nucleotide for interacting with and inhibiting TG2. In addition, sequence comparison provided information describing the evolutionary scenario of GTP usage for controlling the activity of TG2. PMID:25192068

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

    NASA Astrophysics Data System (ADS)

    Wang, Jianghua

    1999-07-01

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

  9. A Method to Determine 18O Kinetic Isotope Effects in the Hydrolysis of Nucleotide Triphosphates

    PubMed Central

    Du, Xinlin; Ferguson, Kurt; Sprang, Stephen R.

    2007-01-01

    A method to determine 18O kinetic isotope effects (KIE) in the hydrolysis of GTP is described that is generally applicable to reactions involving other nucleotide triphosphates. Internal competition, wherein the substrate of the reaction is a mixture of 18O-labeled and unlabeled nucleotides, is employed and the change in relative abundance of the two species in the course of the reaction is used to calculate KIE. The nucleotide labeled with 18O at sites of mechanistic interest also contains 13C at all carbon positions, while the 16O-nucleotide is depleted of 13C. The relative abundance of the labeled and unlabeled substrates or products is reflected in the carbon isotope ratio (13C/12C) in GTP or GDP, which is determined by use of a liquid chromatography-coupled isotope ratio mass spectrometer (LC-coupled IRMS). The LC is coupled to the IRMS by an Isolink™ interface (ThermoFinnigan). Carbon isotope ratios can be determined with accuracy and precision greater than 0.04%, and are consistent over an order of magnitude in sample amount. KIE values for Ras/NF1333-catalyzed hydrolysis of [β18O3,13C]GTP were determined by change in the isotope ratio of GTP or GDP or the ratio of the isotope ratio of GDP to that of GTP. KIE values computed in the three ways agree within 0.1%, although the method using the ratio of isotope ratios of GDP and GTP gives superior precision (< 0.1%). A single KIE measurement can be conducted in 25 minutes with less than 5 μg nucleotide reaction product. PMID:17963711

  10. Crystal structure of Rab6A'(Q72L) mutant reveals unexpected GDP/Mg²⁺ binding with opened GTP-binding domain.

    PubMed

    Shin, Young-Cheul; Yoon, Jong Hwan; Jang, Tae-Ho; Kim, Seo Yun; Heo, Won Do; So, Insuk; Jeon, Ju-Hong; Park, Hyun Ho

    2012-07-27

    The Ras small G protein-superfamily is a family of GTP hydrolases whose activity is regulated by GTP/GDP binding states. Rab6A, a member of the Ras superfamily, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, biosynthesis, secretion, cell differentiation and cell growth. Rab6A exists in two isoforms, termed RabA and Rab6A'. Substitution of Gln72 to Leu72 (Q72L) at Rab6 family blocks GTP hydrolysis activity and this mutation usually causes the Rab6 protein to be constitutively in an active form. Here, we report the crystal structure of the human Rab6A'(Q72L) mutant form at 1.9Å resolution. Unexpectedly, we found that Rab6A'(Q72L) possesses GDP/Mg(2+) in the GTP binding pockets, which is formed by a flexible switch I and switch II. Large conformational changes were also detected in the switch I and switch II regions. Our structure revealed that the non-hydrolysable, constitutively active form of Rab6A' can accommodate GDP/Mg(2+) in the open conformation.

  11. 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...

  12. The hydrolysis of polyimides

    NASA Technical Reports Server (NTRS)

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

    1973-01-01

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

  13. Insights into nucleotide recognition by cell division protein FtsZ from a mant-GTP competition assay and molecular dynamics.

    PubMed

    Schaffner-Barbero, Claudia; Gil-Redondo, Rubén; Ruiz-Avila, Laura B; Huecas, Sonia; Läppchen, Tilman; den Blaauwen, Tanneke; Diaz, J Fernando; Morreale, Antonio; Andreu, Jose M

    2010-12-14

    Essential cell division protein FtsZ forms the bacterial cytokinetic ring and is a target for new antibiotics. FtsZ monomers bind GTP and assemble into filaments. Hydrolysis to GDP at the association interface between monomers leads to filament disassembly. We have developed a homogeneous competition assay, employing the fluorescence anisotropy change of mant-GTP upon binding to nucleotide-free FtsZ, which detects compounds binding to the nucleotide site in FtsZ monomers and measures their affinities within the millimolar to 10 nM range. We have employed this method to determine the apparent contributions of the guanine, ribose, and the α-, β-, and γ-phosphates to the free energy change of nucleotide binding. Similar relative contributions have also been estimated through molecular dynamics and binding free energy calculations, employing the crystal structures of FtsZ-nucleotide complexes. We find an energetically dominant contribution of the β-phosphate, comparable to the whole guanosine moiety. GTP and GDP bind with similar observed affinity to FtsZ monomers. Loss of the regulatory γ-phosphate results in a predicted accommodation of GDP which has not been observed in the crystal structures. The binding affinities of a series of C8-substituted GTP analogues, known to inhibit FtsZ but not eukaryotic tubulin assembly, correlate with their inhibitory capacity on FtsZ polymerization. Our methods permit testing of FtsZ inhibitors targeting its nucleotide site, as well as compounds from virtual screening of large synthetic libraries. Our results give insight into the FtsZ-nucleotide interactions, which could be useful in the rational design of new inhibitors, especially GTP phosphate mimetics.

  14. The GTP binding motif: variations on a theme.

    PubMed

    Kjeldgaard, M; Nyborg, J; Clark, B F

    1996-10-01

    GTP binding proteins (G-proteins) have wide-ranging functions in biology, being involved in cell proliferation, signal transduction, protein synthesis, and protein targeting. Common to their functioning is that they are active in the GTP-bound form and inactive in the GDP-bound form. The protein synthesis elongation factor EF-Tu was the first G-protein whose nucleotide binding domain was solved structurally by X-ray crystallography to yield a structural definition of the GDP-bound form, but a still increasing number of new structures of G-proteins are appearing in the literature, in both GDP and GTP bound forms. A common structural core for nucleotide binding is present in all these structures, and this core has long been known to include common consensus sequence elements involved in binding of the nucleotide. Nevertheless, subtle changes in the common sequences reflect functional differences. Therefore, it becomes increasingly important to focus on how these differences are reflected in the structures, and how these structural differences are related to function. The aim of this review is to describe to what extent this structural motif for GDP/GTP binding is common to other known structures of this class of proteins. We first describe the common structural core of the G-proteins. Next, examples are based on information available on the Ras protein superfamily, the targeting protein ARF, elongation factors EF-Tu and EF-G, and the heterotrimeric G-proteins. Finally, we discuss the important structures of complexes between GTP binding proteins and their substrates that have appeared in the literature recently.

  15. Progressing batch hydrolysis process

    DOEpatents

    Wright, J.D.

    1985-01-10

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

  16. Ribavirin-induced intracellular GTP depletion activates transcription elongation in coagulation factor VII gene expression.

    PubMed

    Suzuki, Atsuo; Miyawaki, Yuhri; Okuyama, Eriko; Murata, Moe; Ando, Yumi; Kato, Io; Takagi, Yuki; Takagi, Akira; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

    2013-01-01

    Coagulation FVII (Factor VII) is a vitamin K-dependent glycoprotein synthesized in hepatocytes. It was reported previously that FVII gene (F7) expression was up-regulated by ribavirin treatment in hepatitis C virus-infected haemophilia patients; however, its precise mechanism is still unknown. In the present study, we investigated the molecular mechanism of ribavirin-induced up-regulation of F7 expression in HepG2 (human hepatoma cell line). We found that intracellular GTP depletion by ribavirin as well as other IMPDH (inosine-5'-monophosphate dehydrogenase) inhibitors, such as mycophenolic acid and 6-mercaptopurine, up-regulated F7 expression. FVII mRNA transcription was mainly enhanced by accelerated transcription elongation, which was mediated by the P-TEFb (positive-transcription elongation factor b) complex, rather than by promoter activation. Ribavirin unregulated ELL (eleven-nineteen lysine-rich leukaemia) 3 mRNA expression before F7 up-regulation. We observed that ribavirin enhanced ELL3 recruitment to F7, whereas knockdown of ELL3 diminished ribavirin-induced FVII mRNA up-regulation. Ribavirin also enhanced recruitment of CDK9 (cyclin-dependent kinase 9) and AFF4 to F7. These data suggest that ribavirin-induced intracellular GTP depletion recruits a super elongation complex containing P-TEFb, AFF4 and ELL3, to F7, and modulates FVII mRNA transcription elongation. Collectively, we have elucidated a basal mechanism for ribavirin-induced FVII mRNA up-regulation by acceleration of transcription elongation, which may be crucial in understanding its pleiotropic functions in vivo.

  17. Phosphate-modified analogues of m(7)GTP and m(7)Gppppm(7)G-Synthesis and biochemical properties.

    PubMed

    Ziemniak, Marcin; Kowalska, Joanna; Lukaszewicz, Maciej; Zuberek, Joanna; Wnek, Katarzyna; Darzynkiewicz, Edward; Jemielity, Jacek

    2015-09-01

    The synthesis and biochemical properties of 17 new mRNA cap analogues are reported. Six of these nucleotides are m(7)GTP derivatives, whereas 11 are 'two headed' tetraphosphate dinucleotides based on a m(7)Gppppm(7)G structure. The compounds contain either a boranophosphate or phosphorothioate moiety in the nucleoside neighbouring position(s) and some of them possess an additional methylene group between β and γ phosphorus atoms. The compounds were prepared by divalent metal chloride-mediated coupling of an appropriate m(7)GMP analogue with a given P(1),P(2)-di(1-imidazolyl) derivative. The analogues were evaluated as tools for studying cap-dependent processes in a number of biochemical assays, including determination of affinity to eukaryotic initiation factor eIF4E, susceptibility to enzymatic hydrolysis, and translational efficiency in vitro. The results indicate that modification in the phosphate chain can increase binding to cap-interacting proteins and provides higher resistance to degradation. Furthermore, modified derivatives of m(7)GTP were found to be potent inhibitors of cap-dependent translation in cell free systems.

  18. Immunochemical similarity of GTP-binding proteins from different systems

    SciTech Connect

    Kalinina, S.N.

    1986-06-20

    It was found that antibodies against the GTP-binding proteins of bovine retinal photoreceptor membranes blocked the inhibitory effect of estradiol on phosphodiesterase from rat and human uterine cytosol and prevented the cumulative effect of catecholamines and guanylyl-5'-imidodiphosphate on rat skeletal muscle adenylate cyclase. It was established by means of double radial immunodiffusion that these antibodies form a precipitating complex with purified bovine brain tubulin as well as with retinal preparations obtained from eyes of the bull, pig, rat, frog, some species of fish, and one reptile species. Bands of precipitation were not observed with these antibodies when retinal preparations from invertebrates (squid and octopus) were used as the antigens. The antibodies obtained interacted with the ..cap alpha..- and ..beta..-subunits of GTP-binding proteins from bovine retinal photoreceptor membranes.

  19. Yrb4p, a yeast ran-GTP-binding protein involved in import of ribosomal protein L25 into the nucleus.

    PubMed Central

    Schlenstedt, G; Smirnova, E; Deane, R; Solsbacher, J; Kutay, U; Görlich, D; Ponstingl, H; Bischoff, F R

    1997-01-01

    Gsp1p, the essential yeast Ran homologue, is a key regulator of transport across the nuclear pore complex (NPC). We report the identification of Yrb4p, a novel Gsp1p binding protein. The 123 kDa protein was isolated from Saccharomyces cerevisiae cells and found to be related to importin-beta, the mediator of nuclear localization signal (NLS)-dependent import into the nucleus, and to Pse1p. Like importin-beta, Yrb4p and Pse1p specifically bind to Gsp1p-GTP, protecting it from GTP hydrolysis and nucleotide exchange. The GTPase block of Gsp1p complexed to Yrb4p or Pse1p is released by Yrb1p, which contains a Gsp1p binding domain distinct from that of Yrb4p. This might reflect an in vivo function for Yrb1p. Cells disrupted for YRB4 are defective in nuclear import of ribosomal protein L25, but show no defect in the import of proteins containing classical NLSs. Expression of a Yrb4p mutant deficient in Gsp1p-binding is dominant-lethal and blocks bidirectional traffic across the NPC in wild-type cells. L25 binds to Yrb4p and Pse1p and is released by Gsp1p-GTP. Consistent with its putative role as an import receptor for L25-like proteins, Yrb4p localizes to the cytoplasm, the nucleoplasm and the NPC. PMID:9321403

  20. Mutagenesis in the switch IV of the helical domain of the human Gsalpha reduces its GDP/GTP exchange rate.

    PubMed

    Echeverría, V; Hinrichs, M V; Torrejón, M; Ropero, S; Martinez, J; Toro, M J; Olate, J

    2000-01-01

    The Galpha subunits of heterotrimeric G proteins are constituted by a conserved GTPase "Ras-like" domain (RasD) and by a unique alpha-helical domain (HD). Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes. Switch I, II, and III are located in RasD and switch IV in HD. All Galpha known functions, such as GTPase activity and receptor, effector, and Gbetagamma interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region. Through the construction of chimeras between human and Xenopus Gsalpha we have previously identified a HD region, encompassing helices alphaA, alphaB, and alphaC, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett 340:249-254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gsalpha proteins, in the present work we constructed two human Gsalpha mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGsalphaalphaS133N, M135P, P138K, P143S) and M17 (hGsalphaalphaS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTPgammaS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTPgammaS binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested. Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF(4)(-), but a decreased activation with GTPgammaS, which is consistent with the lower GDP dissociating rate they displayed. These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gsalpha subunit. PMID

  1. Progressing batch hydrolysis process

    DOEpatents

    Wright, John D.

    1986-01-01

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

  2. Acid hydrolysis of cellulose

    SciTech Connect

    Salazar, H.

    1980-12-01

    One of the alternatives to increase world production of etha nol is by the hydrolysis of cellulose content of agricultural residues. Studies have been made on the types of hydrolysis: enzimatic and acid. Data obtained from the sulphuric acid hydrolysis of cellulose showed that this process proceed in two steps, with a yield of approximately 95% glucose. Because of increases in cost of alternatives resources, the high demand of the product and the more economic production of ethanol from cellulose materials, it is certain that this technology will be implemented in the future. At the same time further studies on the disposal and reuse of the by-products of this production must be undertaken.

  3. Effect of Ultrasonic Frequency on Enzymatic Hydrolysis of Cellulose

    NASA Astrophysics Data System (ADS)

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

    2010-07-01

    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.

  4. GTP activator and dNTP substrates of HIV-1 restriction factor SAMHD1 generate a long-lived activated state

    PubMed Central

    Hansen, Erik C.; Seamon, Kyle J.; Cravens, Shannen L.; Stivers, James T.

    2014-01-01

    The HIV-1 restriction factor sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) is a tetrameric protein that catalyzes the hydrolysis of all dNTPs to the deoxynucleoside and tripolyphosphate, which effectively depletes the dNTP substrates of HIV reverse transcriptase. Here, we establish that SAMHD1 is activated by GTP binding to guanine-specific activator sites (A1) as well as coactivation by substrate dNTP binding to a distinct set of nonspecific activator sites (A2). Combined activation by GTP and dNTPs results in a long-lived tetrameric form of SAMHD1 that persists for hours, even after activating nucleotides are withdrawn from the solution. These results reveal an ordered model for assembly of SAMHD1 tetramer from its inactive monomer and dimer forms, where GTP binding to the A1 sites generates dimer and dNTP binding to the A2 and catalytic sites generates active tetramer. Thus, cellular regulation of active SAMHD1 is not determined by GTP alone but instead, the levels of all dNTPs and the generation of a persistent tetramer that is not in equilibrium with free activators. The significance of the long-lived activated state is that SAMHD1 can remain active long after dNTP pools have been reduced to a level that would lead to inactivation. This property would be important in resting CD4+ T cells, where dNTP pools are reduced to nanomolar levels to restrict infection by HIV-1. PMID:24753578

  5. SAXS analysis of the tRNA-modifying enzyme complex MnmE/MnmG reveals a novel interaction mode and GTP-induced oligomerization

    PubMed Central

    Fislage, Marcus; Brosens, Elke; Deyaert, Egon; Spilotros, Alessandro; Pardon, Els; Loris, Remy; Steyaert, Jan; Garcia-Pino, Abel; Versées, Wim

    2014-01-01

    Transfer ribonucleic acid (tRNA) modifications, especially at the wobble position, are crucial for proper and efficient protein translation. MnmE and MnmG form a protein complex that is implicated in the carboxymethylaminomethyl modification of wobble uridine (cmnm5U34) of certain tRNAs. MnmE is a G protein activated by dimerization (GAD), and active guanosine-5'-triphosphate (GTP) hydrolysis is required for the tRNA modification to occur. Although crystal structures of MnmE and MnmG are available, the structure of the MnmE/MnmG complex (MnmEG) and the nature of the nucleotide-induced conformational changes and their relevance for the tRNA modification reaction remain unknown. In this study, we mainly used small-angle X-ray scattering to characterize these conformational changes in solution and to unravel the mode of interaction between MnmE, MnmG and tRNA. In the nucleotide-free state MnmE and MnmG form an unanticipated asymmetric α2β2 complex. Unexpectedly, GTP binding promotes further oligomerization of the MnmEG complex leading to an α4β2 complex. The transition from the α2β2 to the α4β2 complex is fast, reversible and coupled to GTP binding and hydrolysis. We propose a model in which the nucleotide-induced changes in conformation and oligomerization of MnmEG form an integral part of the tRNA modification reaction cycle. PMID:24634441

  6. Identification of the guanosine interacting peptide of the GTP binding site of. beta. -tubulin using 8N/sub 3/GTP

    SciTech Connect

    Kim, H.; Ponstingl, H.; Haley, B.E.

    1987-05-01

    It has been shown earlier that 8N/sub 3/GTP is an effective biological mimic of GTP in that it interacts at the exchangeable GTP binding site and causes tubulin to polymerize in a fashion quite similar to GTP. Also, using ..cap alpha.. and ..gamma..(/sup 32/P)8N/sub 3/GTP it is now possible to selectively and effectively photolabel the ..beta..-subunit. A complete sequence of the ..cap alpha.. and ..beta..-subunits of porcine brain tubulin has been reported. They now report on conditions which optimize the photoinsertion of (/sup 32/P)8N/sub 3/GTP selectively into the ..beta..-subunit of tubulin and the use of cation-exchange column chromatography to assist in the purification of the photolabeled peptide. Using two slightly different procedures they have isolated the 8N/sub 3/GTP photolabeled peptide and identified its partial sequence as A-I-L-V-D-L-E-P-G-T which agrees with the highly conserved region of residues 63-72 of ..beta..-tubulin.

  7. 6-Acetyldihydrohomopterin and sepiapterin affect some GTP cyclohydrolase I's and not others

    SciTech Connect

    Jacobson, K.B.; Manos, R.E.

    1988-01-01

    The first enzyme in pteridine biosynthesis, GTP cyclohydrolase I, is a likely site for regulation of pteridine biosynthesis to occur. GTP cyclohydrolase I responds to hormonal treatment and is found altered in a variety of mice with genetically based neurological and immunological disorders. Genetic loci can greatly modify the activity of GTP cyclohydrolase: Punch mutant in Drosophila hph-1 in mouse and atypical phenylketonuria in human. This report examines the ability of Ahp and sepiapterin to alter the activity of GTP cyclohydrolase I from mouse liver, rat liver and Drosophila head. 20 refs., 2 tabs.

  8. Simplified /sup 14/CO/sub 2/-trapping microassay for GTP cyclohydrolases I and II

    SciTech Connect

    Shen, R.S.; Abell, C.W.

    1986-05-01

    GTP cyclohydrolases (GTP-CH) I and II catalyze the removal of the C/sub 8/ atom from GTP as formate. The reaction product of GTP-CH I is D-erythro-7,8-dihydroneopterin triphosphate, a key intermediate leading to the biosynthesis of folic acid in microorganisms and of tetrahydrobiopterin in mammals and microorganisms, and that of GTP-CH II is 2,5-diamino-6-hydroxy-4-(ribosylamino)pyrimidine 5'-phosphate, a key intermediate in the biosynthesis of riboflavin in microorganisms. They have simplified the /sup 14/CO/sub 2/-trapping assay of Burg and Brown for determining GTP-CH I and II activities. The assay consists of two consecutive steps which are carried out in a 2 ml tube. The first reaction yields formate from GTP (37/sup 0/C, 10 min). The reaction mixture contains 1 mM (8-/sup 14/C)-GTP (0.5 ..mu..Ci/..mu..mol), 50 mM Tris-HCl buffer (pH 8.2 for GTP-CH II and 7.7 for GTP-CH I), 0.2 M MgCl/sub 2/ for GTP-CH II or 0.3 M KCl and 1 mM EDTA for GTP-CH I, and enzyme in a final volume of 0.2 ml. The second reaction is the oxidation of /sup 14/C-formate to /sup 14/CO/sub 2/ (95/sup 0/C, 20 min) in the presence of 5% TCA and 1 mM formate (final volume 0.3 ml). Liberated /sup 14/CO/sub 2/ is trapped by the folded filter paper strip (1 x 4 cm), that has been placed inside the top of each tube and impregnated with 30 ..mu..l Protosol. This method is fast, comparable to the HPLC-fluorometric method for the assay of GTP-CH I activity, and ideal for performing a large number of determinations. Human and rat liver express both GTP-CH I and II activities. GTP-CH II is the predominant enzyme in both tissues and exists in multiple forms.

  9. Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii

    PubMed Central

    Singarapu, Kiran K.; Otte, Michele M.; Tonelli, Marco; Westler, William M.; Escalante-Semerena, Jorge C.; Markley, John L.

    2015-01-01

    GTP:adenosylcobinamide-phosphate (AdoCbi-P) guanylyl transferase (CobY) is an enzyme that transfers the GMP moiety of GTP to AdoCbi yielding AdoCbi-GDP in the late steps of the assembly of Ado-cobamides in archaea. The failure of repeated attempts to crystallize ligand-free (apo) CobY prompted us to explore its 3D structure by solution NMR spectroscopy. As reported here, the solution structure has a mixed α/β fold consisting of seven β-strands and five α-helices, which is very similar to a Rossmann fold. Titration of apo-CobY with GTP resulted in large changes in amide proton chemical shifts that indicated major structural perturbations upon complex formation. However, the CobY:GTP complex as followed by 1H-15N HSQC spectra was found to be unstable over time: GTP hydrolyzed and the protein converted slowly to a species with an NMR spectrum similar to that of apo-CobY. The variant CobYG153D, whose GTP complex was studied by X-ray crystallography, yielded NMR spectra similar to those of wild-type CobY in both its apo- state and in complex with GTP. The CobYG153D:GTP complex was also found to be unstable over time. PMID:26513744

  10. CaMKII uses GTP as a phosphate donor for both substrate and autophosphorylation

    PubMed Central

    Bostrom, S. Lynn; Dore, Justin; Griffith, Leslie C.

    2009-01-01

    The vast majority of serine/threonine protein kinases have a strong preference for ATP over GTP as a phosphate donor. CK2 (Casein kinase 2) is an exception to this rule and in this study we investigate whether calcium/calmodulin-dependent protein kinase II (CaMKII) has the same extended nucleotide range. Using the Drosophila enzyme, we have shown that CaMKII uses Mg2+GTP with a higher Km and Vmax compared to Mg2+ATP. Substitution of Mn2+ for Mg2+ resulted in a much lower Km for GTP, while nearly abolishing the ability of CaMKII to use ATP. These similar results were obtained with rat αCaMKII, showing the ability to use GTP to be a general property of CaMKII. The Vmax difference between Mg2+ATP and Mg2+GTP was found to be due to the fact that ADP is a potent inhibitor of phosphorylation, while GDP has modest effects. There were no differences found between sites autophosphorylated by ATP and GTP, either by partial proteolysis or mass spectrometry. Phosphorylation of fly head extract revealed that similar proteins are substrates for CaMKII whether using Mg2+ATP or Mg2+GTP. This new information confirms that CaMKII can use both ATP and GTP, and opens new avenues for the study of regulation of this kinase. PMID:19857459

  11. Autophagy in Saccharomyces cerevisiae requires the monomeric GTP-binding proteins, Arl1 and Ypt6

    PubMed Central

    Yang, Shu; Rosenwald, Anne G.

    2016-01-01

    ABSTRACT Macroautophagy/autophagy is a cellular degradation process that sequesters organelles or proteins into a double-membrane structure called the phagophore; this transient compartment matures into an autophagosome, which then fuses with the lysosome or vacuole to allow hydrolysis of the cargo. Factors that control membrane traffic are also essential for each step of autophagy. Here we demonstrate that 2 monomeric GTP-binding proteins in Saccharomyces cerevisiae, Arl1 and Ypt6, which belong to the Arf/Arl/Sar protein family and the Rab family, respectively, and control endosome-trans-Golgi traffic, are also necessary for starvation-induced autophagy under high temperature stress. Using established autophagy-specific assays we found that cells lacking either ARL1 or YPT6, which exhibit synthetic lethality with one another, were unable to undergo autophagy at an elevated temperature, although autophagy proceeds normally at normal growth temperature; specifically, strains lacking one or the other of these genes are unable to construct the autophagosome because these 2 proteins are required for proper traffic of Atg9 to the phagophore assembly site (PAS) at the restrictive temperature. Using degron technology to construct an inducible arl1Δ ypt6Δ double mutant, we demonstrated that cells lacking both genes show defects in starvation-inducted autophagy at the permissive temperature. We also found Arl1 and Ypt6 participate in autophagy by targeting the Golgi-associated retrograde protein (GARP) complex to the PAS to regulate the anterograde trafficking of Atg9. Our data show that these 2 membrane traffic regulators have novel roles in autophagy. PMID:27462928

  12. Discovery of Widespread GTP-Binding Motifs in Genomic DNA and RNA

    PubMed Central

    Curtis, Edward A.; Liu, David R.

    2013-01-01

    SUMMARY Biological RNAs that bind small-molecules have been implicated in a variety of regulatory and catalytic processes. Inspired by these examples, we used in vitro selection to search a pool of genomeencoded RNA fragments for naturally occurring GTP aptamers. Several classes of aptamers were identified, including one ("the G motif") with a G-quadruplex structure. Further analysis revealed that most RNA and DNA G-quadruplexes bind GTP. The G motif is abundant in eukaryotes, and the human genome contains ∼75,000 examples with dissociation constants comparable to the GTP concentration of a eukaryotic cell (∼300 µM). G-quadruplexes play roles in diverse cellular processes, and our findings raise the possibility that GTP may play a role in the function of these elements. Consistent with this possibility, the sequence requirements of several classes of regulatory G-quadruplexes parallel those of GTP binding. PMID:23601641

  13. Evidence that a single monolayer tubulin-GTP cap is both necessary and sufficient to stabilize microtubules.

    PubMed Central

    Caplow, M; Shanks, J

    1996-01-01

    Evidence that 13 or 14 contiguous tubulin-GTP subunits are sufficient to cap and stabilize a microtubule end and that loss of only one of these subunits results in the transition to rapid disassembly(catastrophe) was obtained using the slowly hydrolyzable GTP analogue guanylyl-(a,b)-methylene-diphosphonate (GMPCPP). The minus end of microtubules assembled with GTP was transiently stabilized against dilution-induced disassembly by reaction with tubulin-GMPCPP subunits for a time sufficient to cap the end with an average 40 subunits. The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was estimated from an observed 25- to 2000-s lifetime of the GMPCPP-stabilized microtubules following dilution with buffer and from the time required for loss of a single tubulin-GMPCPP subunit from the microtubule end (found to be 15 s). Rather than assuming that the 25- to 2000-s dispersion in cap lifetime results from an unlikely 80-fold range in the number of tubulin-GMPCpP subunits added in the 25-s incubation, it is proposed that this results because the minimum stable cap contains 13 to 14 tubulin-GMPCPP subunits. As a consequence, a microtubule capped with 13-14 tubulin-GMPCPP subunits switches to disassembly after only one dissociation event (in about 15 s), whereas the time required for catastrophe of a microtubule with only six times as many subunits (84 subunits) corresponds to 71 dissociation events (84-13). The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was also estimated with microtubules in which a GMPCPP-cap was formed by allowing chance to result in the accumulation of multiple contiguous tubulin-GMPCPP subunits at the end, during the disassembly of microtubules containing both GDP and GMPCPP. Our observation that the disassembly rate was inhibited in proportion to the 13-14th power of the fraction of subunits containing GMPCPP again suggests that a minimum cap contains 13-14 tubulin-GMPCPP subunits. A remeasurement of

  14. Evidence for a GTP-dependent increase in membrane permeability for calcium in NG108-15 microsomes.

    PubMed

    Jean, T; Heppel, L A; Klee, C B

    1989-11-15

    The effect of GTP on Ca2+ uptake and release was studied in a microsomal fraction isolated from neuroblastoma x glioma hybrid NG108-15 cells. GTP did not alter the ATP-dependent initial uptake of Ca2+ but markedly enhanced the efflux of Ca2+ from microsomes. GTP-dependent Ca2+ release requires the presence of millimolar concentration of Mg2+. The effect of GTP was not mimicked by other nucleotides and was competitively blocked by the thiophosphate analogue of GTP, GTP gamma S but not by the non-hydrolyzable nucleotide GMP-PNP. Addition of an inhibiting concentration of GTP gamma S after completion of GTP-induced calcium release did not result in a re-uptake of Ca2+, showing the irreversibility of the releasing effect of GTP. Our data are consistent with the hypothesis of Ca2+-dependent GTP-induced opening of a channel responsible for vectorial transport of Ca2+ ions from one intracellular compartment to another. A model is proposed suggesting that the GTP-binding protein is a GTP-specific diacylglycerol kinase.

  15. Hydrolysis of biomass material

    DOEpatents

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

    2004-02-17

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

  16. GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

    PubMed

    Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

    2015-01-01

    Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

  17. Study of microwave effects on the lipase-catalyzed hydrolysis.

    PubMed

    Chen, Chia-Chen; Reddy, P Muralidhar; Devi, C Shobha; Chang, Po-Chi; Ho, Yen-Peng

    2016-01-01

    The effect of microwave heating on lipase-catalyzed reaction remains controversial. It is not clear whether the reaction rate enhancements are purely due to thermal/heating effects or to non-thermal effects. Therefore, quantitative mass spectrometry was used to conduct accurate kinetic analysis of lipase-catalyzed hydrolysis of triolein by microwave and conventional heating. Commercial lipases from Candida rugosa (CRL), Porcine Pancreas (PPL), and Burkholderia cepacia (BCL) were used. Hydrolysis reactions were performed at various temperatures and pH levels, along with various amounts of buffer and enzymes. Hydrolysis product yields at each time point using an internal-standard method showed no significant difference between microwave and conventional heating conditions when the reaction was carried out at the same temperature. CRL showed optimum catalytic activity at 37 °C, while PPL and BCL had better activities at 50 °C. The phosphate buffer was found to give a better hydrolysis yield than the Tris-HCl buffer. Overall results prove that a non-thermal effect does not exist in microwave-assisted lipase hydrolysis of triolein. Therefore, conventional heating at high temperatures (e.g., 50 °C) can be also used to accelerate hydrolysis reactions.

  18. Study of microwave effects on the lipase-catalyzed hydrolysis.

    PubMed

    Chen, Chia-Chen; Reddy, P Muralidhar; Devi, C Shobha; Chang, Po-Chi; Ho, Yen-Peng

    2016-01-01

    The effect of microwave heating on lipase-catalyzed reaction remains controversial. It is not clear whether the reaction rate enhancements are purely due to thermal/heating effects or to non-thermal effects. Therefore, quantitative mass spectrometry was used to conduct accurate kinetic analysis of lipase-catalyzed hydrolysis of triolein by microwave and conventional heating. Commercial lipases from Candida rugosa (CRL), Porcine Pancreas (PPL), and Burkholderia cepacia (BCL) were used. Hydrolysis reactions were performed at various temperatures and pH levels, along with various amounts of buffer and enzymes. Hydrolysis product yields at each time point using an internal-standard method showed no significant difference between microwave and conventional heating conditions when the reaction was carried out at the same temperature. CRL showed optimum catalytic activity at 37 °C, while PPL and BCL had better activities at 50 °C. The phosphate buffer was found to give a better hydrolysis yield than the Tris-HCl buffer. Overall results prove that a non-thermal effect does not exist in microwave-assisted lipase hydrolysis of triolein. Therefore, conventional heating at high temperatures (e.g., 50 °C) can be also used to accelerate hydrolysis reactions. PMID:26672464

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

    PubMed

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

    2016-06-30

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

  20. Alkali hydrolysis of trinitrotoluene.

    PubMed

    Karasch, Christian; Popovic, Milan; Qasim, Mohamed; Bajpai, Rakesh K

    2002-01-01

    Data for alkali hydrolysis of 2,4,6-trinitrotoluene (TNT) in aqueous solution at pH 12.0 under static (pH-controlled) as well as dynamic (pH-uncontrolled) conditions are reported. The experiments were conducted at two different molar ratios of TNT to hydroxyl ions at room temperature. The TNT disappeared rapidly from the solution as a first-order reaction. The complete disappearance of aromatic structure from the aqueous solution within 24 h was confirmed by the ultraviolet-visible (UV-VIS) spectra of the samples. Cuvet experiments in a UV-VIS spectrophotometer demonstrated the formation of Meisenheimer complex, which slowly disappeared via formation of aromatic compounds with fewer nitro groups. The known metabolites of TNT were found to accumulate only in very small quantities in the liquid phase.

  1. External GTP alters the motility and elicits an oscillating membrane depolarization in Paramecium tetraurelia.

    PubMed

    Clark, K D; Hennessey, T M; Nelson, D L

    1993-05-01

    Paramecium, a unicellular ciliated protist, alters its motility in response to various stimuli. Externally added GTP transiently induced alternating forward and backward swimming interspersed with whirling at a concentration as low as 0.1 microM. ATP was 1000-fold less active, whereas CTP and UTP produced essentially no response. The response to the nonhydrolyzable GTP analogs guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta, gamma-imido]triphosphate was indistinguishable from that to GTP. This behavioral response was correlated with an unusual transient and oscillating membrane depolarization in both wild-type cells and the mutant pawn B, which is defective in the voltage-dependent Ca2+ current required for action potentials. This is a specific effect of external GTP on the excitability of a eukaryotic cell and, to our knowledge, is the first purinergic effect to be discovered in a microorganism.

  2. Phytochrome regulates GTP-binding protein activity in the envelope of pea nuclei

    NASA Technical Reports Server (NTRS)

    Clark, G. B.; Memon, A. R.; Thompson, G. A. Jr; Roux, S. J.

    1993-01-01

    Three GTP-binding proteins with apparent molecular masses of 27, 28 and 30 kDa have been detected in isolated nuclei of etiolated pea plumules. After LDS-PAGE and transfer to nitrocellulose these proteins bind [32P]GTP in the presence of excess ATP, suggesting that they are monomeric G proteins. When nuclei are disrupted, three proteins co-purify with the nuclear envelope fraction and are highly enriched in this fraction. The level of [32P]GTP-binding for all three protein bands is significantly increased when harvested pea plumules are irradiated by red light, and this effect is reversed by far-red light. The results indicate that GTP-binding activity associated with the nuclear envelope of plant cells is photoreversibly regulated by the pigment phytochrome.

  3. GTP binding to the. beta. -subunit of tubulin is greatly reduced in Alzheimers disease

    SciTech Connect

    Khatoon, S.; Slevin, J.T.; Haley, B.E.

    1987-05-01

    A decrease occurs (80-100%) in the (/sup 32/P)8N/sub 3/GTP photoinsertion into a cytosolic protein (55K M/sub r/) of Alzheimer's (AD) brain, tentatively identified as the ..beta..-subunit of tubulin (co-migration with purified tubulin, concentration dependence of interaction with GTP, ATP and their 8-azido photoprobes, and similar effects of Ca/sup 2 +/ and EDTA on photoinsertion). This agrees with prior observations of (/sup 32/P)8N/sub 3/GTP interactions with brain tubulin and a recent report on faulty microtubular assembly in AD brain. The decrease in (/sup 32/P)8N/sub 3/GTP photoinsertion into the 55K M/sub r/ protein of AD brain was in contrast with other photolabeled proteins, which remained at equal levels in AD and age-matched normal brain tissues. The 55K and 45K M/sub r/ were the two major (/sup 32/P)8N/sub 3/GTP photoinsertion species in non-AD brain. Of 5 AD brains, the photoinsertion of (/sup 32/P)8N/sub 3/GTP into the 55K M/sub r/ region was low or absent in 4 (55K/45K=0.1); one was 75% below normals (55K/45K=0.24). Total protein migrating at 55K M/sub r/ was similar in AD and controls. AD brain tubulin, while present, has its exchangeable GTP binding site on ..beta..-tubulin blocked/modified such that (/sup 32/P)8N/sub 3/GTP cannot interact normally with this site.

  4. Cardiomyocyte GTP Cyclohydrolase 1 Protects the Heart Against Diabetic Cardiomyopathy.

    PubMed

    Wu, Hsiang-En; Baumgardt, Shelley L; Fang, Juan; Paterson, Mark; Liu, Yanan; Du, Jianhai; Shi, Yang; Qiao, Shigang; Bosnjak, Zeljko J; Warltier, David C; Kersten, Judy R; Ge, Zhi-Dong

    2016-01-01

    Diabetic cardiomyopathy increases the risk of heart failure and death. At present, there are no effective approaches to preventing its development in the clinic. Here we report that reduction of cardiac GTP cyclohydrolase 1 (GCH1) degradation by genetic and pharmacological approaches protects the heart against diabetic cardiomyopathy. Diabetic cardiomyopathy was induced in C57BL/6 wild-type mice and transgenic mice with cardiomyocyte-specific overexpression of GCH1 with streptozotocin, and control animals were given citrate buffer. We found that diabetes-induced degradation of cardiac GCH1 proteins contributed to adverse cardiac remodeling and dysfunction in C57BL/6 mice, concomitant with decreases in tetrahydrobiopterin, dimeric and phosphorylated neuronal nitric oxide synthase, sarcoplasmic reticulum Ca(2+) handling proteins, intracellular [Ca(2+)]i, and sarcoplasmic reticulum Ca(2+) content and increases in phosphorylated p-38 mitogen-activated protein kinase and superoxide production. Interestingly, GCH-1 overexpression abrogated these detrimental effects of diabetes. Furthermore, we found that MG 132, an inhibitor for 26S proteasome, preserved cardiac GCH1 proteins and ameliorated cardiac remodeling and dysfunction during diabetes. This study deepens our understanding of impaired cardiac function in diabetes, identifies GCH1 as a modulator of cardiac remodeling and function, and reveals a new therapeutic target for diabetic cardiomyopathy. PMID:27295516

  5. Cardiomyocyte GTP Cyclohydrolase 1 Protects the Heart Against Diabetic Cardiomyopathy

    PubMed Central

    Wu, Hsiang-En; Baumgardt, Shelley L.; Fang, Juan; Paterson, Mark; Liu, Yanan; Du, Jianhai; Shi, Yang; Qiao, Shigang; Bosnjak, Zeljko J.; Warltier, David C.; Kersten, Judy R.; Ge, Zhi-Dong

    2016-01-01

    Diabetic cardiomyopathy increases the risk of heart failure and death. At present, there are no effective approaches to preventing its development in the clinic. Here we report that reduction of cardiac GTP cyclohydrolase 1 (GCH1) degradation by genetic and pharmacological approaches protects the heart against diabetic cardiomyopathy. Diabetic cardiomyopathy was induced in C57BL/6 wild-type mice and transgenic mice with cardiomyocyte-specific overexpression of GCH1 with streptozotocin, and control animals were given citrate buffer. We found that diabetes-induced degradation of cardiac GCH1 proteins contributed to adverse cardiac remodeling and dysfunction in C57BL/6 mice, concomitant with decreases in tetrahydrobiopterin, dimeric and phosphorylated neuronal nitric oxide synthase, sarcoplasmic reticulum Ca2+ handling proteins, intracellular [Ca2+]i, and sarcoplasmic reticulum Ca2+ content and increases in phosphorylated p-38 mitogen-activated protein kinase and superoxide production. Interestingly, GCH-1 overexpression abrogated these detrimental effects of diabetes. Furthermore, we found that MG 132, an inhibitor for 26S proteasome, preserved cardiac GCH1 proteins and ameliorated cardiac remodeling and dysfunction during diabetes. This study deepens our understanding of impaired cardiac function in diabetes, identifies GCH1 as a modulator of cardiac remodeling and function, and reveals a new therapeutic target for diabetic cardiomyopathy. PMID:27295516

  6. Aquaporin 1 regulates GTP-induced rapid gating of water in secretory vesicles.

    PubMed

    Cho, Sang-Joon; Sattar, A K M Abdus; Jeong, Eun-Hwan; Satchi, Mylan; Cho, Jin Ah; Dash, Sudhansu; Mayes, Mary Sue; Stromer, Marvin H; Jena, Bhanu P

    2002-04-01

    The swelling of secretory vesicles has been implicated in exocytosis, but the underlying mechanism of vesicle swelling remains largely unknown. Zymogen granules (ZGs), the membrane-bound secretory vesicles in exocrine pancreas, swell in response to GTP mediated by a G(alpha)i3 protein. Evidence is presented here that the water channel aquaporin-1 (AQP1) is present in the ZG membrane and participates in rapid GTP-induced vesicular water gating and swelling. Isolated ZGs exhibit low basal water permeability. However, exposure of granules to GTP results in a marked potentiation of water entry. Treatment of ZGs with the known water channel inhibitor Hg2+ is accompanied by a reversible loss in both the basal and GTP-stimulatable water entry and vesicle swelling. Introduction of AQP1-specific antibody raised against the carboxyl-terminal domain of AQP1 blocks GTP-stimulable swelling of vesicles. Our results demonstrate that AQP1 associated at the ZG membrane is involved in basal as well as GTP-induced rapid gating of water in ZGs of the exocrine pancreas.

  7. Interaction of a novel fluorescent GTP analogue with the small G-protein K-Ras.

    PubMed

    Iwata, Seigo; Masuhara, Kaori; Umeki, Nobuhisa; Sako, Yasushi; Maruta, Shinsaku

    2016-01-01

    A novel fluorescent guanosine 5'-triphosphate (GTP) analogue, 2'(3')-O-{6-(N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino) hexanoic}-GTP (NBD-GTP), was synthesized and utilized to monitor the effect of mutations in the functional region of mouse K-Ras. The effects of the G12S, A59T and G12S/A59T mutations on GTPase activity, nucleotide exchange rates were compared with normal Ras. Mutation at A59T resulted in reduction of the GTPase activity by 0.6-fold and enhancement of the nucleotide exchange rate by 2-fold compared with normal Ras. On the other hand, mutation at G12S only slightly affected the nucleotide exchange rate and did not affect the GTPase activity. We also used NBD-GTP to study the effect of these mutations on the interaction between Ras and SOS1, a guanine nucleotide exchange factor. The mutation at A59T abolished the interaction with SOS1. The results suggest that the fluorescent GTP analogue, NBD-GTP, is applicable to the kinetic studies for small G-proteins.

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  10. Competition between ammonia derived from internal glutamine hydrolysis and hydroxylamine present in the solution for incorporation into UTP as catalysed by Lactococcus lactis CTP synthase.

    PubMed

    Willemoës, Martin

    2004-04-01

    CTP synthase catalyses the reaction: glutamine+UTP+ATP --> glutamate+CTP+ADP+P(i). The reaction is greatly stimulated by the allosteric binding of GTP. In addition to glutamine that is hydrolysed by the enzyme to ammonia and glutamate, CTP synthase will also utilise external sources of amino donors such as NH(4)Cl. This reaction is no longer dependent on allosteric activation by GTP. Hydroxylamine is also a substrate for Lactococcus lactis CTP synthase and results in the formation of N4-OH CTP. This product has the feature that it absorbs at 300nm where CTP absorption was shown to be greatly reduced and enabled the determination of N4-OH CTP formation in the presence of CTP synthesis derived from glutamine hydrolysis. Differences in initial rates determined for the hydroxylamine dependent reaction at 291nm in the presence and absence of glutamine and GTP were ascribed to simultaneous CTP and N4-OH CTP synthesis in the presence of these compounds. A characterisation of the apparent inhibition by GTP and glutamine of N4-OH CTP synthesis determined at 300nm showed that glutamine dependent CTP synthesis occurs at a rate of about 60% of that in the absence of hydroxylamine. GTP dependent inhibition of the ammonium chloride dependent reaction of L. lactis CTP synthase by the glutamine analog glutamate gamma-semialdehyde showed a partial inhibition with a maximum inhibition of about 60%. These results are interpreted in terms of a "half of the sites" mechanism for glutamine hydrolysis on CTP synthase.

  11. Hydrolysis reactor for hydrogen production

    DOEpatents

    Davis, Thomas A.; Matthews, Michael A.

    2012-12-04

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

  12. Importin {beta}-type nuclear transport receptors have distinct binding affinities for Ran-GTP

    SciTech Connect

    Hahn, Silvia; Schlenstedt, Gabriel

    2011-03-18

    Highlights: {yields} Determination of binding properties of nuclear transport receptor/Ran-GTP complexes. {yields} Biosensor measurements provide constants for dissociation, on-rates, and off-rates. {yields} The affinity of receptors for Ran-GTP is widely divergent. {yields} Dissociation constants differ for three orders of magnitude. {yields} The cellular concentration of yeast Ran is not limiting. -- Abstract: Cargos destined to enter or leave the cell nucleus are typically transported by receptors of the importin {beta} family to pass the nuclear pore complex. The yeast Saccharomyces cerevisiae comprises 14 members of this protein family, which can be divided in importins and exportins. The Ran GTPase regulates the association and dissociation of receptors and cargos as well as the transport direction through the nuclear pore. All receptors bind to Ran exclusively in its GTP-bound state and this event is restricted to the nuclear compartment. We determined the Ran-GTP binding properties of all yeast transport receptors by biosensor measurements and observed that the affinity of importins for Ran-GTP differs significantly. The dissociation constants range from 230 pM to 270 nM, which is mostly based on a variability of the off-rate constants. The divergent affinity of importins for Ran-GTP suggests the existence of a novel mode of nucleocytoplasmic transport regulation. Furthermore, the cellular concentration of {beta}-receptors and of other Ran-binding proteins was determined. We found that the number of {beta}-receptors altogether about equals the amounts of yeast Ran, but Ran-GTP is not limiting in the nucleus. The implications of our results for nucleocytoplasmic transport mechanisms are discussed.

  13. Structure and Mutational Analysis of the Archaeal GTP:AdoCbi-P Guanylyltransferase (CobY) from Methanocaldococcus jannaschii: Insights into GTP Binding and Dimerization

    SciTech Connect

    Newmister, Sean A.; Otte, Michele M.; Escalante-Semerena, Jorge C.; Rayment, Ivan

    2012-02-08

    In archaea and bacteria, the late steps in adenosylcobalamin (AdoCbl) biosynthesis are collectively known as the nucleotide loop assembly (NLA) pathway. In the archaeal and bacterial NLA pathways, two different guanylyltransferases catalyze the activation of the corrinoid. Structural and functional studies of the bifunctional bacterial guanylyltransferase that catalyze both ATP-dependent corrinoid phosphorylation and GTP-dependent guanylylation are available, but similar studies of the monofunctional archaeal enzyme that catalyzes only GTP-dependent guanylylation are not. Herein, the three-dimensional crystal structure of the guanylyltransferase (CobY) enzyme from the archaeon Methanocaldococcus jannaschii (MjCobY) in complex with GTP is reported. The model identifies the location of the active site. An extensive mutational analysis was performed, and the functionality of the variant proteins was assessed in vivo and in vitro. Substitutions of residues Gly8, Gly153, or Asn177 resulted in {ge}94% loss of catalytic activity; thus, variant proteins failed to support AdoCbl synthesis in vivo. Results from isothermal titration calorimetry experiments showed that MjCobY{sup G153D} had 10-fold higher affinity for GTP than MjCobY{sup WT} but failed to bind the corrinoid substrate. Results from Western blot analyses suggested that the above-mentioned substitutions render the protein unstable and prone to degradation; possible explanations for the observed instability of the variants are discussed within the framework of the three-dimensional crystal structure of MjCobY{sup G153D} in complex with GTP. The fold of MjCobY is strikingly similar to that of the N-terminal domain of Mycobacterium tuberculosis GlmU (MtbGlmU), a bifunctional acetyltransferase/uridyltransferase that catalyzes the formation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc).

  14. The Lipid Kinase PI5P4Kβ Is an Intracellular GTP Sensor for Metabolism and Tumorigenesis.

    PubMed

    Sumita, Kazutaka; Lo, Yu-Hua; Takeuchi, Koh; Senda, Miki; Kofuji, Satoshi; Ikeda, Yoshiki; Terakawa, Jumpei; Sasaki, Mika; Yoshino, Hirofumi; Majd, Nazanin; Zheng, Yuxiang; Kahoud, Emily Rose; Yokota, Takehiro; Emerling, Brooke M; Asara, John M; Ishida, Tetsuo; Locasale, Jason W; Daikoku, Takiko; Anastasiou, Dimitrios; Senda, Toshiya; Sasaki, Atsuo T

    2016-01-21

    While cellular GTP concentration dramatically changes in response to an organism's cellular status, whether it serves as a metabolic cue for biological signaling remains elusive due to the lack of molecular identification of GTP sensors. Here we report that PI5P4Kβ, a phosphoinositide kinase that regulates PI(5)P levels, detects GTP concentration and converts them into lipid second messenger signaling. Biochemical analyses show that PI5P4Kβ preferentially utilizes GTP, rather than ATP, for PI(5)P phosphorylation, and its activity reflects changes in direct proportion to the physiological GTP concentration. Structural and biological analyses reveal that the GTP-sensing activity of PI5P4Kβ is critical for metabolic adaptation and tumorigenesis. These results demonstrate that PI5P4Kβ is the missing GTP sensor and that GTP concentration functions as a metabolic cue via PI5P4Kβ. The critical role of the GTP-sensing activity of PI5P4Kβ in cancer signifies this lipid kinase as a cancer therapeutic target. PMID:26774281

  15. Serum gamma-GTP levels by type and quantity of alcohol consumed--the 'whisky hypothesis' refuted.

    PubMed

    Robinson, D; Takiwaki, S; Allaway, S; Sekihara, K

    1987-12-01

    Serum gamma-GTP measurements in 11,755 Japanese men were used to test the hypothesis that drinking whisky had little or no effect on the serum level of this enzyme. We found that regular drinking was associated with significantly increased mean levels and raised percentages of high values of gamma-GTP, irrespective of the type of alcohol consumed. Moreover, heavier and more frequent drinking were associated with proportionately greater increases in gamma-GTP levels. Our data therefore refute the hypothesis that whisky drinking is not accompanied by adverse changes in the level of serum gamma-GTP.

  16. Role of GTP-CHI links PAH and TH in melanin synthesis in silkworm, Bombyx mori.

    PubMed

    Chen, Ping; Wang, Jiying; Li, Haiyin; Li, Yan; Chen, Peng; Li, Tian; Chen, Xi; Xiao, Junjie; Zhang, Liang

    2015-08-10

    In insects, pigment patterns are formed by melanin, ommochromes, and pteridines. Here, the effects of pteridine synthesis on melanin formation were studied using 4th instar larvae of a wild-type silkworm strain, dazao (Bombyx mori), with normal color and markings. Results from injected larvae and in vitro integument culture indicated that decreased activity of guanosine triphosphate cyclohydrolase I (GTP-CH I, a rate-limiting enzyme for pteridine synthesis), lowers BH4 (6R-l-erythro-5,6,7,8-tetrahydrobiopterin, a production correlated with GTP-CH I activity) levels and eliminates markings and coloration. The conversion of phenylalanine and tyrosine to melanin was prevented when GTP-CH I was inhibited. When BH4 was added, phenylalanine was converted to tyrosine, and the tyrosine concentration increased. Tyrosine was then converted to melanin to create normal markings and coloration. Decreasing GTP-CH I activity did not affect L-DOPA (3,4-l-dihydroxyphenylalanine). GTP-CH I affected melanin synthesis by generating the BH4 used in two key reaction steps: (1) conversion of phenylalanine to tyrosine by PAH (phenylalanine hydroxylase) and (2) conversion of tyrosine to L-DOPA by TH (tyrosine hydroxylase). Expression profiles of BmGTPCH Ia, BmGTPCH Ib, BmTH, and BmPAH in the integument were consistent with the current findings.

  17. Hydrolysis of tRNA(sup Phe) on Suspensions of Amino Acids

    NASA Technical Reports Server (NTRS)

    Gao, Kui; Orgel, Leslie E.

    2001-01-01

    RNA is adsorbed strongly on suspensions of many moderately soluble organic solids. In some cases, the hydrolysis of tRNA(sup Phe) is greatly accelerated by adsorption, and the major sites of hydrolysis are changed from those that are important in homogeneous solution. Here we show that the hydrolysis is greatly accelerated by suspensions of aspartic acid and beta-glutamic acid but not by suspensions of alpha-glutamic acid, asparagine, or glutamine. The non-enzymatic hydrolysis of RNA has been studied extensively, especially because of its relevance to the mechanisms of action of ribozymes and to biotechnology and therapy. Many ribonucleases, ribozymes, and non-biological catalysts function via acid-base catalysis of an intramolecular transesterification mechanism in which the 2'-OH group attacks the adjacent phosphate group. The pentacoordinated phosphorane intermediate may collapse back to starting material, or yield isomerized or cleaved products.

  18. Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

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

    2011-08-22

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

  19. Structural and functional analysis of a FeoB A143S G5 loop mutant explains the accelerated GDP release rate.

    PubMed

    Guilfoyle, Amy P; Deshpande, Chandrika N; Vincent, Kimberley; Pedroso, Marcelo M; Schenk, Gerhard; Maher, Megan J; Jormakka, Mika

    2014-05-01

    GTPases (G proteins) hydrolyze the conversion of GTP to GDP and free phosphate, comprising an integral part of prokaryotic and eukaryotic signaling, protein biosynthesis and cell division, as well as membrane transport processes. The G protein cycle is brought to a halt after GTP hydrolysis, and requires the release of GDP before a new cycle can be initiated. For eukaryotic heterotrimeric Gαβγ proteins, the interaction with a membrane-bound G protein-coupled receptor catalyzes the release of GDP from the Gα subunit. Structural and functional studies have implicated one of the nucleotide binding sequence motifs, the G5 motif, as playing an integral part in this release mechanism. Indeed, a Gαs G5 mutant (A366S) was shown to have an accelerated GDP release rate, mimicking a G protein-coupled receptor catalyzed release state. In the present study, we investigate the role of the equivalent residue in the G5 motif (residue A143) in the prokaryotic membrane protein FeoB from Streptococcus thermophilus, which includes an N-terminal soluble G protein domain. The structure of this domain has previously been determined in the apo and GDP-bound states and in the presence of a transition state analogue, revealing conformational changes in the G5 motif. The A143 residue was mutated to a serine and analyzed with respect to changes in GTPase activity, nucleotide release rate, GDP affinity and structural alterations. We conclude that the identity of the residue at this position in the G5 loop plays a key role in the nucleotide release rate by allowing the correct positioning and hydrogen bonding of the nucleotide base.

  20. [Effect of iron, actinomycin D and cycloheximide on the GTP-cyclohydrolase synthesis in flavinogenic yeasts].

    PubMed

    Logvinenko, E M; Shavlovskiĭ, G M; Zakal'skiĭ, A E; Zakhodylo, I V

    1982-01-01

    The effect of Fe on the GTP-cyclohydrolase activity of the yeasts Pichia guilliermondii ATCC 9058 and Torulopsis candida BKM 13 whose flavinogenesis is controlled by Fe was investigated. The GTP-cyclohydrolase activity of yeast cells grown in an iron-deficient medium was 40-50 times that of the cells grown in an iron-rich medium. In the latter case the incubation of cells with alpha, alpha'-dipyridyl or 8-oxyquinoline also increased the enzyme activity. Cycloheximide prevented the rise in the cyclohydrolase activity in both cases, thus suggesting the participation of Fe in the control of the enzyme synthesis. Actinomycin D inhibited the enzyme derepression induced by alpha, alpha1-dipyridyl or 8-oxyquinoline in the P. guilliermondii MS1-37 mutant possessing a high sensitivity to this antibiotic. It is assumed that Fe is involved in the control of GTP-cyclohydrolase synthesis in flavinogenic yeasts at the transcription level. PMID:7199939

  1. Lowering GTP level increases survival of amino acid starvation but slows growth rate for Bacillus subtilis cells lacking (p)ppGpp.

    PubMed

    Bittner, Alycia N; Kriel, Allison; Wang, Jue D

    2014-06-01

    Bacterial cells sense external nutrient availability to regulate macromolecular synthesis and consequently their growth. In the Gram-positive bacterium Bacillus subtilis, the starvation-inducible nucleotide (p)ppGpp negatively regulates GTP levels, both to resist nutritional stress and to maintain GTP homeostasis during growth. Here, we quantitatively investigated the relationship between GTP level, survival of amino acid starvation, and growth rate when GTP synthesis is uncoupled from its major homeostatic regulator, (p)ppGpp. We analyzed growth and nucleotide levels in cells that lack (p)ppGpp and found that their survival of treatment with a nonfunctional amino acid analog negatively correlates with both growth rate and GTP level. Manipulation of GTP levels modulates the exponential growth rate of these cells in a positive dose-dependent manner, such that increasing the GTP level increases growth rate. However, accumulation of GTP levels above a threshold inhibits growth, suggesting a toxic effect. Strikingly, adenine counteracts GTP stress by preventing GTP accumulation in cells lacking (p)ppGpp. Our results emphasize the importance of maintaining appropriate levels of GTP to maximize growth: cells can survive amino acid starvation by decreasing GTP level, which comes at a cost to growth, while (p)ppGpp enables rapid adjustment to nutritional stress by adjusting GTP level, thus maximizing fitness.

  2. Continued protein synthesis at low [ATP] and [GTP] enables cell adaptation during energy limitation.

    PubMed

    Jewett, Michael C; Miller, Mark L; Chen, Yvonne; Swartz, James R

    2009-02-01

    One of biology's critical ironies is the need to adapt to periods of energy limitation by using the energy-intensive process of protein synthesis. Although previous work has identified the individual energy-requiring steps in protein synthesis, we still lack an understanding of the dependence of protein biosynthesis rates on [ATP] and [GTP]. Here, we used an integrated Escherichia coli cell-free platform that mimics the intracellular, energy-limited environment to show that protein synthesis rates are governed by simple Michaelis-Menten dependence on [ATP] and [GTP] (K(m)(ATP), 27 +/- 4 microM; K(m)(GTP), 14 +/- 2 microM). Although the system-level GTP affinity agrees well with the individual affinities of the GTP-dependent translation factors, the system-level K(m)(ATP) is unexpectedly low. Especially under starvation conditions, when energy sources are limited, cells need to replace catalysts that become inactive and to produce new catalysts in order to effectively adapt. Our results show how this crucial survival priority for synthesizing new proteins can be enforced after rapidly growing cells encounter energy limitation. A diminished energy supply can be rationed based on the relative ATP and GTP affinities, and, since these affinities for protein synthesis are high, the cells can adapt with substantial changes in protein composition. Furthermore, our work suggests that characterization of individual enzymes may not always predict the performance of multicomponent systems with complex interdependencies. We anticipate that cell-free studies in which complex metabolic systems are activated will be valuable tools for elucidating the behavior of such systems.

  3. Different effects of guanine nucleotides (GDP and GTP) on protein-mediated mitochondrial proton leak.

    PubMed

    Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

    2014-01-01

    In this study, we compared the influence of GDP and GTP on isolated mitochondria respiring under conditions favoring oxidative phosphorylation (OXPHOS) and under conditions excluding this process, i.e., in the presence of carboxyatractyloside, an adenine nucleotide translocase inhibitor, and/or oligomycin, an FOF1-ATP synthase inhibitor. Using mitochondria isolated from rat kidney and human endothelial cells, we found that the action of GDP and GTP can differ diametrically depending on the conditions. Namely, under conditions favoring OXPHOS, both in the absence and presence of linoleic acid, an activator of uncoupling proteins (UCPs), the addition of 1 mM GDP resulted in the state 4 (non-phosphorylating respiration)-state 3 (phosphorylating respiration) transition, which is characteristic of ADP oxidative phosphorylation. In contrast, the addition of 1 mM GTP resulted in a decrease in the respiratory rate and an increase in the membrane potential, which is characteristic of UCP inhibition. The stimulatory effect of GDP, but not GTP, was also observed in inside-out submitochondrial particles prepared from rat kidney mitochondria. However, the effects of GDP and GTP were more similar in the presence of OXPHOS inhibitors. The importance of these observations in connection with the action of UCPs, adenine nucleotide translocase (or other carboxyatractyloside-sensitive carriers), carboxyatractyloside- and purine nucleotide-insensitive carriers, as well as nucleoside-diphosphate kinase (NDPK) are considered. Because the measurements favoring oxidative phosphorylation better reflect in vivo conditions, our study strongly supports the idea that GDP cannot be considered a significant physiological inhibitor of UCP. Moreover, it appears that, under native conditions, GTP functions as a more efficient UCP inhibitor than GDP and ATP.

  4. Site-directed mutagenesis of the GTP-binding domain of beta-tubulin.

    PubMed

    Farr, G W; Sternlicht, H

    1992-09-01

    Tubulin binds guanine nucleotides with high affinity and specificity. GTP, an allosteric effector of microtubule assembly, requires Mg2+ for its interaction with beta-tubulin and binds as the MgGTP complex. In contrast, GDP binding does not require Mg2+. The structural basis for this difference is not understood but may be of fundamental importance for microtubule assembly. We investigated the interaction of beta-tubulin with guanine nucleotides using site-directed mutagenesis. Acidic amino acid residues have been shown to interact with nucleotide in numerous nucleotide-binding proteins. In this study, we mutated seven highly conserved aspartic acid residues and one highly conserved glutamic acid residue in the putative GTP-binding domain of beta-tubulin (N-terminal 300 amino acids) to asparagine and glutamine, respectively. The mutants were synthesized in vitro using rabbit reticulocyte lysates, and their affinities for nucleotide determined by an h.p.l.c.-based assay. Our results indicate that the mutations can be placed in six separate categories on the basis of their effects on nucleotide binding. These categories range from having no effect on nucleotide binding to a mutation that apparently abolishes nucleotide binding. One mutation at Asp224 reduced the affinity of beta-tubulin for GTP in the presence but not in the absence of Mg2+. The specific effect of this mutation on nucleotide binding is consistent with an interaction of this amino acid with the Mg2+ moiety of MgGTP. This residue is in a region sharing sequence homology with the putative Mg2+ site in myosin and other ATP-binding proteins. As a result, tubulin belongs to a distinct class of GTP-binding proteins which may be evolutionarily related to the ATP-binding proteins.

  5. Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance.

    PubMed

    Kriel, Allison; Bittner, Alycia N; Kim, Sok Ho; Liu, Kuanqing; Tehranchi, Ashley K; Zou, Winnie Y; Rendon, Samantha; Chen, Rui; Tu, Benjamin P; Wang, Jue D

    2012-10-26

    Cells constantly adjust their metabolism in response to environmental conditions, yet major mechanisms underlying survival remain poorly understood. We discover a posttranscriptional mechanism that integrates starvation response with GTP homeostasis to allow survival, enacted by the nucleotide (p)ppGpp, a key player in bacterial stress response and persistence. We reveal that (p)ppGpp activates global metabolic changes upon starvation, allowing survival by regulating GTP. Combining metabolomics with biochemical demonstrations, we find that (p)ppGpp directly inhibits the activities of multiple GTP biosynthesis enzymes. This inhibition results in robust and rapid GTP regulation in Bacillus subtilis, which we demonstrate is essential to maintaining GTP levels within a range that supports viability even in the absence of starvation. Correspondingly, without (p)ppGpp, gross GTP dysregulation occurs, revealing a vital housekeeping function of (p)ppGpp; in fact, loss of (p)ppGpp results in death from rising GTP, a severe and previously unknown consequence of GTP dysfunction.

  6. Direct Regulation of GTP Homeostasis by (p)ppGpp: A Critical Component of Viability and Stress Resistance

    PubMed Central

    Kriel, Allison; Bittner, Alycia N.; Kim, Sok Ho; Liu, Kuanqing; Tehranchi, Ashley K.; Zou, Winnie Y.; Rendon, Samantha; Chen, Rui; Tu, Benjamin P.; Wang, Jue D.

    2012-01-01

    Summary Cells constantly adjust their metabolism in response to environmental conditions, yet major mechanisms underlying survival remain poorly understood. We discover a post-transcriptional mechanism that integrates starvation response with GTP homeostasis to allow survival, enacted by the nucleotide (p)ppGpp, a key player in bacterial stress response and persistence. We reveal that (p)ppGpp activates global metabolic changes upon starvation, allowing survival by regulating GTP. Combining metabolomics with biochemical demonstrations, we find that (p)ppGpp directly inhibits the activities of multiple GTP biosynthesis enzymes. This inhibition results in robust and rapid GTP regulation in Bacillus subtilis, which we demonstrate is essential to maintaining GTP levels within a range that supports viability even in the absence of starvation. Correspondingly, without (p)ppGpp, gross GTP dysregulation occurs, revealing a vital housekeeping function of (p)ppGpp; in fact, loss of (p)ppGpp results in death from rising GTP, a severe and previously unknown consequence of GTP dysfunction. PMID:22981860

  7. Extracellular enzyme activities during regulated hydrolysis of high-solid organic wastes.

    PubMed

    Zhang, Bo; He, Pin-Jing; Lü, Fan; Shao, Li-Ming; Wang, Pei

    2007-11-01

    The hydrolysis process, where the complex insoluble organic materials are hydrolyzed by extracellular enzymes, is a rate-limiting step for anaerobic digestion of high-solid organic solid wastes. Recirculating the leachate from hydrolysis reactor and recycling the effluent from methanogenic reactor to hydrolysis reactor in the two-stage solid-liquid anaerobic digestion process could accelerate degradation of organic solid wastes. To justify the influencing mechanism of recirculation and recycling on hydrolysis, the relationship of hydrolysis to the synthesis and locations of extracellular enzymes was evaluated by regulating the dilution rate of the methanogenic effluent recycle. The results showed that the hydrolysis could be enhanced by increasing the dilution rate, resulting from improved total extracellular enzyme activities. About 15%, 25%, 37%, 56% and 92% of carbon, and about 9%, 18%, 27%, 45% and 80% of nitrogen were converted from the solid phase to the liquid phase at dilution rates of 0.09, 0.25, 0.5, 0.9 and 1.8d(-1), respectively. The hydrolysis of organic wastes was mainly attributable to cell-free enzyme, followed by biofilm-associated enzyme. Increasing the dilution rate afforded cell-free extracellular enzymes with more opportunity to access the surface of organic solid waste, which ensured a faster renewal of niche where extracellular enzymes functioned actively. Meanwhile, the increment of biofilm-associated enzyme was promoted concomitantly, and therefore improved the hydrolysis of organic solid wastes.

  8. Linear Accelerators

    SciTech Connect

    Sidorin, Anatoly

    2010-01-05

    In linear accelerators the particles are accelerated by either electrostatic fields or oscillating Radio Frequency (RF) fields. Accordingly the linear accelerators are divided in three large groups: electrostatic, induction and RF accelerators. Overview of the different types of accelerators is given. Stability of longitudinal and transverse motion in the RF linear accelerators is briefly discussed. The methods of beam focusing in linacs are described.

  9. Structure of a GTP-dependent Bacterial PEP-carboxykinase from Corynebacterium glutamicum

    SciTech Connect

    Aich, Sanjukta; Prasad, Lata; Delbaere, Louis T.J.

    2008-06-23

    GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2{sub 1} with four molecules per asymmetric unit. The 2.3 {angstrom} resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guanine binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.

  10. Measuring Ras-family GTP levels in vivo--running hot and cold.

    PubMed

    Castro, Ariel F; Rebhun, John F; Quilliam, Lawrence A

    2005-10-01

    The detection of Ras-family GTPase activity is important in the determination of cell signaling events elicited by numerous ligands and cellular processes. This has been made much easier in recent years by the use of glutathione S-transferase (GST)-fused Ras binding domains. These domains from downstream effectors such as Raf and RalGDS preferentially bind the GTP-bound Ras proteins enabling their extraction and subsequent quantification by immunoblotting. Despite this advance, effectors that efficiently discriminate between GTP- and GDP-bound states are not available for many Ras-family members. While this hampers the ability to detect activity in tissue specimens, it is still possible to metabolically label cells with (32)Pi to load the GTP/GDP pool with labeled nucleotides, immunoprecipitate the Ras protein and detect the bound label following thin layer chromatographic separation and exposure to film or a phosphorimager. Using a transfection system and antibodies that recognize epitope tags one can test the ability of a protein to work as a GEF or GAP for a certain GTPase. Alternatively, if an immunoprecipitating antibody is available to the target GTPase, then analysis of endogenous GTP/GDP ratio is possible. Here we describe the detection of M-Ras and Rap1 activity by GST-RBD pull-down as well as that of Rheb and epitope-tagged R-Ras by classical metabolic labeling and immunoprecipitation.

  11. The RanGTP Pathway: From Nucleo-Cytoplasmic Transport to Spindle Assembly and Beyond

    PubMed Central

    Cavazza, Tommaso; Vernos, Isabelle

    2016-01-01

    The small GTPase Ran regulates the interaction of transport receptors with a number of cellular cargo proteins. The high affinity binding of the GTP-bound form of Ran to import receptors promotes cargo release, whereas its binding to export receptors stabilizes their interaction with the cargo. This basic mechanism linked to the asymmetric distribution of the two nucleotide-bound forms of Ran between the nucleus and the cytoplasm generates a switch like mechanism controlling nucleo-cytoplasmic transport. Since 1999, we have known that after nuclear envelope breakdown (NEBD) Ran and the above transport receptors also provide a local control over the activity of factors driving spindle assembly and regulating other aspects of cell division. The identification and functional characterization of RanGTP mitotic targets is providing novel insights into mechanisms essential for cell division. Here we review our current knowledge on the RanGTP system and its regulation and we focus on the recent advances made through the characterization of its mitotic targets. We then briefly review the novel functions of the pathway that were recently described. Altogether, the RanGTP system has moonlighting functions exerting a spatial control over protein interactions that drive specific functions depending on the cellular context. PMID:26793706

  12. Sensitive assay of GTP cyclohydrolase I activity in rat and human tissues using radioimmunoassay of neopterin

    SciTech Connect

    Sawada, M.; Horikoshi, T.; Masada, M.; Akino, M.; Sugimoto, T.; Matsuura, S.; Nagatsu, T.

    1986-04-01

    A highly sensitive and simple assay for the activity of GTP cyclohydrolase I (EC 3.5.4.16) was established using a newly developed radioimmunoassay. D-erythro-7,8-Dihydroneopterin triphosphate formed from GTP by GTP cyclohydrolase I was oxidized by iodine and dephosphorylated by alkaline phosphatase to D-erythro-neopterin, and quantified by a radioimmunoassay for D-erythro-neopterin. This method was highly sensitive and required only 0.2 mg of rat liver tissues for the measurement of the activity. It was reproducible and can be applied for the simultaneous assay of many samples. The activity of GTP cyclohydrolase I was measured in several rat tissues. For example, the enzyme activity in rat striatum (n = 5) was 13.7 +/- 1.5 pmol/mg protein per hour (mean +/- SE), and agreed well with those obtained by high-performance liquid chromatography with fluorescence detection. The activity in the autopsy human brains (caudate nucleus) was measured by this new method for the first time. The activity in the caudate nucleus from parkinsonian patients (n = 6) was 0.82 +/- 0.56 pmol/mg protein per hour which was significantly lower than the control value, 4.22 +/- 0.43 pmol/mg protein per hour (n = 10).

  13. Occurrence and Ecological Significance of GTP in the Ocean and in Microbial Cells

    PubMed Central

    Karl, D. M.

    1978-01-01

    A comparison between the ATP concentrations based on peak height light emission values (0 to 3 s) and integrated light flux determinations (15 to 75 s) for a variety of seawater samples revealed that the integrated method of light detection consistently yielded higher ATP concentrations, ranging from 1.38 to 2.35 times larger than the corresponding peak ATP values. A significant correlation (r = 0.923) was observed for a plot of ΔADP (i.e., integrated ATP - peak ATP) versus GTP + UTP, suggesting that the analytical interference on the ATP assay was the result of the presence of non-adenine nucleotide triphosphates. Size-fractionation studies revealed an enrichment of the non-adenine nucleotide triphosphates, relative to ATP, in the smallest size fraction analyzed (<10 μm). Investigations were conducted with 20 species of unicellular marine algae to determine their intracellular nucleotide concentrations, and these determinations were compared to the levels measured in lab cultures of the marine bacterium Serratia marinorubra. These results indicated that the intracellular GTP/ATP ratios in S. marinorubra increase in direct proportion to the rate of cell growth, and that the GTP/ATP ratios in bacteria are much greater than in growing algae, presumably due to the differences in rates of cellular biosynthesis. It is concluded that quantitative determinations of GTP/ATP ratios in environmental sample extracts may be useful for measuring microbial growth. PMID:16345313

  14. Linkages between the effects of taxol, colchicine, and GTP on tubulin polymerization.

    PubMed

    Howard, W D; Timasheff, S N

    1988-01-25

    A comparative study has been carried out of the effects of taxol on the polymerizations into microtubules of microtubule-associated protein-free tubulin, prepared by the modified Weisenberg procedure, and of the tubulin-colchicine complex into large aggregates. Taxol enhances, to a much greater extent, the stability of microtubules than that of the tubulin-colchicine polymers so that, with highly purified tubulin, assembly into microtubules takes place at 10 degrees C, even in the absence of exogenous GTP. The polymerization of tubulin-colchicine requires both heat and GTP, and the process is reversed by cooling. These results indicate that in both systems polymerization is linked to interactions with taxol and GTP, the interplay of linkage free energies imparting the observed polymer stabilities. In the case of microtubule formation, the linkage free energy provided by taxol binding is approximately -3.0 kcal/mol of alpha-beta-tubulin dimer, whereas this quantity is reduced to approximately -0.5 kcal/mol in tubulin-colchicine, indicating the expenditure of much more binding free energy in the latter case for overcoming unfavorable factors, such as steric hindrance and geometric strain. The difference in the effect of GTP on the two polymerization processes reflects the respective abilities of the bindings of taxol to the two states of tubulin to overcome the loss of the linkage free energy of GTP binding. Analysis of the linkages leads to the conclusions that taxol need not change qualitatively the mechanism of microtubule assembly and that tubulin with the E-site unoccupied by nucleotide should have the capacity to form microtubules, the reaction being extremely weak.

  15. Extracellular Loop II Modulates GTP Sensitivity of the Prostaglandin EP3 Receptor

    PubMed Central

    Natarajan, Chandramohan; Hata, Aaron N.; Hamm, Heidi E.; Zent, Roy

    2013-01-01

    Unlike the majority of G protein–coupled receptors, the prostaglandin E2 (PGE2) E-prostanoid 3 (EP3) receptor binds agonist with high affinity that is insensitive to the presence of guanosine 5[prime]-O-(3-thio)triphosphate (GTPγS). We report the identification of mutations that confer GTPγS sensitivity to agonist binding. Seven point mutations were introduced into the conserved motif in the second extracellular loop (ECII) of EP3, resulting in acquisition of GTP-sensitive agonist binding. One receptor mutation W203A was studied in detail. Loss of agonist binding was observed on intact human embryonic kidney 293 cells expressing the W203A receptor, conditions where high GTP levels are present; however, high affinity binding [3H]PGE2 was observed in broken cell preparations washed free of GTP. The [3H]PGE2 binding of W203A in broken cell membrane fractions was inhibited by addition of GTPγS (IC50 21 ± 1.8 nM). Taken together, these results suggest that the wild-type EP3 receptor displays unusual characteristics of the complex coupled equilibria between agonist-receptor and receptor–G protein interaction. Moreover, mutation of ECII can alter this coupled equilibrium from GTP-insensitive agonist binding to more conventional GTP-sensitive binding. This suggests that for the mutant receptors, ECII plays a critical role in linking the agonist bound receptor conformation to the G protein nucleotide bound state. PMID:23087260

  16. Hippurate hydrolysis by Legionella pneumophila.

    PubMed Central

    Hébert, G A

    1981-01-01

    Strains of Legionella pneumophila were shown to hydrolyze sodium hippurate in an overnight test system, but strains of L. bozemanii, L. micdadei, L. dumoffii, and some other organisms similar to the legionellae did not. Although only a small number of strains of legionellae other than L. pneumophila have been classified and tested, the results indicate that the hippurate hydrolysis test may prove useful for differentiating among Legionella species. PMID:7462418

  17. Thermodynamics of the GTP-GDP-operated conformational switch of selenocysteine-specific translation factor SelB.

    PubMed

    Paleskava, Alena; Konevega, Andrey L; Rodnina, Marina V

    2012-08-10

    SelB is a specialized translation factor that binds GTP and GDP and delivers selenocysteyl-tRNA (Sec-tRNA(Sec)) to the ribosome. By analogy to elongation factor Tu (EF-Tu), SelB is expected to control the delivery and release of Sec-tRNA(Sec) to the ribosome by the structural switch between GTP- and GDP-bound conformations. However, crystal structures of SelB suggested a similar domain arrangement in the apo form and GDP- and GTP-bound forms of the factor, raising the question of how SelB can fulfill its delivery function. Here, we studied the thermodynamics of guanine nucleotide binding to SelB by isothermal titration calorimetry in the temperature range between 10 and 25 °C using GTP, GDP, and two nonhydrolyzable GTP analogs, guanosine 5'-O-(γ-thio)triphosphate (GTPγS) and guanosine 5'-(β,γ-imido)-triphosphate (GDPNP). The binding of SelB to either guanine nucleotide is characterized by a large heat capacity change (-621, -467, -235, and -275 cal × mol(-1) × K(-1), with GTP, GTPγS, GDPNP, and GDP, respectively), associated with compensatory changes in binding entropy and enthalpy. Changes in heat capacity indicate a large decrease of the solvent-accessible surface area in SelB, amounting to 43 or 32 amino acids buried upon binding of GTP or GTPγS, respectively, and 15-19 amino acids upon binding GDP or GDPNP. The similarity of the GTP and GDP forms in the crystal structures can be attributed to the use of GDPNP, which appears to induce a structure of SelB that is more similar to the GDP than to the GTP-bound form.

  18. Can Accelerators Accelerate Learning?

    NASA Astrophysics Data System (ADS)

    Santos, A. C. F.; Fonseca, P.; Coelho, L. F. S.

    2009-03-01

    The 'Young Talented' education program developed by the Brazilian State Funding Agency (FAPERJ) [1] makes it possible for high-schools students from public high schools to perform activities in scientific laboratories. In the Atomic and Molecular Physics Laboratory at Federal University of Rio de Janeiro (UFRJ), the students are confronted with modern research tools like the 1.7 MV ion accelerator. Being a user-friendly machine, the accelerator is easily manageable by the students, who can perform simple hands-on activities, stimulating interest in physics, and getting the students close to modern laboratory techniques.

  19. Can Accelerators Accelerate Learning?

    SciTech Connect

    Santos, A. C. F.; Fonseca, P.; Coelho, L. F. S.

    2009-03-10

    The 'Young Talented' education program developed by the Brazilian State Funding Agency (FAPERJ)[1] makes it possible for high-schools students from public high schools to perform activities in scientific laboratories. In the Atomic and Molecular Physics Laboratory at Federal University of Rio de Janeiro (UFRJ), the students are confronted with modern research tools like the 1.7 MV ion accelerator. Being a user-friendly machine, the accelerator is easily manageable by the students, who can perform simple hands-on activities, stimulating interest in physics, and getting the students close to modern laboratory techniques.

  20. GTP-binding peptide of beta-tubulin. Localization by direct photoaffinity labeling and comparison with nucleotide-binding proteins

    SciTech Connect

    Linse, K.; Mandelkow, E.M.

    1988-10-15

    The binding site of the guanine moiety of GTP on beta-tubulin was located within the peptide consisting of residues 63-77, AILVDLEPGTMDSVR. The result was obtained using direct photoaffinity labeling, peptide sequencing, and limited proteolysis. Peptides were identified by end-labeling with a monoclonal antibody against beta-tubulin whose epitope was located between 3 and 4 kDa from the C terminus. The sequence of the GTP-binding site is consistent with predictions from other GTP-binding proteins such as elongation factor Tu or ras p21.

  1. The Acid Hydrolysis Mechanism of Acetals Catalyzed by a Supramolecular Assembly in Basic Solution

    SciTech Connect

    Pluth, Michael D.; Bergman, Robert G.; Raymond, Kenneth N.

    2008-09-24

    A self-assembled supramolecular host catalyzes the hydrolysis of acetals in basic aqueous solution. The mechanism of hydrolysis is consistent with the Michaelis-Menten kinetic model. Further investigation of the rate limiting step of the reaction revealed a negative entropy of activation ({Delta}S{double_dagger} = -9 cal mol{sup -1}K{sup -1}) and an inverse solvent isotope effect (k(H{sub 2}O)/k(D{sub 2}O) = 0.62). These data suggest that the mechanism of hydrolysis that takes place inside the assembly proceeds through an A-2 mechanism, in contrast to the A-1 mechanism operating in the uncatalyzed reaction. Comparison of the rates of acetal hydrolysis in the assembly with the rate of the reaction of unencapsulated substrates reveals rate accelerations of up to 980 over the background reaction for the substrate diethoxymethane.

  2. HYDROLYSIS

    EPA Science Inventory

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

  3. Membrane reactor for enzymatic hydrolysis of cellobiose

    SciTech Connect

    Hong, J.; Tsao, G.T.; Wankat, P.C.

    1981-07-01

    A pressurized, stirred vessel attached with an ultrafiltration membrane was used as a membrane reactor. Cellobiose hydrolysis by cellobiase was carried out and theoretically analyzed in terms of steady-state conversion and flow rate through the membrane. When the flow rate exceeds a critical value, a significant fraction of the enzyme inside the reactor is localized in the concentration polarization layer where shear from stirring is high. Consequently, enzyme deactivation inside the concentration polarization layer is accelerated and the conversion decreased due to an exchange of active enzyme in bulk with deactivated enzyme in the polarization layer via convection and back diffusion. Successful operation can be obtained at flow rates lower than the critical point to avoid the polarization and thus the deactivation. It is shown that 6.5 L of 2mg/mL of cellobiose solution is hydrolyzed to glucose with a conversion of 91% in 20 hours with 1.617 mg of cellobiase enzyme in a reactor attached with a PM 10 membrane of an effective surface area of 39.2 sq.cm. (Refs. 17).

  4. Canoe binds RanGTP to promote Pins(TPR)/Mud-mediated spindle orientation.

    PubMed

    Wee, Brett; Johnston, Christopher A; Prehoda, Kenneth E; Doe, Chris Q

    2011-10-31

    Regulated spindle orientation maintains epithelial tissue integrity and stem cell asymmetric cell division. In Drosophila melanogaster neural stem cells (neuroblasts), the scaffolding protein Canoe (Afadin/Af-6 in mammals) regulates spindle orientation, but its protein interaction partners and mechanism of action are unknown. In this paper, we use our recently developed induced cell polarity system to dissect the molecular mechanism of Canoe-mediated spindle orientation. We show that a previously uncharacterized portion of Canoe directly binds the Partner of Inscuteable (Pins) tetratricopeptide repeat (TPR) domain. The Canoe-Pins(TPR) interaction recruits Canoe to the cell cortex and is required for activation of the Pins(TPR)-Mud (nuclear mitotic apparatus in mammals) spindle orientation pathway. We show that the Canoe Ras-association (RA) domains directly bind RanGTP and that both the Canoe(RA) domains and RanGTP are required to recruit Mud to the cortex and activate the Pins/Mud/dynein spindle orientation pathway.

  5. Modified 3-alkyl-1,8-dibenzylxanthines as GTP-competitive inhibitors of phosphoenolpyruvate carboxykinase.

    PubMed

    Foley, Louise H; Wang, Ping; Dunten, Pete; Ramsey, Gwendolyn; Gubler, Mary-Lou; Wertheimer, Stanley J

    2003-10-20

    The first non-substrate like inhibitors of human cytosolic phosphoenolpyruvate carboxykinase (PEPCK) competitive with GTP are reported. An effort to discover orally active compounds that improve glucose homeostasis in Type 2 diabetics by reversibly inhibiting PEPCK led to the discovery of 1-allyl-3-butyl-8-methylxanthine (5). We now report modifications at N-1 and C-8 that improved the in vitro activity of the initial xanthine HTS hit by 100-fold and a developing SAR for this class of inhibitor.

  6. Assembly of the nuclear pore: biochemically distinct steps revealed with NEM, GTP gamma S, and BAPTA

    PubMed Central

    1996-01-01

    A key event in nuclear formation is the assembly of functional nuclear pores. We have used a nuclear reconstitution system derived from Xenopus eggs to examine the process of nuclear pore assembly in vitro. With this system, we have identified three reagents which interfere with nuclear pore assembly, NEM, GTP gamma S, and the Ca++ chelator, BAPTA. These reagents have allowed us to determine that the assembly of a nuclear pore requires the prior assembly of a double nuclear membrane. Inhibition of nuclear vesicle fusion by pretreatment of the membrane vesicle fraction with NEM blocks pore complex assembly. In contrast, NEM treatment of already fused double nuclear membranes does not block pore assembly. This indicates that NEM inhibits a single step in pore assembly--the initial fusion of vesicles required to form a double nuclear membrane. The presence of GTP gamma S blocks pore assembly at two distinct steps, first by preventing fusion between nuclear vesicles, and second by blocking a step in pore assembly that occurs on already fused double nuclear membranes. Interestingly, when the Ca2+ chelator BAPTA is added to a nuclear assembly reaction, it only transiently blocks nuclear vesicle fusion, but completely blocks nuclear pore assembly. This results in the formation of a nucleus surrounded by a double nuclear membrane, but devoid of nuclear pores. To order the positions at which GTP gamma S and BAPTA interfere with pore assembly, a novel anchored nuclear assembly assay was developed. This assay revealed that the BAPTA-sensitive step in pore assembly occurs after the second GTP gamma S-sensitive step. Thus, through use of an in vitro nuclear reconstitution system, it has been possible to biochemically define and order multiple steps in nuclear pore assembly. PMID:8567730

  7. GTP-binding proteins of the Rho/Rac family: regulation, effectors and functions in vivo

    PubMed Central

    Bustelo, Xosé R.; Sauzeau, Vincent; Berenjeno, Inmaculada M.

    2007-01-01

    Summary Rho/Rac proteins constitute a subgroup of the Ras superfamily of GTP hydrolases. Although originally implicated in the control of cytoskeletal events, it is currently known that these GTPases coordinate diverse cellular functions, including cell polarity, vesicular trafficking, the cell cycle and transcriptomal dynamics. In this review, we will provide an overview on the recent advances in this field regarding the mechanism of regulation and signaling, and the roles in vivo of this important GTPase family. PMID:17373658

  8. Multiple GTP-binding proteins regulate vesicular transport from the ER to Golgi membranes

    PubMed Central

    1992-01-01

    Using indirect immunofluorescence we have examined the effects of reagents which inhibit the function of ras-related rab small GTP- binding proteins and heterotrimeric G alpha beta gamma proteins in ER to Golgi transport. Export from the ER was inhibited by an antibody towards rab1B and an NH2-terminal peptide which inhibits ARF function (Balch, W. E., R. A. Kahn, and R. Schwaninger. 1992. J. Biol. Chem. 267:13053-13061), suggesting that both of these small GTP-binding proteins are essential for the transport vesicle formation. Export from the ER was also potently inhibited by mastoparan, a peptide which mimics G protein binding regions of seven transmembrane spanning receptors activating and uncoupling heterotrimeric G proteins from their cognate receptors. Consistent with this result, purified beta gamma subunits inhibited the export of VSV-G from the ER suggesting an initial event in transport vesicle assembly was regulated by a heterotrimeric G protein. In contrast, incubation in the presence of GTP gamma S or AIF(3-5) resulted in the accumulation of transported protein in different populations of punctate pre-Golgi intermediates distributed throughout the cytoplasm of the cell. Finally, a peptide which is believed to antagonize the interaction of rab proteins with putative downstream effector molecules inhibited transport at a later step preceding delivery to the cis Golgi compartment, similar to the site of accumulation of transported protein in the absence of NSF or calcium (Plutner, H., H. W. Davidson, J. Saraste, and W. E. Balch. 1992. J. Cell Biol. 119:1097-1116). These results are consistent with the hypothesis that multiple GTP-binding proteins including a heterotrimeric G protein(s), ARF and rab1 differentially regulate steps in the transport of protein between early compartments of the secretory pathway. The concept that G protein-coupled receptors gate the export of protein from the ER is discussed. PMID:1447289

  9. De novo GTP Biosynthesis Is Critical for Virulence of the Fungal Pathogen Cryptococcus neoformans

    PubMed Central

    Morrow, Carl A.; Valkov, Eugene; Stamp, Anna; Chow, Eve W. L.; Lee, I. Russel; Wronski, Ania; Williams, Simon J.; Hill, Justine M.; Djordjevic, Julianne T.; Kappler, Ulrike; Kobe, Bostjan; Fraser, James A.

    2012-01-01

    We have investigated the potential of the GTP synthesis pathways as chemotherapeutic targets in the human pathogen Cryptococcus neoformans, a common cause of fatal fungal meningoencephalitis. We find that de novo GTP biosynthesis, but not the alternate salvage pathway, is critical to cryptococcal dissemination and survival in vivo. Loss of inosine monophosphate dehydrogenase (IMPDH) in the de novo pathway results in slow growth and virulence factor defects, while loss of the cognate phosphoribosyltransferase in the salvage pathway yielded no phenotypes. Further, the Cryptococcus species complex displays variable sensitivity to the IMPDH inhibitor mycophenolic acid, and we uncover a rare drug-resistant subtype of C. gattii that suggests an adaptive response to microbial IMPDH inhibitors in its environmental niche. We report the structural and functional characterization of IMPDH from Cryptococcus, revealing insights into the basis for drug resistance and suggesting strategies for the development of fungal-specific inhibitors. The crystal structure reveals the position of the IMPDH moveable flap and catalytic arginine in the open conformation for the first time, plus unique, exploitable differences in the highly conserved active site. Treatment with mycophenolic acid led to significantly increased survival times in a nematode model, validating de novo GTP biosynthesis as an antifungal target in Cryptococcus. PMID:23071437

  10. Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane.

    PubMed

    Vijayaraghavan, Balaje; Jafferali, Mohammed Hakim; Figueroa, Ricardo A; Hallberg, Einar

    2016-07-01

    Samp1 is a transmembrane protein of the inner nuclear membrane (INM), which interacts with the nuclear lamina and the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex in interphase and during mitosis, it localizes to the mitotic spindle. Samp1 was recently found to coprecipitate a protein complex containing Ran, a GTPase with fundamental regulatory functions both in interphase and in mitosis. To investigate the interaction between Samp1 and Ran in further detail, we have designed and expressed recombinant fusion proteins of the Chaetomium thermophilum homolog of Samp1 (Ct.Samp1) and human Ran. Pulldown experiments show that Samp1 binds directly to Ran and that Samp1 binds better to RanGTP compared to RanGDP. Samp1 also preferred RanGTP over RanGDP in living tsBN2 cells. We also show that the Ran binding domain is located between amino acids 75-135 in the nucleoplasmically exposed N-terminal tail of Samp1. This domain is unique for Samp1, without homology in any other proteins in fungi or metazoa. Samp1 is the first known transmembrane protein that binds to Ran and could provide a unique local binding site for RanGTP in the INM. Samp1 overexpression resulted in increased Ran concentrations in the nuclear periphery supporting this idea. PMID:27541860

  11. A naturally occurring, noncanonical GTP aptamer made of simple tandem repeats.

    PubMed

    Curtis, Edward A; Liu, David R

    2014-01-01

    Recently, we used in vitro selection to identify a new class of naturally occurring GTP aptamer called the G motif. Here we report the discovery and characterization of a second class of naturally occurring GTP aptamer, the "CA motif." The primary sequence of this aptamer is unusual in that it consists entirely of tandem repeats of CA-rich motifs as short as three nucleotides. Several active variants of the CA motif aptamer lack the ability to form consecutive Watson-Crick base pairs in any register, while others consist of repeats containing only cytidine and adenosine residues, indicating that noncanonical interactions play important roles in its structure. The circular dichroism spectrum of the CA motif aptamer is distinct from that of A-form RNA and other major classes of nucleic acid structures. Bioinformatic searches indicate that the CA motif is absent from most archaeal and bacterial genomes, but occurs in at least 70 percent of approximately 400 eukaryotic genomes examined. These searches also uncovered several phylogenetically conserved examples of the CA motif in rodent (mouse and rat) genomes. Together, these results reveal the existence of a second class of naturally occurring GTP aptamer whose sequence requirements, like that of the G motif, are not consistent with those of a canonical secondary structure. They also indicate a new and unexpected potential biochemical activity of certain naturally occurring tandem repeats.

  12. Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

    SciTech Connect

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li -Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-06-16

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.

  13. dGTP Starvation in Escherichia coli Provides New Insights into the Thymineless-Death Phenomenon

    PubMed Central

    Itsko, Mark; Schaaper, Roel M.

    2014-01-01

    Starvation of cells for the DNA building block dTTP is strikingly lethal (thymineless death, TLD), and this effect is observed in all organisms. The phenomenon, discovered some 60 years ago, is widely used to kill cells in anticancer therapies, but many questions regarding the precise underlying mechanisms have remained. Here, we show for the first time that starvation for the DNA precursor dGTP can kill E. coli cells in a manner sharing many features with TLD. dGTP starvation is accomplished by combining up-regulation of a cellular dGTPase with a deficiency of the guanine salvage enzyme guanine-(hypoxanthine)-phosphoribosyltransferase. These cells, when grown in medium without an exogenous purine source like hypoxanthine or adenine, display a specific collapse of the dGTP pool, slow-down of chromosomal replication, the generation of multi-branched nucleoids, induction of the SOS system, and cell death. We conclude that starvation for a single DNA building block is sufficient to bring about cell death. PMID:24810600

  14. Ras-GTP dimers activate the Mitogen-Activated Protein Kinase (MAPK) pathway

    PubMed Central

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li-Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-01-01

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors. PMID:26080442

  15. Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane

    PubMed Central

    Vijayaraghavan, Balaje; Jafferali, Mohammed Hakim; Figueroa, Ricardo A.; Hallberg, Einar

    2016-01-01

    ABSTRACT Samp1 is a transmembrane protein of the inner nuclear membrane (INM), which interacts with the nuclear lamina and the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex in interphase and during mitosis, it localizes to the mitotic spindle. Samp1 was recently found to coprecipitate a protein complex containing Ran, a GTPase with fundamental regulatory functions both in interphase and in mitosis. To investigate the interaction between Samp1 and Ran in further detail, we have designed and expressed recombinant fusion proteins of the Chaetomium thermophilum homolog of Samp1 (Ct.Samp1) and human Ran. Pulldown experiments show that Samp1 binds directly to Ran and that Samp1 binds better to RanGTP compared to RanGDP. Samp1 also preferred RanGTP over RanGDP in living tsBN2 cells. We also show that the Ran binding domain is located between amino acids 75–135 in the nucleoplasmically exposed N-terminal tail of Samp1. This domain is unique for Samp1, without homology in any other proteins in fungi or metazoa. Samp1 is the first known transmembrane protein that binds to Ran and could provide a unique local binding site for RanGTP in the INM. Samp1 overexpression resulted in increased Ran concentrations in the nuclear periphery supporting this idea. PMID:27541860

  16. Strain-dependent occurrence of functional GTP:AMP phosphotransferase (AK3) in Saccharomyces cerevisiae.

    PubMed

    Schricker, R; Magdolen, V; Strobel, G; Bogengruber, E; Breitenbach, M; Bandlow, W

    1995-12-29

    The gene for yeast GTP:AMP phosphotransferase (PAK3) was found to encode a nonfunctional protein in 10 laboratory strains and one brewers' strain. The protein product showed high similarity to vertebrate AK3 and was located exclusively in the mitochondrial matrix. The deduced amino acid sequence revealed a protein that was shorter at the carboxyl terminus than all other known adenylate kinases. Introduction of a +1 frameshift into the 3'-terminal region of the gene extended homology of the deduced amino acid sequence to other members of the adenylate kinase family including vertebrate AK3. Frameshift mutations obtained after in vitro and in vivo mutagenesis were capable of complementing the adk1 temperature-conditional deficiency in Escherichia coli, indicating that the frameshift led to the expression of a protein that could phosphorylate AMP. Some yeasts, however, including strain D273-10B, two wine yeasts, and two more distantly related yeast genera, harbored an active allele, named AKY3, which contained a +1 frameshift close to the carboxyl terminus as compared with the laboratory strains. The encoded protein exhibited GTP:AMP and ITP:AMP phosphotransferase activities but did not accept ATP as phosphate donor. Although single copy in the haploid genome, disruption of the AKY3 allele displayed no phenotype, excluding the possibility that laboratory and brewers' strains had collected second site suppressors. It must be concluded that yeast mitochondria can completely dispense with GTP:AMP phosphotransferase activity.

  17. Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway

    DOE PAGES

    Nan, Xiaolin; Tamgüney, Tanja M.; Collisson, Eric A.; Lin, Li -Jung; Pitt, Cameron; Galeas, Jacqueline; Lewis, Sophia; Gray, Joe W.; McCormick, Frank; Chu, Steven

    2015-06-16

    Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referredmore » to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRasG12D, a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRasG12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.« less

  18. Acid hydrolysis of cellulose to yield glucose

    DOEpatents

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

    1979-01-01

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

  19. Simulating GTP:Mg and GDP:Mg with a simple force field: a structural and thermodynamic analysis.

    PubMed

    Simonson, Thomas; Satpati, Priyadarshi

    2013-04-01

    Di- and tri-phosphate nucleotides are essential cofactors for many proteins, usually in an Mg(2+) -bound form. Proteins like GTPases often detect the difference between NDP and NTP and respond by changing conformations. To study such complexes, simple, fixed charge force fields have been used, which allow long simulations and precise free energy calculations. The preference for NTP or NDP binding depends on many factors, including ligand structure and Mg(2+) coordination and the changes they undergo upon binding. Here, we use a simple force field to examine two Mg(2+) coordination modes for the unbound GDP and GTP: direct, or "Inner Sphere" (IS) coordination by one or more phosphate oxygens and indirect, "Outer Sphere" (OS) coordination involving one or more bridging waters. We compare GTP: and GDP:Mg binding with OS and IS coordination; combining the results with experimental data then indicates that GTP prefers the latter. We also examine different kinds of IS coordination and their sensitivity to a key force field parameter: the optimal Mg:oxygen van der Waals distance Rmin . Increasing Rmin improves the Mg:oxygen distances, the GTP: and GDP:Mg binding affinities, and the fraction of GTP:Mg with β + γ phosphate coordination, but does not improve or change the GTP/GDP affinity difference, which remains much larger than experiment. It has no effect on the free energy of GDP binding to a GTPase.

  20. Acid Hydrolysis of Trioxalatocobaltate (III) Ion

    ERIC Educational Resources Information Center

    Wiggans, P. W.

    1975-01-01

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

  1. GDP beta S enhances the activation of phospholipase C caused by thrombin in human platelets: evidence for involvement of an inhibitory GTP-binding protein

    SciTech Connect

    Oberdisse, E.; Lapetina, E.G.

    1987-05-14

    Guanosine 5'-O-thiotriphosphate (GTP gamma S) and thrombin stimulate the activity of phospholipase C in platelets that have been permeabilized with saponin and whose inositol phospholipids have been prelabeled with (/sup 3/H)inositol. Ca/sup 2 +/ has opposite effects on the formation of (/sup 3/H)inositol phosphates induced by thrombin or GTP gamma S. While the action of GTP gamma S on the formation of (/sup 3/H)inositol phosphates is inhibited by Ca/sup 2 +/, action of thrombin is stimulated by Ca/sup 2 +/. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which inhibits the function of GTP-binding proteins, also inhibits the effect of GTP gamma S on phospholipase C stimulation but, surprisingly, increases the effect of thrombin. Ca/sup 2 +/ increases the inhibitory effect of GDP beta S on GTP gamma S activation of phospholipase C, but Ca/sup 2 +/ further enhances the stimulatory effect of GDP beta S on the thrombin activation of phospholipase C. This indicates that two mechanisms are responsible for the activation of phospholipase C in platelets. A GTP-binding protein is responsible for regulation of phospholipase C induced by GTP gamma S, while the effect of thrombin on the stimulation of phospholipase C is independent of GTP-binding proteins. However, the effect of thrombin may be modulated by the action of an inhibitory GTP-binding protein.

  2. Endo-exo Synergism in Cellulose Hydrolysis Revisited*

    PubMed Central

    Jalak, Jürgen; Kurašin, Mihhail; Teugjas, Hele; Väljamäe, Priit

    2012-01-01

    Synergistic cooperation of different enzymes is a prerequisite for efficient degradation of cellulose. The conventional mechanistic interpretation of the synergism between randomly acting endoglucanases (EGs) and chain end-specific processive cellobiohydrolases (CBHs) is that EG-generated new chain ends on cellulose surface serve as starting points for CBHs. Here we studied the hydrolysis of bacterial cellulose (BC) by CBH TrCel7A and EG TrCel5A from Trichoderma reesei under both single-turnover and “steady state” conditions. Unaccountable by conventional interpretation, the presence of EG increased the rate constant of TrCel7A-catalyzed hydrolysis of BC in steady state. At optimal enzyme/substrate ratios, the “steady state” rate of synergistic hydrolysis became limited by the velocity of processive movement of TrCel7A on BC. A processivity value of 66 ± 7 cellobiose units measured for TrCel7A on 14C-labeled BC was close to the leveling off degree of polymerization of BC, suggesting that TrCel7A cannot pass through the amorphous regions on BC and stalls. We propose a mechanism of endo-exo synergism whereby the degradation of amorphous regions by EG avoids the stalling of TrCel7A and leads to its accelerated recruitment. Hydrolysis of pretreated wheat straw suggested that this mechanism of synergism is operative also in the degradation of lignocellulose. Although both mechanisms of synergism are used in parallel, the contribution of conventional mechanism is significant only at high enzyme/substrate ratios. PMID:22733813

  3. Comparing the catalytic strategy of ATP hydrolysis in biomolecular motors.

    PubMed

    Kiani, Farooq Ahmad; Fischer, Stefan

    2016-07-27

    ATP-driven biomolecular motors utilize the chemical energy obtained from the ATP hydrolysis to perform vital tasks in living cells. Understanding the mechanism of enzyme-catalyzed ATP hydrolysis reaction has substantially progressed lately thanks to combined quantum/classical molecular mechanics (QM/MM) simulations. Here, we present a comparative summary of the most recent QM/MM results for myosin, kinesin and F1-ATPase motors. These completely different motors achieve the acceleration of ATP hydrolysis through a very similar catalytic mechanism. ATP hydrolysis has high activation energy because it involves the breaking of two strong bonds, namely the Pγ-Oβγ bond of ATP and the H-O bond of lytic water. The key to the four-fold decrease in the activation barrier by the three enzymes is that the breaking of the Pγ-Oβγ bond precedes the deprotonation of the lytic water molecule, generating a metaphosphate hydrate complex. The resulting singly charged trigonal planar PγO3(-) metaphosphate is a better electrophilic target for attack by an OaH(-) hydroxyl group. The formation of this OaH(-) is promoted by a strong polarization of the lytic water: in all three proteins, this water is forming a hydrogen-bond with a backbone carbonyl group and interacts with the carboxylate group of glutamate (either directly or via an intercalated water molecule). This favors the shedding of one proton by the attacking water. The abstracted proton is transferred to the γ-phosphate via various proton wires, resulting in a H2PγO4(-)/ADP(3-) product state. This catalytic strategy is so effective that most other nucleotide hydrolyzing enzymes adopt a similar approach, as suggested by their very similar triphosphate binding sites. PMID:27296627

  4. GTP cyclohydrolase I prevents diabetic-impaired endothelial progenitor cells and wound healing by suppressing oxidative stress/thrombospondin-1.

    PubMed

    Tie, Lu; Chen, Lu-Yuan; Chen, Dan-Dan; Xie, He-Hui; Channon, Keith M; Chen, Alex F

    2014-05-15

    Endothelial progenitor cell (EPC) dysfunction is a key contributor to diabetic refractory wounds. Endothelial nitric oxide synthase (eNOS), which critically regulates the mobilization and function of EPCs, is uncoupled in diabetes due to decreased cofactor tetrahydrobiopterin (BH4). We tested whether GTP cyclohydrolase I (GTPCH I), the rate-limiting enzyme of BH4 synthesis, preserves EPC function in type 1 diabetic mice. Type 1 diabetes was induced in wild-type (WT) and GTPCH I transgenic (Tg-GCH) mice by intraperitoneal injection of streptozotocin (STZ). EPCs were isolated from the peripheral blood and bone marrow of WT, Tg-GCH, and GTPCH I-deficient hph-1 mice. The number of EPCs was significantly lower in STZ-WT mice and hph-1 mice and was rescued in STZ Tg-GCH mice. Furthermore, GTPCH I overexpression improved impaired diabetic EPC migration and tube formation. EPCs from WT, Tg-GCH, and STZ-Tg-GCH mice were administered to diabetic excisional wounds and accelerated wound healing significantly, with a concomitant augmentation of angiogenesis. Flow cytometry measurements showed that intracellular nitric oxide (NO) levels were reduced significantly in STZ-WT and hph-1 mice, paralleled by increased superoxide anion levels; both were rescued in STZ-Tg-GCH mice. Western blot analysis revealed that thrombospondin-1 (TSP-1) was significantly upregulated in the EPCs of STZ-WT mice and hph-1 mice and suppressed in STZ-treated Tg-GCH mice. Our results demonstrate that the GTPCH I/BH4 pathway is critical to preserve EPC quantity, function, and regenerative capacity during wound healing in type 1 diabetic mice at least partly through the attenuation of superoxide and TSP-1 levels and augmentation of NO level.

  5. Microwave Pretreatment For Hydrolysis Of Cellulose

    NASA Technical Reports Server (NTRS)

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

    1993-01-01

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

  6. Rate of Hydrolysis of Tertiary Halogeno Alkanes

    ERIC Educational Resources Information Center

    Pritchard, D. R.

    1978-01-01

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

  7. A Novel GTP-Binding Inhibitor, FX2149, Attenuates LRRK2 Toxicity in Parkinson’s Disease Models

    PubMed Central

    Thomas, Joseph M.; Yang, Dejun; Zhong, Shijun; Xue, Fengtian; Smith, Wanli W.

    2015-01-01

    Leucine-rich repeat kinase-2 (LRRK2), a cytoplasmic protein containing both GTP binding and kinase activities, has emerged as a highly promising drug target for Parkinson’s disease (PD). The majority of PD-linked mutations in LRRK2 dysregulate its GTP binding and kinase activities, which may contribute to neurodegeneration. While most known LRRK2 inhibitors are developed to target the kinase domain, we have recently identified the first LRRK2 GTP binding inhibitor, 68, which not only inhibits LRRK2 GTP binding and kinase activities with high potency in vitro, but also reduces neurodegeneration. However, the in vivo effects of 68 are low due to its limited brain penetration. To address this problem, we reported herein the design and synthesis of a novel analog of 68, FX2149, aimed at increasing the in vivo efficacy. Pharmacological characterization of FX2149 exhibited inhibition of LRRK2 GTP binding activity by ~90% at a concentration of 10 nM using in vitro assays. Furthermore, FX2149 protected against mutant LRRK2-induced neurodegeneration in SH-SY5Y cells at 50-200 nM concentrations. Importantly, FX2149 at 10 mg/kg (i.p.) showed significant brain inhibition efficacy equivalent to that of 68 at 20 mg/kg (i.p.), determined by mouse brain LRRK2 GTP binding and phosphorylation assays. Furthermore, FX2149 at 10 mg/kg (i.p.) attenuated lipopolysaccharide (LPS)-induced microglia activation and LRRK2 upregulation in a mouse neuroinflammation model comparable to 68 at 20 mg/kg (i.p.). Our results highlight a novel GTP binding inhibitor with better brain efficacy, which represents a new lead compound for further understanding PD pathogenesis and therapeutic studies. PMID:25816252

  8. Future accelerators (?)

    SciTech Connect

    John Womersley

    2003-08-21

    I describe the future accelerator facilities that are currently foreseen for electroweak scale physics, neutrino physics, and nuclear structure. I will explore the physics justification for these machines, and suggest how the case for future accelerators can be made.

  9. The integrated global temperature change potential (iGTP) and relationships between emission metrics

    NASA Astrophysics Data System (ADS)

    Peters, Glen P.; Aamaas, Borgar; Berntsen, Terje; Fuglestvedt, Jan S.

    2011-12-01

    The Kyoto Protocol compares greenhouse gas emissions (GHGs) using the global warming potential (GWP) with a 100 yr time-horizon. The GWP was developed, however, to illustrate the difficulties in comparing GHGs. In response, there have been many critiques of the GWP and several alternative emission metrics have been proposed. To date, there has been little focus on understanding the linkages between, and interpretations of, different emission metrics. We use an energy balance model to mathematically link the absolute GWP, absolute global temperature change potential (AGTP), absolute ocean heat perturbation (AOHP), and integrated AGTP. For pulse emissions, energy conservation requires that AOHP = AGWP - iAGTP/λ and hence AGWP and iAGTP are closely linked and converge as AOHP decays to zero. When normalizing the metrics with CO2 (GWP, GTP, and iGTP), we find that the iGTP and GWP are similar numerically for a wide range of GHGs and time-horizons, except for very short-lived species. The similarity between the iGTPX and GWPX depends on how well a pulse emission of CO2 can substitute for a pulse emission of X across a range of time-horizons. The ultimate choice of emission metric(s) and time-horizon(s) depends on policy objectives. To the extent that limiting integrated temperature change over a specific time-horizon is consistent with the broader objectives of climate policy, our analysis suggests that the GWP represents a relatively robust, transparent and policy-relevant emission metric.

  10. Hg sup 2+ induces GTP-tubulin interactions in rat brain similar to those observed in Alzheimer's disease

    SciTech Connect

    Duhr, E.; Pendergrass, C.; Kasarskis, E.; Slevin, J.; Haley, B. )

    1991-03-11

    The pathogenesis of Alzheimer's Disease (AD) is unknown. Using SDS-PAGE and autoradiography the authors' laboratory has shown: (1) that the tubulin in AD brain is less photolabeled by ({sup 32}P)8N{sub 3}GTP than is tubulin from control brain and (2) that low {mu}M levels of preformed Hg{sup 2+}EDTA specifically blocked interactions of tubulin-({sup 32}P)8N{sub 3}GTP in control human brain homogenates giving a photolabeling profile identical to AD brain. Elevated levels of Hg{sup 2+} have been reported in AD brain by others. Earlier work using ({sup 32}P)8N{sub 3}GTP with Al{sup 3+} treated rat and rabbit brain showed no differences from control with regards to tubulin photolabeling. However, our latest data show that brain samples from Hg{sup 2+} fed rats display an abolished GTP-tubulin interaction similar to AD brain samples as determined by ({sup 32}P)8N{sub 3}GTP photolabeling profiles. Removal of Hg{sup 2+} from treated rats did not reverse the effect. These results suggest that certain complexed forms of Hg{sup 2+} must be considered as a potential source for the etiology of AD.

  11. Conformational changes in dynamin on GTP binding and oligomerization reported by intrinsic and extrinsic fluorescence.

    PubMed

    Solomaha, Elena; Palfrey, H Clive

    2005-11-01

    The effects of guanine nucleotides on the intrinsic and extrinsic fluorescence properties of dynamin were assessed. The intrinsic Trp (tryptophan) fluorescence spectra of purified recombinant dynamin-1 and -2 were very similar, with a maximum at 332 nm. Collisional quenching by KI was weak (approximately 30%), suggesting that the majority of Trp residues are buried. Binding of guanine nucleotides decreased intrinsic fluorescence by 15-20%. Titration of the effects showed that GTP and GDP bound to a single class of non-interacting sites in dynamin tetramers with apparent dissociation constants (K(d)) values of 5.4 and 7.4 microM (dynamin-1) and 13.2 and 7.1 microM (dynamin-2) respectively. Similar dissociation constant values for both nucleotides were obtained by titrating the quenching of IAEDANS [N-iodoacetyl-N'-(5-sulpho-1-naphthyl)ethylenediamine]-labelled dynamin-2. Despite the similar binding affinities, GTP and GDP result in different conformations of the protein, as revealed by sensitivity to proteinase K fragmentation. Dynamins contain five Trp residues, of which four are in the PH domain (pleckstrin homology domain) and one is in the C-terminal PRD (proline/arginine-rich domain). Guanine nucleotides quenched fluorescence emission from a truncated (DeltaPRD) mutant dynamin-1 to the same extent as in the full-length protein, suggesting conformational coupling between the G (groove)-domain and the PH domain. Efficient resonance energy transfer from PH domain Trp residues to bound mant-GTP [where mant stands for 2'-(3')-O-(N-methylanthraniloyl)] suggests that the G-domain and PH domain are in close proximity (5-6 nm). Promotion of dynamin-2 oligomerization, by reduction in ionic strength or increasing protein concentration, had little effect on intrinsic dynamin fluorescence. However, fluorescence emission from IAEDANS.dynamin-2 showed a significant spectral shift on oligomerization. In addition, energy transfer was observed when oligomerization was promoted

  12. Structural insights into how Yrb2p accelerates the assembly of the Xpo1p nuclear export complex.

    PubMed

    Koyama, Masako; Shirai, Natsuki; Matsuura, Yoshiyuki

    2014-11-01

    Proteins and ribonucleoproteins containing a nuclear export signal (NES) assemble with the exportin Xpo1p (yeast CRM1) and Gsp1p-GTP (yeast Ran-GTP) in the nucleus and exit through the nuclear pore complex. In the cytoplasm, Yrb1p (yeast RanBP1) displaces NES from Xpo1p. Efficient export of NES-cargoes requires Yrb2p (yeast RanBP3), a primarily nuclear protein containing nucleoporin-like phenylalanine-glycine (FG) repeats and a low-affinity Gsp1p-binding domain (RanBD). Here, we show that Yrb2p strikingly accelerates the association of Gsp1p-GTP and NES to Xpo1p. We have solved the crystal structure of the Xpo1p-Yrb2p-Gsp1p-GTP complex, a key assembly intermediate that can bind cargo rapidly. Although the NES-binding cleft of Xpo1p is closed in this intermediate, our data suggest that preloading of Gsp1p-GTP onto Xpo1p by Yrb2p, conformational flexibility of Xpo1p, and the low affinity of RanBD enable active displacement of Yrb2p RanBD by NES to occur effectively. The structure also reveals the major binding sites for FG repeats on Xpo1p.

  13. Structure of BipA in GTP form bound to the ratcheted ribosome

    PubMed Central

    Kumar, Veerendra; Chen, Yun; Ero, Rya; Ahmed, Tofayel; Tan, Jackie; Li, Zhe; Wong, Andrew See Weng; Bhushan, Shashi; Gao, Yong-Gui

    2015-01-01

    BPI-inducible protein A (BipA) is a member of the family of ribosome-dependent translational GTPase (trGTPase) factors along with elongation factors G and 4 (EF-G and EF4). Despite being highly conserved in bacteria and playing a critical role in coordinating cellular responses to environmental changes, its structures (isolated and ribosome bound) remain elusive. Here, we present the crystal structures of apo form and GTP analog, GDP, and guanosine-3′,5′-bisdiphosphate (ppGpp)-bound BipA. In addition to having a distinctive domain arrangement, the C-terminal domain of BipA has a unique fold. Furthermore, we report the cryo-electron microscopy structure of BipA bound to the ribosome in its active GTP form and elucidate the unique structural attributes of BipA interactions with the ribosome and A-site tRNA in the light of its possible function in regulating translation. PMID:26283392

  14. Purification, crystallization and preliminary X-ray characterization of the human GTP fucose pyrophosphorylase

    SciTech Connect

    Quirk, Stephen; Seley-Radtke, Katherine L.

    2006-04-01

    The human GTP fucose pyrophosphohydrolase protein has been crystallized via the hanging-drop technique over a reservoir of polyethylene glycol (MW 8000) and ethylene glycol. The orthorhombic crystals diffract to 2.8 Å resolution. The human nucleotide-sugar metabolizing enzyme GTP fucose pyrophosphorylase (GFPP) has been purified to homogeneity by an affinity chromatographic procedure that utilizes a novel nucleoside analog. This new purification regime results in a protein preparation that produces significantly better crystals than traditional purification methods. The purified 66.6 kDa monomeric protein has been crystallized via hanging-drop vapor diffusion at 293 K. Crystals of the native enzyme diffract to 2.8 Å and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}. There is a single GFPP monomer in the asymmetric unit, giving a Matthews coefficient of 2.38 Å{sup 3} Da{sup −1} and a solvent content of 48.2%. A complete native data set has been collected as a first step in determining the three-dimensional structure of this enzyme.

  15. Measurement of Rab35 activity with the GTP-Rab35 trapper RBD35.

    PubMed

    Kobayashi, Hotaka; Etoh, Kan; Marubashi, Soujiro; Ohbayashi, Norihiko; Fukuda, Mitsunori

    2015-01-01

    Small GTPase Rab35 functions as a molecular switch for membrane trafficking, specifically for endocytic recycling, by cycling between a GTP-bound active form and a GDP-bound inactive form. Although Rab35 has been shown to regulate various cellular processes, including cytokinesis, cell migration, and neurite outgrowth, its precise roles in these processes are not fully understood. Since a molecular tool that could be used to measure Rab35 activity would be useful for identifying the mechanisms by which Rab35 mediates membrane trafficking, we recently used a RUN domain-containing region of RUSC2 to develop an active Rab35 trapper, and we named it RBD35 (Rab-binding domain specific for Rab35). Because RBD35 specifically interacts with the GTP-bound active form of Rab35 and does not interact with any of the other 59 Rab proteins identified in humans and mice, RBD35 is a useful tool for measuring the level of active Rab35 by pull-down assays and for inhibiting the function of Rab35 by overexpression. In this chapter, we describe the assay procedures for analyzing Rab35 with RBD35.

  16. Association of the GTP-binding protein Rab3A with bovine adrenal chromaffin granules

    SciTech Connect

    Darchen, F.; Hammel, F.; Monteils, M.P.; Scherman, D. ); Zahraoui, A.; Tavitian, A. )

    1990-08-01

    The Rab3A protein belongs to a large family of small GTP-binding proteins that are present in eukaryotic cells and that share amino acid identities with the Ras proteins (products of the ras protooncogenes). Rab3A, which is specifically located in nervous and endocrine tissues, is suspected to play a key role in secretion. Its localization was investigated in bovine adrenal gland by using a polyclonal antibody. Rab3A was detected in adrenal medulla but not in adrenal cortex. In cultured adrenal medulla cells, Rab3A was specifically expressed in the catecholamine-secreting chromaffin cells. Subcellular fractionation suggested that Rab3A is about 30% cytosolic and that particulate Rab3A is associated with the membrane of chromaffin granules (the catecholamine storage organelles) and with a second compartment likely to be the plasma membrane. The Rab3A localization on chromaffin granule membranes was confirmed by immunoadsorption with an antibody against dopamine {beta}-hydroxylase. Rab3A was not extracted from this membrane by NaCl or KBr but was partially extracted by urea and totally solubilized by Triton X-100, suggesting either an interaction with an intrinsic protein or a membrane association through fatty acid acylation. This study suggests that Rab3A, which may also be located on other secretory vesicles containing noncharacterized small GTP-binding proteins, is involved in their biogenesis or in the regulated secretion process.

  17. Structure of Escherichia coli dGTP triphosphohydrolase: a hexameric enzyme with DNA effector molecules.

    PubMed

    Singh, Deepa; Gawel, Damian; Itsko, Mark; Hochkoeppler, Alejandro; Krahn, Juno M; London, Robert E; Schaaper, Roel M

    2015-04-17

    The Escherichia coli dgt gene encodes a dGTP triphosphohydrolase whose detailed role still remains to be determined. Deletion of dgt creates a mutator phenotype, indicating that the dGTPase has a fidelity role, possibly by affecting the cellular dNTP pool. In the present study, we have investigated the structure of the Dgt protein at 3.1-Å resolution. One of the obtained structures revealed a protein hexamer that contained two molecules of single-stranded DNA. The presence of DNA caused significant conformational changes in the enzyme, including in the catalytic site of the enzyme. Dgt preparations lacking DNA were able to bind single-stranded DNA with high affinity (Kd ∼ 50 nM). DNA binding positively affected the activity of the enzyme: dGTPase activity displayed sigmoidal (cooperative) behavior without DNA but hyperbolic (Michaelis-Menten) kinetics in its presence, consistent with a specific lowering of the apparent Km for dGTP. A mutant Dgt enzyme was also created containing residue changes in the DNA binding cleft. This mutant enzyme, whereas still active, was incapable of DNA binding and could no longer be stimulated by addition of DNA. We also created an E. coli strain containing the mutant dgt gene on the chromosome replacing the wild-type gene. The mutant also displayed a mutator phenotype. Our results provide insight into the allosteric regulation of the enzyme and support a physiologically important role of DNA binding.

  18. Catalysis of GTP hydrolysis by small GTPases at atomic detail by integration of X-ray crystallography, experimental, and theoretical IR spectroscopy.

    PubMed

    Rudack, Till; Jenrich, Sarah; Brucker, Sven; Vetter, Ingrid R; Gerwert, Klaus; Kötting, Carsten

    2015-10-01

    Small GTPases regulate key processes in cells. Malfunction of their GTPase reaction by mutations is involved in severe diseases. Here, we compare the GTPase reaction of the slower hydrolyzing GTPase Ran with Ras. By combination of time-resolved FTIR difference spectroscopy and QM/MM simulations we elucidate that the Mg(2+) coordination by the phosphate groups, which varies largely among the x-ray structures, is the same for Ran and Ras. A new x-ray structure of a Ran·RanBD1 complex with improved resolution confirmed this finding and revealed a general problem with the refinement of Mg(2+) in GTPases. The Mg(2+) coordination is not responsible for the much slower GTPase reaction of Ran. Instead, the location of the Tyr-39 side chain of Ran between the γ-phosphate and Gln-69 prevents the optimal positioning of the attacking water molecule by the Gln-69 relative to the γ-phosphate. This is confirmed in the RanY39A·RanBD1 crystal structure. The QM/MM simulations provide IR spectra of the catalytic center, which agree very nicely with the experimental ones. The combination of both methods can correlate spectra with structure at atomic detail. For example the FTIR difference spectra of RasA18T and RanT25A mutants show that spectral differences are mainly due to the hydrogen bond of Thr-25 to the α-phosphate in Ran. By integration of x-ray structure analysis, experimental, and theoretical IR spectroscopy the catalytic center of the x-ray structural models are further refined to sub-Å resolution, allowing an improved understanding of catalysis.

  19. Catalysis of GTP Hydrolysis by Small GTPases at Atomic Detail by Integration of X-ray Crystallography, Experimental, and Theoretical IR Spectroscopy*

    PubMed Central

    Rudack, Till; Jenrich, Sarah; Brucker, Sven; Vetter, Ingrid R.; Gerwert, Klaus; Kötting, Carsten

    2015-01-01

    Small GTPases regulate key processes in cells. Malfunction of their GTPase reaction by mutations is involved in severe diseases. Here, we compare the GTPase reaction of the slower hydrolyzing GTPase Ran with Ras. By combination of time-resolved FTIR difference spectroscopy and QM/MM simulations we elucidate that the Mg2+ coordination by the phosphate groups, which varies largely among the x-ray structures, is the same for Ran and Ras. A new x-ray structure of a Ran·RanBD1 complex with improved resolution confirmed this finding and revealed a general problem with the refinement of Mg2+ in GTPases. The Mg2+ coordination is not responsible for the much slower GTPase reaction of Ran. Instead, the location of the Tyr-39 side chain of Ran between the γ-phosphate and Gln-69 prevents the optimal positioning of the attacking water molecule by the Gln-69 relative to the γ-phosphate. This is confirmed in the RanY39A·RanBD1 crystal structure. The QM/MM simulations provide IR spectra of the catalytic center, which agree very nicely with the experimental ones. The combination of both methods can correlate spectra with structure at atomic detail. For example the FTIR difference spectra of RasA18T and RanT25A mutants show that spectral differences are mainly due to the hydrogen bond of Thr-25 to the α-phosphate in Ran. By integration of x-ray structure analysis, experimental, and theoretical IR spectroscopy the catalytic center of the x-ray structural models are further refined to sub-Å resolution, allowing an improved understanding of catalysis. PMID:26272610

  20. Cotton cellulose: enzyme adsorption and enzymic hydrolysis

    SciTech Connect

    Beltrame, P.L.; Carniti, P.; Focher, B.; Marzetti, A.; Cattaneo, M.

    1982-01-01

    The adsorption of a crude cellulase complex from Trichoderma viride on variously pretreated cotton cellulose samples was studied in the framework of the Langmuir approach at 2-8 degrees. The saturation amount of adsorbed enzyme was related to the susceptibility of the substrates to hydrolysis. In every case the adsorption process was faster by 2-3 orders of magnitude than the hydrolysis step to give end products. For ZnCl/sub 2/-treated cotton cellulose the Langmuir parameters correlated fairly well with the value of the Michaelis constant, measured for its enzymic hydrolysis, and the adsorptive complex was indistinguishable from the complex of the Michaelis-Menten model for the hydrolysis.

  1. Continuous steam hydrolysis of tulip poplar

    SciTech Connect

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

    1982-01-01

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

  2. Hydrolysis of dicalcium phosphate dihydrate to hydroxyapatite.

    PubMed

    Fulmer, M T; Brown, P W

    1998-04-01

    Dicalcium phosphate dihydrate (DCPD) was hydrolysed in water and in 1 M Na2HPO4 solution at temperatures from 25-60 degrees C. Hydrolysis was incomplete in water. At 25 degrees C, DCPD partially hydrolysed to hydroxyapatite (HAp). Formation of HAp is indicative of incongruent DCPD dissolution. At the higher temperatures, hydrolysis to HAp was more extensive and was accompanied by the formation of anhydrous dicalcium phosphate (DCP). Both of these processes are endothermic. When hydrolysis was carried out in 1 M Na2HPO4 solution, heat absorption was greater at any given temperature than for hydrolysis in water. Complete hydrolysis to HAp occurred in this solution. The hydrolysis of DCPD to HAp in sodium phosphate solution was also endothermic. The complete conversion of DCPD to HAp in sodium phosphate solution would not be expected if the only effect of this solution was to cause DCPD dissolution to become congruent. Because of the buffering capacity of a dibasic sodium phosphate solution, DCPD hydrolysed completely to HAp. Complete conversion to HAp was accompanied by the conversion of dibasic sodium phosphate to monobasic sodium phosphate. The formation of DCP was not observed indicating that the sodium phosphate solution precluded the DCPD-to-DCP dehydration reaction. In addition to affecting the extent of hydrolysis, reaction in the sodium phosphate solution also caused a morphological change in the HAp which formed. HAp formed by hydrolysis in water was needle-like to globular while that formed in the sodium phosphate solution exhibited a florette-like morphology.

  3. Enzymes involved in triglyceride hydrolysis.

    PubMed

    Taskinen, M R; Kuusi, T

    1987-08-01

    The lipolytic enzymes LPL and HL play important roles in the metabolism of lipoproteins and participate in lipoprotein interconversions. LPL was originally recognized to be the key enzyme in the hydrolysis of chylomicrons and triglyceride, but it also turned out to be one determinant of HDL concentration in plasma. When LPL activity is high, chylomicrons and VLDL are rapidly removed from circulation and a concomitant rise of the HDL2 occurs. In contrast, low LPL activity impedes the removal of triglyceride-rich particles, resulting in the elevation of serum triglycerides and a decrease of HDL (HDL2). Concordant changes of this kind in LPL and HDL2 are induced by many physiological and pathological perturbations. Finally, the operation of LPL is also essential for the conversion of VLDL to LDL. This apparently clear-cut role of LPL in lipoprotein interconversions is contrasted with the enigmatic actions of HL. The enzyme was originally thought to participate in the catalyses of chylomicron and VLDL remnants generated in the LPL reaction. However, substantial in vitro and in vivo data indicate that HL is a key enzyme in the degradation of plasma HDL (HDL2) in a manner which opposes LPL. A scheme is presented for the complementary actions of the two enzymes in plasma HDL metabolism. In addition, recent studies have attributed a role to HL in the catabolism of triglyceride-rich lipoproteins, particularly those containing apo E. However, this function becomes clinically important only under conditions where the capacity of the LPL-mediated removal system is exceeded. Such a situation may arise when the input of triglyceride-rich particles (chylomicrons and/or VLDL) is excessive or LPL activity is decreased or absent.

  4. Cloning and molecular characterization of the salt-regulated jojoba ScRab cDNA encoding a small GTP-binding protein.

    PubMed

    Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

    2002-10-01

    Salt stress results in a massive change in gene expression. An 837 bp cDNA designated ScRab was cloned from shoot cultures of the salt tolerant jojoba (Simmondsia chinesis). The cloned cDNA encodes a full length 200 amino acid long polypeptide that bears high homology to the Rab subfamily of small GTP binding proteins, particularly, the Rab5 subfamily. ScRab expression is reduced in shoots grown in the presence of salt compared to shoots from non-stressed cultures. His6-tagged ScRAB protein was expressed in E. coli, and purified to homogeneity. The purified protein bound radiolabelled GTP. The unlabelled guanine nucleotides GTP, GTP gamma S and GDP but not ATP, CTP or UTP competed with GTP binding.

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

    PubMed

    Zhang, Junhua; Pakarinen, Annukka; Viikari, Liisa

    2013-02-01

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

  6. Mg2+-linked self-assembly of FtsZ in the presence of GTP or a GTP analog involves the concerted formation of a narrow size distribution of oligomeric species†

    PubMed Central

    Monterroso, Begoña; Ahijado-Guzmán, Rubén; Reija, Belén; Alfonso, Carlos; Zorrilla, Silvia; Minton, Allen P.; Rivas, Germán

    2012-01-01

    The assembly of the bacterial cell division FtsZ protein in the presence of constantly replenished GTP was studied as a function of Mg2+ concentration (at neutral pH and 0.5 M potassium) under steady-state conditions by sedimentation velocity, concentration-gradient light scattering, fluorescence correlation spectroscopy and dynamic light scattering. Sedimentation velocity measurements confirmed previous results indicating cooperative appearance of a narrow size distribution of finite oligomers with increasing protein concentration. The concentration dependence of light scattering and diffusion coefficients independently verified the cooperative appearance of a narrow distribution of high molecular weight oligomers, and in addition provided a measurement of the average size of these species, which corresponds to 100 ± 20 FtsZ protomers at millimolar Mg2+ concentration. Parallel experiments on solutions containing GMPCPP, a slowly hydrolysable analog of GTP, in place of GTP, likewise indicated the concerted formation of a narrow size distribution of fibrillar oligomers with a larger average mass (corresponding to 160 ± 20 FtsZ monomers). The closely similar behavior of FtsZ in the presence of both GTP and GMPCPP suggests that the observations reflect equilibrium rather than non-equilibrium steady-state properties of both solutions and exhibit parallel manifestations of a common association scheme. PMID:22568594

  7. Dephosphorylation of cofilin in stimulated platelets: roles for a GTP-binding protein and Ca2+.

    PubMed Central

    Davidson, M M; Haslam, R J

    1994-01-01

    In human platelets, thrombin not only stimulates the phosphorylation of pleckstrin (P47) and of myosin P-light chains, but also induces the dephosphorylation of an 18-19 kDa phosphoprotein (P18) [Imaoka, Lynham and Haslam (1983) J. Biol. Chem. 258, 11404-11414]. We have now studied this protein in detail. The thrombin-induced dephosphorylation reaction did not begin until the phosphorylation of myosin P-light chains and the secretion of dense-granule 5-hydroxytryptamine were nearly complete, but did parallel the later stages of platelet aggregation. Experiments with ionophore A23187 and phorbol 12-myristate 13-acetate indicated that dephosphorylation of P18 was stimulated by Ca2+, but not by protein kinase C. Two-dimensional analysis of platelet proteins, using non-equilibrium pH gradient electrophoresis followed by SDS/PAGE, showed that thrombin decreased the amount of phosphorylated P18 in platelets by up to 70% and slightly increased the amount of a more basic unlabelled protein that was present in 3-fold excess of P18 in unstimulated platelets. These two proteins were identified as the phosphorylated and non-phosphorylated forms of the pH-sensitive actin-depolymerizing protein, cofilin, by sequencing of peptide fragments and immunoblotting with a monoclonal antibody specific for cofilin. The molar concentration of cofilin in platelets was approx. 10% that of actin. Platelet cofilin was phosphorylated exclusively on serine. Experiments with electropermeabilized platelets showed that dephosphorylation of cofilin could be stimulated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in the absence of Ca2+ or by a free Ca2+ concentration of 10 microM. This GTP[S]-induced dephosphorylation reaction was inhibited by 1-naphthyl phosphate, but not by okadaic acid. Our results add cofilin to the actin-binding proteins that may regulate the platelet cytoskeleton, and suggest that platelet cofilin can be activated by dephosphorylation reactions initiated either by a GTP

  8. GTP synthases. Proton pumping and phosphorylation in ligand-receptor-G alpha-protein complexes.

    PubMed

    Nederkoorn, P H; Timmerman, H; Donné-Op Den Kelder, G M; Timms, D; Wilkinson, A J; Kelly, D R; Broadley, K J; Davies, R H

    1996-01-01

    A structural model for a ligand-receptor-Gs alpha-protein complex to function as a GTP synthase is presented. The mechanism which is dependent on the movement and rotation of the G alpha-protein alpha 2-helix is seen to involve the delivery of, at least, one proton to the phosphorylation site in the rotation of this helix. The cycle is driven by a ligand-mediated proton pump through the alpha-helices of the receptor, attachment of the conserved Tyr-Arg-Tyr receptor proton shuttle being made to an aspartate group on the Gs alpha-protein terminal sidechain, which is itself linked to the Asn-Gln interaction known to control movement and rotation of the alpha 2-helix between .GDP and .GTP structures. The energetics of proton transfer through the shuttle mechanism and delivery of a proton to the aspartate group are shown to be sufficient to rupture this controlling interaction and its associated backbone bond. The complex leads to full spatial and energetic definition of the receptor proton shuttle mechanism, while there is a striking association of further Tyrosine and Arginine residues in the vicinity of the Gs alpha-protein Asn-Gln interaction. Calculations at the HF 6-31G** level confirm that a critical balance between ion pair and neutral forms of Tyr-Arg interactions under multiply hydrogen bonded conditions in a hydrophobic environment controls proton transfer and recovery mechanisms. The intrinsic preference of the neutral Tyr-Arg form over the ion-pair is 14.0 kcal/mol. Activation of the Tyrosine oxygen atom in the neutral form by single-NH or -OH groups reduces this difference by some 6.4-8.6 kcal/mol but the dominance of the neutral form is maintained. The expected slight overestimates are consistent with the maximum activation enthalpy of 11.0-12.0 kcal/ mol required to initiate proton transfer through the shuttle. The extended form of the shuttle with the Arginine acting competitively between the two Tyrosine residues allows interpretation of observed

  9. [Display of 8-hydroxy-GTP substrate properties of UTP in the reaction of polynucleotide synthesis catalyzed by RNA-polymerase from Escherichia coli in the presence of poly[d(AT).d(AT)] template].

    PubMed

    Bruskov, V I; Kuklina, O V

    1988-01-01

    8-oxy-GTP was obtained via reaction of GTP with ascorbic acid and addition of hydrogen peroxide. 8-oxy-GTP is recognized and displays substrate properties of UTP on substitution of 8-oxy-GTP for UTP in polynucleotide synthesis catalyzed by E. coli RNA polymerase on a poly[d(A-T)].poly[d(A-T)] template. Such incorporation does not take place at equimolar quantities of GTP and 8-Br-GTP. The incorporation of 8-oxy-GTP instead of UTP, is 2.5-3 times higher upon replacement of Mg2+ by Mn2+ ions. The dinucleotide ApU serving as an initiator rises the incorporation level of 8-oxy-GTP both for Mg2+ and Mn2+ ions. 8-oxy-GTP slightly inhibits poly[r(A-U)] synthesis, but UTP strongly inhibits the incorporation of 8-oxy-GTP. [alpha-32P] 8-oxy-GTP is incorporated mainly instead of UTP, but it can be incorporated also during the substitution of 8-oxy-GTP for ATP.

  10. [Role of the adaptins, dynamin like GTP-ases and Rab proteins in metabolic disorders and various infections].

    PubMed

    Kierczak, Marcin; Surmacz, Liliana; Wiejak, Jolanta; Wyroba, Elzbieta

    2003-01-01

    Numerous metabolic and genetic diseases are due to mutations in adaptins, dynamin-like GTP-ases or disorders in trafficking machinery mediated by Rab proteins. A great number of pathogenes including viruses (HIV, SIV), bacteria and protozoa use various elements of endocytic/trafficking machinery to get into the host cells and to make their infection successful. Their different strategies are discussed.

  11. Molecular Mechanism for Conformational Dynamics of Ras·GTP Elucidated from In-Situ Structural Transition in Crystal

    PubMed Central

    Matsumoto, Shigeyuki; Miyano, Nao; Baba, Seiki; Liao, Jingling; Kawamura, Takashi; Tsuda, Chiemi; Takeda, Azusa; Yamamoto, Masaki; Kumasaka, Takashi; Kataoka, Tohru; Shima, Fumi

    2016-01-01

    Ras•GTP adopts two interconverting conformational states, state 1 and state 2, corresponding to inactive and active forms, respectively. However, analysis of the mechanism for state transition was hampered by the lack of the structural information on wild-type Ras state 1 despite its fundamental nature conserved in the Ras superfamily. Here we solve two new crystal structures of wild-type H-Ras, corresponding to state 1 and state 2. The state 2 structure seems to represent an intermediate of state transition and, intriguingly, the state 1 crystal is successfully derived from this state 2 crystal by regulating the surrounding humidity. Structural comparison enables us to infer the molecular mechanism for state transition, during which a wide range of hydrogen-bonding networks across Switch I, Switch II and the α3-helix interdependently undergo gross rearrangements, where fluctuation of Tyr32, translocation of Gln61, loss of the functional water molecules and positional shift of GTP play major roles. The NMR-based hydrogen/deuterium exchange experiments also support this transition mechanism. Moreover, the unveiled structural features together with the results of the biochemical study provide a new insight into the physiological role of state 1 as a stable pool of Ras•GTP in the GDP/GTP cycle of Ras. PMID:27180801

  12. Enzymatic saccharification of pretreated wheat straw: comparison of solids-recycling, sequential hydrolysis and batch hydrolysis.

    PubMed

    Pihlajaniemi, Ville; Sipponen, Satu; Sipponen, Mika H; Pastinen, Ossi; Laakso, Simo

    2014-02-01

    In the enzymatic hydrolysis of lignocellulose materials, the recycling of the solid residue has previously been considered within the context of enzyme recycling. In this study, a steady state investigation of a solids-recycling process was made with pretreated wheat straw and compared to sequential and batch hydrolysis at constant reaction times, substrate feed and liquid and enzyme consumption. Compared to batch hydrolysis, the recycling and sequential processes showed roughly equal hydrolysis yields, while the volumetric productivity was significantly increased. In the 72h process the improvement was 90% due to an increased reaction consistency, while the solids feed was 16% of the total process constituents. The improvement resulted primarily from product removal, which was equally efficient in solids-recycling and sequential hydrolysis processes. No evidence of accumulation of enzymes beyond the accumulation of the substrate was found in recycling. A mathematical model of solids-recycling was constructed, based on a geometrical series.

  13. Proton-in-Flight Mechanism for the Spontaneous Hydrolysis of N-Methyl O-Phenyl Sulfamate: Implications for the Design of Steroid Sulfatase Inhibitors

    PubMed Central

    Edwards, David R.; Wolfenden, Richard

    2012-01-01

    The hydrolysis of N-methyl O-phenyl sulfamate (1) has been studied as a model for steroid sulfatase inhibitors such as Coumate, 667 Coumate and EMATE. At neutral pH, simulating physiological conditions, hydrolysis of 1 involves an intramolecular proton transfer from nitrogen to the bridging oxygen atom of the leaving group. Remarkably, this proton transfer is estimated to accelerate the decomposition of 1 by a factor of 1011. Examination of existing kinetic data reveals that the sulfatase PaAstA catalyzes the hydrolysis of sulfamate esters with moderate efficiencies of ~104; whereas, the catalytic rate acceleration generated by the enzyme for its cognate substrate is on the order of ~1015. Rate constants for hydrolysis of a wide range of sulfuryl esters, ArOSO2X−, are shown to be correlated by a two parameter equation based on pKaArOH and pKaArOSO2XH. PMID:22486328

  14. Review: Enzymatic Hydrolysis of Cellulosic Biomass

    SciTech Connect

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

    2011-07-16

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

  15. Ca2+-independent and Ca2+/GTP-binding protein-controlled exocytosis in a plant cell.

    PubMed

    Homann, U; Tester, M

    1997-06-10

    Exocytosis allows the release of secretory products and the delivery of new membrane material to the plasma membrane. So far, little is known about the underlying molecular mechanism and its control in plant cells. We have used the whole-cell patch-clamp technique to monitor changes in membrane capacitance to study exocytosis in barley aleurone protoplasts. To investigate the involvement of Ca2+ and GTP-binding proteins in exocytosis, protoplasts were dialyzed with very low (<2 nM) and high (1 microM) free Ca2+ and nonhydrolyzable guanine nucleotides guanosine 5'-gamma-thio]triphosphate (GTP[gammaS]) or guanosine 5'-[beta-thio]diphosphate (GDP[betaS]). With less than 2 nM cytoplasmic free Ca2+, the membrane capacitance increased significantly over 20 min. This increase was not altered by GTP[gammaS] or GDP[betaS]. In contrast, dialyzing protoplasts with 1 microM free Ca2+ resulted in a large increase in membrane capacitance that was slightly reduced by GTP[gammaS] and strongly inhibited by GDP[betaS]. We conclude that two exocytotic pathways exist in barley aleurone protoplasts: one that is Ca2+-independent and whose regulation is currently not known and another that is stimulated by Ca2+ and modulated by GTP-binding proteins. We suggest that Ca2+-independent exocytosis may be involved in cell expansion in developing protoplasts. Ca2+-stimulated exocytosis may play a role in gibberellic acid-stimulated alpha-amylase secretion in barley aleurone and, more generally, may be involved in membrane resealing in response to cell damage.

  16. C-Terminal glycine-gated radical initiation by GTP 3',8-cyclase in the molybdenum cofactor biosynthesis.

    PubMed

    Hover, Bradley M; Yokoyama, Kenichi

    2015-03-11

    The molybdenum cofactor (Moco) is an essential redox cofactor found in all kingdoms of life. Genetic mutations in the human Moco biosynthetic enzymes lead to a fatal metabolic disorder, Moco deficiency (MoCD). Greater than 50% of all human MoCD patients have mutations in MOCS1A, a radical S-adenosyl-l-methionine (SAM) enzyme involved in the conversion of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin monophosphate. In MOCS1A, one of the frequently affected locations is the GG motif constituted of two consecutive Gly at the C-terminus. The GG motif is conserved among all MOCS1A homologues, but its role in catalysis or the mechanism by which its mutation causes MoCD was unknown. Here, we report the functional characterization of the GG motif using MoaA, a bacterial homologue of MOCS1A, as a model. Our study elucidated that the GG motif is essential for the activity of MoaA to produce 3',8-cH2GTP from GTP (GTP 3',8-cyclase), and that synthetic peptides corresponding to the C-terminal region of wt-MoaA rescue the GTP 3',8-cyclase activity of the GG-motif mutants. Further biochemical characterization suggested that the C-terminal tail containing the GG motif interacts with the SAM-binding pocket of MoaA, and is essential for the binding of SAM and subsequent radical initiation. In sum, these observations suggest that the C-terminal tail of MoaA provides an essential mechanism to trigger the free radical reaction, impairment of which results in the complete loss of catalytic function of the enzyme, and causes MoCD.

  17. Wakefield accelerators

    SciTech Connect

    Simpson, J.D.

    1990-01-01

    The search for new methods to accelerate particle beams to high energy using high gradients has resulted in a number of candidate schemes. One of these, wakefield acceleration, has been the subject of considerable R D in recent years. This effort has resulted in successful proof of principle experiments and in increased understanding of many of the practical aspects of the technique. Some wakefield basics plus the status of existing and proposed experimental work is discussed, along with speculations on the future of wake field acceleration. 10 refs., 6 figs.

  18. LINEAR ACCELERATOR

    DOEpatents

    Colgate, S.A.

    1958-05-27

    An improvement is presented in linear accelerators for charged particles with respect to the stable focusing of the particle beam. The improvement consists of providing a radial electric field transverse to the accelerating electric fields and angularly introducing the beam of particles in the field. The results of the foregoing is to achieve a beam which spirals about the axis of the acceleration path. The combination of the electric fields and angular motion of the particles cooperate to provide a stable and focused particle beam.

  19. ION ACCELERATOR

    DOEpatents

    Bell, J.S.

    1959-09-15

    An arrangement for the drift tubes in a linear accelerator is described whereby each drift tube acts to shield the particles from the influence of the accelerating field and focuses the particles passing through the tube. In one embodiment the drift tube is splii longitudinally into quadrants supported along the axis of the accelerator by webs from a yoke, the quadrants. webs, and yoke being of magnetic material. A magnetic focusing action is produced by energizing a winding on each web to set up a magnetic field between adjacent quadrants. In the other embodiment the quadrants are electrically insulated from each other and have opposite polarity voltages on adjacent quadrants to provide an electric focusing fleld for the particles, with the quadrants spaced sufficienily close enough to shield the particles within the tube from the accelerating electric field.

  20. Acceleration switch

    DOEpatents

    Abbin, J.P. Jr.; Devaney, H.F.; Hake, L.W.

    1979-08-29

    The disclosure relates to an improved integrating acceleration switch of the type having a mass suspended within a fluid filled chamber, with the motion of the mass initially opposed by a spring and subsequently not so opposed.

  1. Acceleration switch

    DOEpatents

    Abbin, Jr., Joseph P.; Devaney, Howard F.; Hake, Lewis W.

    1982-08-17

    The disclosure relates to an improved integrating acceleration switch of the type having a mass suspended within a fluid filled chamber, with the motion of the mass initially opposed by a spring and subsequently not so opposed.

  2. Hydrolysis of iodine: equilibria at high temperatures

    SciTech Connect

    Palmer, D.A.; Ramette, R.W.; Mesmer, R.E.

    1984-01-01

    The hydrolysis (or disproportionation) of molecular iodine to form iodate and iodide ions has been studied by emf measurements over the temperature range, 3.8/sup 0/ to 209.0/sup 0/C. The interpretation of these results required a knowledge of the formation constant for triiodide ion and the acid dissociation constant of iodic acid, both of which were measured as a function of temperature. The resulting thermodynamic data have been incorporated into a general computer model describing the hydrolysis equilibria of iodine as a function of initial concentration, pH and temperature.

  3. LINEAR ACCELERATOR

    DOEpatents

    Christofilos, N.C.; Polk, I.J.

    1959-02-17

    Improvements in linear particle accelerators are described. A drift tube system for a linear ion accelerator reduces gap capacity between adjacent drift tube ends. This is accomplished by reducing the ratio of the diameter of the drift tube to the diameter of the resonant cavity. Concentration of magnetic field intensity at the longitudinal midpoint of the external sunface of each drift tube is reduced by increasing the external drift tube diameter at the longitudinal center region.

  4. Glucose- and GTP-dependent stimulation of the carboxyl methylation of CDC42 in rodent and human pancreatic islets and pure beta cells. Evidence for an essential role of GTP-binding proteins in nutrient-induced insulin secretion.

    PubMed Central

    Kowluru, A; Seavey, S E; Li, G; Sorenson, R L; Weinhaus, A J; Nesher, R; Rabaglia, M E; Vadakekalam, J; Metz, S A

    1996-01-01

    Several GTP-binding proteins (G-proteins) undergo post-translational modifications (isoprenylation and carboxyl methylation) in pancreatic beta cells. Herein, two of these were identified as CDC42 and rap 1, using Western blotting and immunoprecipitation. Confocal microscopic data indicated that CDC42 is localized only in islet endocrine cells but not in acinar cells of the pancreas. CDC42 undergoes a guanine nucleotide-specific membrane association and carboxyl methylation in normal rat islets, human islets, and pure beta (HIT or INS-1) cells. GTPgammaS-dependent carboxyl methylation of a 23-kD protein was also demonstrable in secretory granule fractions from normal islets or beta cells. AFC (a specific inhibitor of prenyl-cysteine carboxyl methyl transferases) blocked the carboxyl methylation of CDC42 in five types of insulin-secreting cells, without blocking GTPgammaS-induced translocation, implying that methylation is a consequence (not a cause) of transfer to membrane sites. High glucose (but not a depolarizing concentration of K+) induced the carboxyl methylation of CDC42 in intact cells, as assessed after specific immunoprecipitation. This effect was abrogated by GTP depletion using mycophenolic acid and was restored upon GTP repletion by coprovision of guanosine. In contrast, although rap 1 was also carboxyl methylated, it was not translocated to the particulate fraction by GTPgammaS; furthermore, its methylation was also stimulated by 40 mM K+ (suggesting a role which is not specific to nutrient stimulation). AFC also impeded nutrient-induced (but not K+-induced) insulin secretion from islets and beta cells under static or perifusion conditions, whereas an inactive structural analogue of AFC failed to inhibit insulin release. These effects were reproduced not only by S-adenosylhomocysteine (another methylation inhibitor), but also by GTP depletion. Thus, the glucose- and GTP-dependent carboxyl methylation of G-proteins such as CDC42 is an obligate step in

  5. Phosphatase hydrolysis of organic phosphorus compounds

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Optimization of dilute acid hydrolysis of Enteromorpha

    NASA Astrophysics Data System (ADS)

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

    2011-11-01

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

  7. Monitoring enzymatic ATP hydrolysis by EPR spectroscopy.

    PubMed

    Hacker, Stephan M; Hintze, Christian; Marx, Andreas; Drescher, Malte

    2014-07-14

    An adenosine triphosphate (ATP) analogue modified with two nitroxide radicals is developed and employed to study its enzymatic hydrolysis by electron paramagnetic resonance spectroscopy. For this application, we demonstrate that EPR holds the potential to complement fluorogenic substrate analogues in monitoring enzymatic activity.

  8. Hydrolysis of DNA by 17 snake venoms.

    PubMed

    de Roodt, Adolfo Rafael; Litwin, Silvana; Angel, Sergio O

    2003-08-01

    DNA hydrolysis caused by venoms of 17 species of snakes was studied by different methodologies. Endonucleolytic activity was tested by incubation of the venoms with the plasmid pBluescript and subsequent visualization of the electrophoretic patterns in 1% agarose gels stained with ethidium bromide. DNA was sequentially degraded, from supercoiled to opened circle, to linear form, in a concentration dependent manner. The highest hydrolytic activity was observed in Bothrops (B.) neuwiedii and Naja (N.) siamensis venoms. Exonucleolytic activity was analyzed on pBluescript digested with SmaI or EcoRI. All venoms caused complete hydrolysis after 2 h of incubation. SDS-PAGE analysis in gels containing calf thymus DNA showed that the hydrolytic bands were located at approximately 30 kDa. DNA degradation was studied by radial hydrolysis in 1% agarose gels containing calf thymus DNA plus ethidium bromide and visualized by UV light. Venom of B. neuwiedii showed the highest activity whereas those of B. ammodytoides and Ovophis okinavensis (P<0.05) showed the lowest activity. Antibodies against venom of B. neuwiedii or N. siamensis neutralized the DNAse activity of both venoms. In conclusion, venom from different snakes showed endo- and exonucleolytic activity on DNA. The inhibition of DNA hydrolysis by EDTA and heterologous antibodies suggests similarities in the structure of the venom components involved.

  9. Kinetics of the alkaline hydrolysis of nitrocellulose.

    PubMed

    Christodoulatos, C; Su, T L; Koutsospyros, A

    2001-01-01

    Cellulose nitrate (nitrocellulose) is an explosive solid substance used in large quantities in various formulations of rocket and gun propellants. Safe destruction of nitrocellulose can be achieved by alkaline hydrolysis, which converts it to biodegradable products that can then be treated by conventional biological processes. The kinetics of the alkaline hydrolysis of munitions-grade nitrocellulose in sodium hydroxide solutions were investigated in completely mixed batch reactors. Experiments were conducted using solutions of alkaline strength ranging from 0.1 to 15% by mass and temperatures in the range of 30 to 90 degrees C. Regression analysis of the kinetic data revealed that alkaline hydrolysis of nitrocellulose is of the order 1.0 and 1.5 with respect to nitrocellulose and hydroxide concentration, respectively. The activation energy of the hydrolysis reaction was found to be 100.9 kJ/mol with a preexponential Arrhenius constant of 4.73 x 10(13). Nitrite and nitrate, in a 3:1 ratio, were the primary nitrogen species present in the posthydrolysis solution. The kinetic information is pertinent to the development and optimization of nitrocellulose chemical-biological treatment systems.

  10. Thioglycoside hydrolysis catalyzed by {beta}-glucosidase

    SciTech Connect

    Shen Hong; Byers, Larry D.

    2007-10-26

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

  11. Influence of alkoxy groups on rates of acetal hydrolysis and tosylate solvolysis: electrostatic stabilization of developing oxocarbenium ion intermediates and neighboring-group participation to form oxonium ions.

    PubMed

    Garcia, Angie; Otte, Douglas A L; Salamant, Walter A; Sanzone, Jillian R; Woerpel, K A

    2015-05-01

    The hydrolysis of 4-alkoxy-substituted acetals was accelerated by about 20-fold compared to that of sterically comparable substrates that do not have an alkoxy group. Rate accelerations are largest when the two functional groups are linked by a flexible cyclic tether. When controlled for the inductive destabilization, an alkoxy group can accelerate acetal hydrolysis by up to 200-fold. The difference in rates of acetal hydrolysis between a substrate where the alkoxy group was tethered to the acetal group by a five-membered ring compared to one where it was tethered by an eight-membered ring was less than 100-fold, suggesting that fused-ring intermediates were not formed. By comparison, the difference in rates of solvolysis of structurally related tosylates were nearly 10(6)-fold between the five- and eight-membered ring series. This observation implicates neighboring-group participation in the solvolysis of tosylates but not in the hydrolysis of acetals. The acceleration of acetal hydrolysis by an alkoxy group is better explained by electrostatic stabilization of intermediates that accumulate positive charge at the acetal carbon atom.

  12. Influence of alkoxy groups on rates of acetal hydrolysis and tosylate solvolysis: electrostatic stabilization of developing oxocarbenium ion intermediates and neighboring-group participation to form oxonium ions.

    PubMed

    Garcia, Angie; Otte, Douglas A L; Salamant, Walter A; Sanzone, Jillian R; Woerpel, K A

    2015-05-01

    The hydrolysis of 4-alkoxy-substituted acetals was accelerated by about 20-fold compared to that of sterically comparable substrates that do not have an alkoxy group. Rate accelerations are largest when the two functional groups are linked by a flexible cyclic tether. When controlled for the inductive destabilization, an alkoxy group can accelerate acetal hydrolysis by up to 200-fold. The difference in rates of acetal hydrolysis between a substrate where the alkoxy group was tethered to the acetal group by a five-membered ring compared to one where it was tethered by an eight-membered ring was less than 100-fold, suggesting that fused-ring intermediates were not formed. By comparison, the difference in rates of solvolysis of structurally related tosylates were nearly 10(6)-fold between the five- and eight-membered ring series. This observation implicates neighboring-group participation in the solvolysis of tosylates but not in the hydrolysis of acetals. The acceleration of acetal hydrolysis by an alkoxy group is better explained by electrostatic stabilization of intermediates that accumulate positive charge at the acetal carbon atom. PMID:25806832

  13. Using porphyritic andesite as a new additive for improving hydrolysis and acidogenesis of solid organic wastes.

    PubMed

    Li, Dawei; Zhou, Tao; Chen, Ling; Jiang, Weizhong; Cheng, Fan; Li, Baoming; Kitamura, Yutaka

    2009-12-01

    The effects of porphyritic andesite on the hydrolysis and acidogenesis of solid organic wastes were investigated by batch and continuous experiments using a rotational drum fermentation system. The results of the batch experiment show that if porphyritic andesite (1%, 3%, and 5% reactants) is added initially, the pH level increases and hydrolysis and acidogenesis are accelerated. The highest surface based hydrolysis constant (26.4x10(-3) kgm(-2) d(-1)) and volatile solid degradation ratio (43.3%) were obtained at a 1% porphyritic andesite addition. In the continuous experiment, porphyritic andesite elevated the first order hydrolysis constant from 13.10x10(-3) d(-1) to 18.82x10(-3) d(-1). A particle mean diameter reduction rate of 33.05 microm/d and a volatile solid degradation rate of 3.53 g/L d(-1) were obtained under the hydraulic retention time of 4, 8, 12 and 16 d.

  14. Using porphyritic andesite as a new additive for improving hydrolysis and acidogenesis of solid organic wastes.

    PubMed

    Li, Dawei; Zhou, Tao; Chen, Ling; Jiang, Weizhong; Cheng, Fan; Li, Baoming; Kitamura, Yutaka

    2009-12-01

    The effects of porphyritic andesite on the hydrolysis and acidogenesis of solid organic wastes were investigated by batch and continuous experiments using a rotational drum fermentation system. The results of the batch experiment show that if porphyritic andesite (1%, 3%, and 5% reactants) is added initially, the pH level increases and hydrolysis and acidogenesis are accelerated. The highest surface based hydrolysis constant (26.4x10(-3) kgm(-2) d(-1)) and volatile solid degradation ratio (43.3%) were obtained at a 1% porphyritic andesite addition. In the continuous experiment, porphyritic andesite elevated the first order hydrolysis constant from 13.10x10(-3) d(-1) to 18.82x10(-3) d(-1). A particle mean diameter reduction rate of 33.05 microm/d and a volatile solid degradation rate of 3.53 g/L d(-1) were obtained under the hydraulic retention time of 4, 8, 12 and 16 d. PMID:19560914

  15. A New Use for a Familiar Fold: the X-Ray Crystal Structure of GTP-Bound GTP Cyclohydrolase III From Methanocaldococcus Jannaschii Reveals a Two Metal Ion Catalytic Mechanism

    SciTech Connect

    Morrison, S.D.; Roberts, S.A.; Zegeer, A.M.; Montfort, W.R.; Bandarian, V.

    2009-05-26

    GTP cyclohydrolase (GCH) III from Methanocaldococcus jannaschii, which catalyzes the conversion of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy), has been shown to require Mg{sup 2+} for catalytic activity and is activated by monovalent cations such as K{sup +} and ammonium [Graham, D. E., Xu, H., and White, R. H. (2002) Biochemistry 41, 15074-15084]. The reaction is formally identical to that catalyzed by a GCH II ortholog (SCO 6655) from Streptomyces coelicolor; however, SCO 6655, like other GCH II proteins, is a zinc-containing protein. The structure of GCH III complexed with GTP solved at 2 {angstrom} resolution clearly shows that GCH III adopts a distinct fold that is closely related to the palm domains of phosphodiesterases, such as DNA polymerase I. GCH III is a tetramer of identical subunits; each monomer is composed of an N- and a C-terminal domain that adopt nearly superimposible structures, suggesting that the protein has arisen by gene duplication. Three metal ions were located in the active site, two of which occupy positions that are analogous to those occupied by divalent metal ions in the structures of a number of palm domain containing proteins, such as DNA polymerase I. Two conserved Asp residues that coordinate the metal ions, which are also found in palm domain containing proteins, are observed in GCH III. Site-directed variants (Asp{yields}Asn) of these residues in GCH III are less active than wild-type. The third metal ion, most likely a potassium ion, is involved in substrate recognition through coordination of O6 of GTP. The arrangement of the metal ions in the active site suggests that GCH III utilizes two metal ion catalysis. The structure of GCH III extends the repertoire of possible reactions with a palm fold to include cyclohydrolase chemistry.

  16. Glycosyl conformational and inductive effects on the acid catalysed hydrolysis of purine nucleosides.

    PubMed Central

    Jordan, F; Niv, H

    1977-01-01

    The log kobs vs. pH profiles were determined in the intermediate acidity region for the glycosyl hydrolysis of guanosine and its 8-amino, 8-monomethylamino, 8-dimethylamino and 8-bromo derivatives. The decreased rate of the 8-amino and enhanced rate of the 8-bromo compound compared to guanosine support an A type mechanism: base protonation followed by glycosyl bond cleavage. All three 8-amino guanosines exhibited log kobs - pH profiles clearly showing that both mono and di-base protonated nucleosides undergo hydrolysis. The 700 fold rate acceleration of 8-N(CH3)-guanosine compared to 8-NHCH3-guanosine and the 110 fold rate acceleration of 8-N(CH3)2-adenosine compared to 8-NHCH3-adenosine could be unequivocally attributed to the fixed syn glycosyl conformation of both 8-dimethylamino compounds and relief of steric compression upon hydrolysis in these molecules. The lack of anomerization of all substrates during the course of the reaction supports an A rather than a Schiff-base mechanism. PMID:17100

  17. Formation of. beta. ,. gamma. -methylene-7,8-dihydroneopterin 3'-triphosphate from. beta. ,. gamma. -methyleneguanosine 5'-triphosphate by GTP cyclohydrolase I of Escherichia coli

    SciTech Connect

    Ferre, J.; Jacobson, K.B.

    1984-01-01

    GTP cyclohydrolase I of Escherichia coli converts (..beta..,..gamma..-methylene)GTP to a fluorescent product that is characterized as (..beta..,..gamma..-methylene)dihydroneopterin triphosphate. Interaction between the GTP analog and the enzyme gave a K/sub i/ of 3.0 ..mu..M, which may be compared to the K/sub m/ of 0.1 ..mu..M for GTP. This new analog of dihydroneopterin triphosphate may, in turn, be converted to the same greenish-yellow pteridines (compounds X, X1, and X2) that are obtained from dihydroneopterin triphosphate. Because of its stability to phosphatase action, this analog may be useful for studies in pteridine metabolism. 14 references, 5 figures.

  18. Biochemical and functional characterization of the ROC domain of DAPK establishes a new paradigm of GTP regulation in ROCO proteins.

    PubMed

    Bialik, Shani; Kimchi, Adi

    2012-10-01

    DAPK (death-associated protein kinase) is a newly recognized member of the mammalian family of ROCO proteins, characterized by common ROC (Ras of complex proteins) and COR (C-terminal of ROC) domains. In the present paper, we review our recent work showing that DAPK is functionally a ROCO protein; its ROC domain binds and hydrolyses GTP. Furthermore, GTP binding regulates DAPK catalytic activity in a novel manner by enhancing autophosphorylation on inhibitory Ser308, thereby promoting the kinase 'off' state. This is a novel mechanism for in cis regulation of kinase activity by the distal ROC domain. The functional similarities between DAPK and the Parkinson's disease-associated protein LRRK2 (leucine-rich repeat protein kinase 2), another member of the ROCO family, are also discussed.

  19. Technical bases for precipitate hydrolysis process operating parameters

    SciTech Connect

    Bannochie, C.J.

    1992-10-05

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

  20. Nucleotide binding interactions modulate dNTP selectivity and facilitate 8-oxo-dGTP incorporation by DNA polymerase lambda

    PubMed Central

    Burak, Matthew J.; Guja, Kip E.; Garcia-Diaz, Miguel

    2015-01-01

    8-Oxo-7,8,-dihydro-2′-deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol λ) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol λ. We have solved high resolution crystal structures showing how Pol λ accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn-conformation, and is stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala510 and Asn513, play differential roles in dNTP selectivity. Specifically, Ala510 and Asn513 facilitate incorporation of 8-oxo-dGMP opposite dA and dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol λ and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases. PMID:26220180

  1. The GTP- and Phospholipid-Binding Protein TTD14 Regulates Trafficking of the TRPL Ion Channel in Drosophila Photoreceptor Cells

    PubMed Central

    Cerny, Alexander C.; Altendorfer, André; Schopf, Krystina; Baltner, Karla; Maag, Nathalie; Sehn, Elisabeth; Wolfrum, Uwe; Huber, Armin

    2015-01-01

    Recycling of signaling proteins is a common phenomenon in diverse signaling pathways. In photoreceptors of Drosophila, light absorption by rhodopsin triggers a phospholipase Cβ-mediated opening of the ion channels transient receptor potential (TRP) and TRP-like (TRPL) and generates the visual response. The signaling proteins are located in a plasma membrane compartment called rhabdomere. The major rhodopsin (Rh1) and TRP are predominantly localized in the rhabdomere in light and darkness. In contrast, TRPL translocates between the rhabdomeral plasma membrane in the dark and a storage compartment in the cell body in the light, from where it can be recycled to the plasma membrane upon subsequent dark adaptation. Here, we identified the gene mutated in trpl translocation defective 14 (ttd14), which is required for both TRPL internalization from the rhabdomere in the light and recycling of TRPL back to the rhabdomere in the dark. TTD14 is highly conserved in invertebrates and binds GTP in vitro. The ttd14 mutation alters a conserved proline residue (P75L) in the GTP-binding domain and abolishes binding to GTP. This indicates that GTP binding is essential for TTD14 function. TTD14 is a cytosolic protein and binds to PtdIns(3)P, a lipid enriched in early endosome membranes, and to phosphatidic acid. In contrast to TRPL, rhabdomeral localization of the membrane proteins Rh1 and TRP is not affected in the ttd14 P75L mutant. The ttd14 P75L mutation results in Rh1-independent photoreceptor degeneration and larval lethality suggesting that other processes are also affected by the ttd14 P75L mutation. In conclusion, TTD14 is a novel regulator of TRPL trafficking, involved in internalization and subsequent sorting of TRPL into the recycling pathway that enables this ion channel to return to the plasma membrane. PMID:26509977

  2. Particle acceleration

    NASA Technical Reports Server (NTRS)

    Vlahos, L.; Machado, M. E.; Ramaty, R.; Murphy, R. J.; Alissandrakis, C.; Bai, T.; Batchelor, D.; Benz, A. O.; Chupp, E.; Ellison, D.

    1986-01-01

    Data is compiled from Solar Maximum Mission and Hinothori satellites, particle detectors in several satellites, ground based instruments, and balloon flights in order to answer fundamental questions relating to: (1) the requirements for the coronal magnetic field structure in the vicinity of the energization source; (2) the height (above the photosphere) of the energization source; (3) the time of energization; (4) transistion between coronal heating and flares; (5) evidence for purely thermal, purely nonthermal and hybrid type flares; (6) the time characteristics of the energization source; (7) whether every flare accelerates protons; (8) the location of the interaction site of the ions and relativistic electrons; (9) the energy spectra for ions and relativistic electrons; (10) the relationship between particles at the Sun and interplanetary space; (11) evidence for more than one acceleration mechanism; (12) whether there is single mechanism that will accelerate particles to all energies and also heat the plasma; and (13) how fast the existing mechanisms accelerate electrons up to several MeV and ions to 1 GeV.

  3. Accelerated Achievement

    ERIC Educational Resources Information Center

    Ford, William J.

    2010-01-01

    This article focuses on the accelerated associate degree program at Ivy Tech Community College (Indiana) in which low-income students will receive an associate degree in one year. The three-year pilot program is funded by a $2.3 million grant from the Lumina Foundation for Education in Indianapolis and a $270,000 grant from the Indiana Commission…

  4. ACCELERATION INTEGRATOR

    DOEpatents

    Pope, K.E.

    1958-01-01

    This patent relates to an improved acceleration integrator and more particularly to apparatus of this nature which is gyrostabilized. The device may be used to sense the attainment by an airborne vehicle of a predetermined velocitv or distance along a given vector path. In its broad aspects, the acceleration integrator utilizes a magnetized element rotatable driven by a synchronous motor and having a cylin drical flux gap and a restrained eddy- current drag cap deposed to move into the gap. The angular velocity imparted to the rotatable cap shaft is transmitted in a positive manner to the magnetized element through a servo feedback loop. The resultant angular velocity of tae cap is proportional to the acceleration of the housing in this manner and means may be used to measure the velocity and operate switches at a pre-set magnitude. To make the above-described dcvice sensitive to acceleration in only one direction the magnetized element forms the spinning inertia element of a free gyroscope, and the outer housing functions as a gimbal of a gyroscope.

  5. Plasma accelerator

    DOEpatents

    Wang, Zhehui; Barnes, Cris W.

    2002-01-01

    There has been invented an apparatus for acceleration of a plasma having coaxially positioned, constant diameter, cylindrical electrodes which are modified to converge (for a positive polarity inner electrode and a negatively charged outer electrode) at the plasma output end of the annulus between the electrodes to achieve improved particle flux per unit of power.

  6. Cex1p facilitates Rna1p-mediated dissociation of the Los1p-tRNA-Gsp1p-GTP export complex.

    PubMed

    McGuire, Andrew T; Mangroo, Dev

    2012-02-01

    Nuclear tRNA export plays an essential role in key cellular processes such as regulation of protein synthesis, cell cycle progression, response to nutrient availability and DNA damage and development. Like other nuclear export processes, assembly of the nuclear tRNA export complex in the nucleus is dependent on Ran-GTP/Gsp1p-GTP, and dissociation of the export receptor-tRNA-Ran-GTP/Gsp1p-GTP complex in the cytoplasm requires RanBP1/Yrb1p and RanGAP/Rna1p to activate the GTPase activity of Ran-GTP/Gsp1p-GTP. The Saccharomyces cerevisiae Cex1p and Human Scyl1 have also been proposed to participate in unloading of the tRNA export receptors at the cytoplasmic face of the nuclear pore complex (NPC). Here, we provide evidence suggesting that Cex1p is required for activation of the GTPase activity of Gsp1p and dissociation of the receptor-tRNA-Gsp1p export complex in S. cerevisiae. The data suggest that Cex1p recruits Rna1p from the cytoplasm to the NPC and facilitates Rna1p activation of the GTPase activity of Gsp1p by enabling Rna1p to gain access to Gsp1p-GTP bound to the export receptor tRNA complex. It is possible that this tRNA unloading mechanism is conserved in evolutionarily diverse organisms and that other Gsp1p-GTP-dependent export processes use a pathway-specific component to recruit Rna1p to the NPC.

  7. Rhizobium meliloti NodP and NodQ form a multifunctional sulfate-activating complex requiring GTP for activity.

    PubMed Central

    Schwedock, J S; Liu, C; Leyh, T S; Long, S R

    1994-01-01

    The nodulation genes nodP and nodQ are required for production of Rhizobium meliloti nodulation (Nod) factors. These sulfated oligosaccharides act as morphogenic signals to alfalfa, the symbiotic host of R. meliloti. In previous work, we have shown that nodP and nodQ encode ATP sulfurylase, which catalyzes the formation of APS (adenosine 5'-phosphosulfate) and PPi. In the subsequent metabolic reaction, APS is converted to PAPS (3'-phosphoadenosine 5'-phosphosulfate) by APS kinase. In Escherichia coli, cysD and cysN encode ATP sulfurylase; cysC encodes APS kinase. Here, we present genetic, enzymatic, and sequence similarity data demonstrating that nodP and nodQ encode both ATP sulfurylase and APS kinase activities and that these enzymes associate into a multifunctional protein complex which we designate the sulfate activation complex. We have previously described the presence of a putative GTP-binding site in the nodQ sequence. The present report also demonstrates that GTP enhances the rate of PAPS synthesis from ATP and sulfate (SO4(2-)) by NodP and NodQ expressed in E. coli. Thus, GTP is implicated as a metabolic requirement for synthesis of the R. meliloti Nod factors. Images PMID:7961471

  8. GTP-binding of ARL-3 is activated by ARL-13 as a GEF and stabilized by UNC-119

    PubMed Central

    Zhang, Qing; Li, Yan; Zhang, Yuxia; Torres, Vicente E.; Harris, Peter C.; Ling, Kun; Hu, Jinghua

    2016-01-01

    Primary cilia are sensory organelles indispensable for organogenesis and tissue pattern formation. Ciliopathy small GTPase ARLs are proposed as prominent ciliary switches, which when disrupted result in dysfunctional cilia, yet how ARLs are activated remain elusive. Here, we discover a novel small GTPase functional module, which contains ARL-3, ARL-13, and UNC-119, localizes near the poorly understood inversin (InV)-like compartment in C. elegans. ARL-13 acts synergistically with UNC-119, but antagonistically with ARL-3, in regulating ciliogenesis. We demonstrate that ARL-3 is a unique small GTPase with unusual high intrinsic GDP release but low intrinsic GTP binding rate. Importantly, ARL-13 acts as a nucleotide exchange factor (GEF) of ARL-3, while UNC-119 can stabilize the GTP binding of ARL-3. We further show that excess inactivated ARL-3 compromises ciliogenesis. The findings reveal a novel mechanism that one ciliopathy GTPase ARL-13, as a GEF, coordinates with UNC-119, which may act as a GTP-binding stabilizing factor, to properly activate another GTPase ARL-3 in cilia, a regulatory process indispensable for ciliogenesis. PMID:27102355

  9. Inhibitory GTP binding protein G/sub i/ regulates US -adrenoceptor affinity towards US -agonists

    SciTech Connect

    Marbach, I.; Levitzki, A.

    1987-05-01

    Treatment of S-49 lymphoma cell membranes with pertussis toxin (PT) causes a three-fold reduction of US -adrenoceptor (US AR) affinity towards isoproterenol. A similar treatment with cholera toxin (CT) does not cause such a modulation. The effects were studied by the detailed analysis of SVI-cyanopindolol (CYP) binding curves in the absence and presence of increasing agonist concentrations. Thus, the authors were able to compare in detail the effects of G/sub s/ and G/sub i/ on the agonist-associated state of the US AR. In contrast to these findings, PT treatment does not have any effect on the displacement of SVI-CYP by (-)isoproterenol. These results demonstrate that the inhibitory GTP protein G/sub i/ modulates the US AR affinity towards US -agonists. This might be due to the association of G/sub i/ with the agonist-bound US AR x G/sub s/ x C complex within the membrane. This hypothesis, as well as others, is under investigation.

  10. Cleanrooms and tissue banking how happy I could be with either GMP or GTP?

    PubMed

    Klykens, J; Pirnay, J-P; Verbeken, G; Giet, O; Baudoux, E; Jashari, R; Vanderkelen, A; Ectors, N

    2013-12-01

    The regulatory framework of tissue banking introduces a number of requirements for monitoring cleanrooms for processing tissue or cell grafts. Although a number of requirements were clearly defined, some requirements are open for interpretation. This study aims to contribute to the interpretation of GMP or GTP guidelines for tissue banking. Based on the experience of the participating centers, the results of the monitoring program were evaluated to determine the feasibility of a cleanroom in tissue banking and the monitoring program. Also the microbial efficacy of a laminar airflow cabinet and an incubator in a cleanroom environment was evaluated. This study indicated that a monitoring program of a cleanroom at rest in combination with (final) product testing is a feasible approach. Although no statistical significance (0.90 < p < 0.95) was found there is a strong indication that a Grade D environment is not the ideal background environment for a Grade A obtained through a laminar airflow cabinet. The microbial contamination of an incubator in a cleanroom is limited but requires closed containers for tissue and cell products.

  11. Erythropoietin prevents endothelial dysfunction in GTP-cyclohydrolase I-deficient hph1 mice.

    PubMed

    dʼUscio, Livius V; Santhanam, Anantha V R; Katusic, Zvonimir S

    2014-12-01

    : In this study, we used the mutant hph1 mouse model, which has deficiency in GTP-cyclohydrolase I (GTPCH I) activity, to test the hypothesis that erythropoietin (EPO) protects aortic wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and tetrahydrobiopterin (BH4) levels were reduced in hph1 mice, whereas 7,8-dihydrobiopterin (7,8-BH2) levels were significantly increased. Furthermore, BH4 deficiency caused increased production of superoxide anion and hydrogen peroxide in the aorta thus resulting in impairment of endothelium-dependent relaxations to acetylcholine. Treatment of hph1 mice with recombinant human EPO (1000 U/kg, subcutaneously for 3 days) significantly decreased superoxide anion production by eNOS and improved BH4 to 7,8-BH2 ratio in aortas. EPO also significantly decreased production of hydrogen peroxide and improved endothelium-dependent relaxations in aortas of hph1 mice. In addition, EPO treatment increased protein expressions of copper-/zinc-superoxide dismutase, manganese-superoxide dismutase, and catalase in the aorta of hph1 mice. Our findings demonstrate that treatment with EPO prevented oxidative stress and endothelial dysfunction caused by eNOS uncoupling. Increased vascular expressions of antioxidants seem to be an important molecular mechanism underlying vascular protection by EPO during chronic BH4 deficiency.

  12. From GTP and G proteins to TRPC channels: a personal account.

    PubMed

    Birnbaumer, Lutz

    2015-09-01

    By serendipity and good fortune, as a postdoctoral fellow in 1967, I landed at the right place at the right time, as I was allowed to investigate the mechanism by which hormones activate the enzyme adenylyl cyclase (then adenyl cyclase) in Martin Rodbell's Laboratory at the NIH in Bethesda, Maryland. The work uncovered first, the existence of receptors separate from the enzyme and then, the existence of transduction mechanisms requiring guanosine-5'-triphosphate (GTP) and Mg(2+). With my laboratory colleagues first and postdoctoral fellows after leaving NIH, I participated in the development of the field "signal transduction by G proteins," uncovered by molecular cloning several G-protein-coupled receptors (GPCRs) and became interested in both the molecular makeup of voltage-gated Ca channels and Ca2+ homeostasis downstream of activation of phospholipase C (PLC) by the Gq/11 signaling pathway. We were able to confirm the hypothesis that there would be mammalian homologues of the Drosophila "transient receptor potential" channel and discovered the existence of six of the seven mammalian genes, now called transient receptor potential canonical (TRPC) channels. In the present article, I summarize from a bird's eye view of what I feel were key findings along this path, not only from my laboratory but also from many others, that allowed for the present knowledge of cell signaling involving G proteins to evolve. Towards the end, I summarize roles of TRPC channels in health and disease. PMID:26377676

  13. Ethylene regulates monomeric GTP-binding protein gene expression and activity in Arabidopsis.

    PubMed

    Moshkov, Igor E; Mur, Luis A J; Novikova, Galina V; Smith, Aileen R; Hall, Michael A

    2003-04-01

    Ethylene rapidly and transiently up-regulates the activity of several monomeric GTP-binding proteins (monomeric G proteins) in leaves of Arabidopsis as determined by two-dimensional gel electrophoresis and autoradiographic analyses. The activation is suppressed by the receptor-directed inhibitor 1-methylcyclopropene. In the etr1-1 mutant, constitutive activity of all the monomeric G proteins activated by ethylene is down-regulated relative to wild type, and ethylene treatment has no effect on the levels of activity. Conversely, in the ctr1-1 mutant, several of the monomeric G proteins activated by ethylene are constitutively up-regulated. However, the activation profile of ctr1-1 does not exactly mimic that of ethylene-treated wild type. Biochemical and molecular evidence suggested that some of these monomeric G proteins are of the Rab class. Expression of the genes for a number of monomeric G proteins in response to ethylene was investigated by reverse transcriptase-PCR. Rab8 and Ara3 expression was increased within 10 min of ethylene treatment, although levels fell back significantly by 40 min. In the etr1-1 mutant, expression of Rab8 was lower than wild type and unaffected by ethylene; in ctr1-1, expression of Rab8 was much higher than wild type and comparable with that seen in ethylene treatments. Expression in ctr1-1 was also unaffected by ethylene. Thus, the data indicate a role for monomeric G proteins in ethylene signal transduction.

  14. Developmentally regulated GTP-binding protein 2 coordinates Rab5 activity and transferrin recycling

    PubMed Central

    Mani, Muralidharan; Lee, Unn Hwa; Yoon, Nal Ae; Kim, Hyo Jeong; Ko, Myoung Seok; Seol, Wongi; Joe, Yeonsoo; Chung, Hun Taeg; Lee, Byung Ju; Moon, Chang Hoon; Cho, Wha Ja; Park, Jeong Woo

    2016-01-01

    The small GTPase Rab5 regulates the early endocytic pathway of transferrin (Tfn), and Rab5 deactivation is required for Tfn recycling. Rab5 deactivation is achieved by RabGAP5, a GTPase-activating protein, on the endosomes. Here we report that recruitment of RabGAP5 is insufficient to deactivate Rab5 and that developmentally regulated GTP-binding protein 2 (DRG2) is required for Rab5 deactivation and Tfn recycling. DRG2 was associated with phosphatidylinositol 3-phosphate–containing endosomes. It colocalized and interacted with EEA1 and Rab5 on endosomes in a phosphatidylinositol 3-kinase–dependent manner. DRG2 depletion did not affect Tfn uptake and recruitment of RabGAP5 and Rac1 to Rab5 endosomes. However, it resulted in impairment of interaction between Rab5 and RabGAP5, Rab5 deactivation on endosomes, and Tfn recycling. Ectopic expression of shRNA-resistant DRG2 rescued Tfn recycling in DRG2-depleted cells. Our results demonstrate that DRG2 is an endosomal protein and a key regulator of Rab5 deactivation and Tfn recycling. PMID:26582392

  15. Sweet tastants stimulate adenylate cyclase coupled to GTP-binding protein in rat tongue membranes.

    PubMed

    Striem, B J; Pace, U; Zehavi, U; Naim, M; Lancet, D

    1989-05-15

    Sucrose and other saccharides, which produce an appealing taste in rats, were found to significantly stimulate the activity of adenylate cyclase in membranes derived from the anterior-dorsal region of rat tongue. In control membranes derived from either tongue muscle or tongue non-sensory epithelium, the effect of sugars on adenylate cyclase activity was either much smaller or absent. Sucrose enhanced adenylate cyclase activity in a dose-related manner, and this activation was dependent on the presence of guanine nucleotides, suggesting the involvement of a GTP-binding protein ('G-protein'). The activation of adenylate cyclase by various mono- and di-saccharides correlated with their electrophysiological potency. Among non-sugar sweeteners, sodium saccharin activated the enzyme, whereas aspartame and neohesperidin dihydrochalcone did not, in correlation with their sweet-taste effectiveness in the rat. Sucrose activation of the enzyme was partly inhibited by Cu2+ and Zn2+, in agreement with their effect on electrophysiological sweet-taste responses. Our results are consistent with a sweet-taste transduction mechanism involving specific receptors, a guanine-nucleotide-binding protein and the cyclic AMP-generating enzyme adenylate cyclase.

  16. RCC1-dependent activation of Ran accelerates cell cycle and DNA repair, inhibiting DNA damage–induced cell senescence

    PubMed Central

    Cekan, Pavol; Hasegawa, Keisuke; Pan, Yu; Tubman, Emily; Odde, David; Chen, Jin-Qiu; Herrmann, Michelle A.; Kumar, Sheetal; Kalab, Petr

    2016-01-01

    The coordination of cell cycle progression with the repair of DNA damage supports the genomic integrity of dividing cells. The function of many factors involved in DNA damage response (DDR) and the cell cycle depends on their Ran GTPase–regulated nuclear–cytoplasmic transport (NCT). The loading of Ran with GTP, which is mediated by RCC1, the guanine nucleotide exchange factor for Ran, is critical for NCT activity. However, the role of RCC1 or Ran⋅GTP in promoting cell proliferation or DDR is not clear. We show that RCC1 overexpression in normal cells increased cellular Ran⋅GTP levels and accelerated the cell cycle and DNA damage repair. As a result, normal cells overexpressing RCC1 evaded DNA damage–induced cell cycle arrest and senescence, mimicking colorectal carcinoma cells with high endogenous RCC1 levels. The RCC1-induced inhibition of senescence required Ran and exportin 1 and involved the activation of importin β–dependent nuclear import of 53BP1, a large NCT cargo. Our results indicate that changes in the activity of the Ran⋅GTP–regulated NCT modulate the rate of the cell cycle and the efficiency of DNA repair. Through the essential role of RCC1 in regulation of cellular Ran⋅GTP levels and NCT, RCC1 expression enables the proliferation of cells that sustain DNA damage. PMID:26864624

  17. On the binding of BODIPY-GTP by the photosensory protein YtvA from the common soil bacterium Bacillus subtilis.

    PubMed

    Nakasone, Yusuke; Hellingwerf, Klaas J

    2011-01-01

    The YtvA protein, which is one of the proteins that comprises the network carrying out the signal transfer inducing the general stress response in Bacillus subtilis, is composed of an N-terminal LOV domain (that binds a flavin [FMN]) and a C-terminal STAS domain. This latter domain shows sequence features typical for a nucleotide (NTP) binding protein. It has been proposed (FEBS Lett., 580 [2006], 3818) that BODIPY-GTP can be used as a reporter for nucleotide binding to this site and that activation of the LOV domain by blue light is reflected in an alteration of the BODIPY-GTP fluorescence. Here we confirm that BODIPY-GTP indeed binds to YtvA, but rather nonspecifically, and not limited to the STAS domain. Blue-light modulation of fluorescence emission of YtvA-bound BODIPY-GTP is observed both in the full-length YtvA protein and in a truncated protein composed of the LOV-domain plus the LOV-STAS linker region (YtvA(1-147)) as a light-induced decrease in fluorescence emission. The isolated LOV domain (i.e. without the linker region) does not show such BODIPY-GTP fluorescence changes. Dialysis experiments have confirmed the blue-light-induced release of BODIPY-GTP from YtvA. PMID:21388385

  18. Synthesis of CDP-diacylglycerol by rat liver rough microsomes as visualized by electron microscopic autoradiography: Relationship to GTP-stimulated membrane fusion

    SciTech Connect

    Jolicoeur, M.; Kan, F.W.; Paiement, J. )

    1991-03-01

    Following conditions of incubation for the analysis of liponucleotide synthesis, we compared GTP-dependent formation of CDP-diacylglycerol (CDP-DG) and membrane fusion in RNA-depleted rough microsomes from rat liver. After incubation of stripped rough microsomes (SRM) in the presence of GTP and (5-3H)-CTP, radioactivity was recovered in lipid extracts and identified by thin-layer chromatography as a single spot which co-migrated with CDP-DG. The nucleotide requirement for CDP-DG synthesis and that for membrane fusion were observed to be identical. We next carried out an electron microscopic autoradiographic analysis on incubated membranes to determine the site of incorporation of (5-3H)-CTP. Silver grains were observed directly over the unilamellar membranes of natural vesicles. In confirmation of the biochemical data, quantitation of silver grain density indicated more grains over membranes incubated in the presence of GTP than over those incubated in the absence of this nucleotide. For membranes incubated in the presence of GTP, the grain density was similar over fused and unfused membranes in the same preparation. When SRM were incubated with the enzyme co-factors required for synthesis of phosphatidylinositol, a GTP-independent membrane fusion was observed by both transmission and freeze-fracture electron microscopy. Together with the biochemical and autoradiographic data, this suggests that phospholipid metabolism may be activated by GTP and lead to the fusion of RER membrane.

  19. Formation of a Trimeric Xpo1-Ran[GTP]-Ded1 Exportin Complex Modulates ATPase and Helicase Activities of Ded1

    PubMed Central

    Hauk, Glenn; Bowman, Gregory D.

    2015-01-01

    The DEAD-box RNA helicase Ded1, which is essential in yeast and known as DDX3 in humans, shuttles between the nucleus and cytoplasm and takes part in several basic processes including RNA processing and translation. A key interacting partner of Ded1 is the exportin Xpo1, which together with the GTP-bound state of the small GTPase Ran, facilitates unidirectional transport of Ded1 out of the nucleus. Here we demonstrate that Xpo1 and Ran[GTP] together reduce the RNA-stimulated ATPase and helicase activities of Ded1. Binding and inhibition of Ded1 by Xpo1 depend on the affinity of the Ded1 nuclear export sequence (NES) for Xpo1 and the presence of Ran[GTP]. Association with Xpo1/Ran[GTP] reduces RNA-stimulated ATPase activity of Ded1 by increasing the apparent KM for the RNA substrate. Despite the increased KM, the Ded1:Xpo1:Ran[GTP] ternary complex retains the ability to bind single stranded RNA, suggesting that Xpo1/Ran[GTP] may modulate the substrate specificity of Ded1. These results demonstrate that, in addition to transport, exportins such as Xpo1 also have the capability to alter enzymatic activities of their cargo. PMID:26120835

  20. Combination of biological pretreatment with liquid hot water pretreatment to enhance enzymatic hydrolysis of Populus tomentosa.

    PubMed

    Wang, Wei; Yuan, Tongqi; Wang, Kun; Cui, Baokai; Dai, Yucheng

    2012-03-01

    A novel stepwise pretreatment of combination of fungal treatment with liquid hot water (LHW) treatment was conducted to enhance the enzymatic hydrolysis of Populus tomentosa. The results showed that lignin and cellulose increased with the elevating temperature, while significant amount of hemicellulose was degraded during the LHW pretreatment. A highest hemicellulose removal of 92.33% was observed by combination of Lenzites betulina C5617 with LHW treatment at 200°C, which was almost 2 times higher than that of sole LHW treatment at the same level. Saccharification of poplar co-treated with L. betulina C5617 and LHW at 200°C resulted in a 2.66-fold increase of glucose yield than that of sole LHW treatment, and an increase (2.25-fold) of glucose yield was obtained by the combination of Trametes ochracea C6888 with LHW. The combination pretreatment performed well at accelerating the enzymatic hydrolysis of poplar wood.

  1. Kinetics and mechanism of the mercury(II)-assisted hydrolysis of methyl iodide.

    PubMed

    Celo, Valbona; Scott, Susannah L

    2005-04-01

    The kinetics and mechanism of the reaction of aqueous Hg(II) with methyl iodide have been investigated. The overall reaction is best described as Hg(II)-assisted hydrolysis, resulting in quantitative formation of methanol and, in the presence of excess methyl iodide, ultimately, HgI2 via the intermediate HgI+. The kinetics are biexponential when methyl iodide is in excess. At 25 degrees C, the acceleration provided by Hg2+ is 7.5 times greater than that caused by HgI+, while assistance of hydrolysis was not observed for HgI2. Thus, the reactions are not catalytic in Hg(II). The kinetics are consistent with an SN2-M+ mechanism involving electrophilic attack at iodide. As expected, methylation of mercury is not a reaction pathway; traces of methylmercury(II) are artifacts of the extraction/preconcentration procedure used for methylmercury analysis.

  2. Pathway of processive ATP hydrolysis by kinesin

    PubMed Central

    Gilbert, Susan P.; Webb, Martin R.; Brune, Martin; Johnson, Kenneth A.

    2007-01-01

    Direct measurement of the kinetics of kinesin dissociation from microtubules, the release of phosphate and ADP from kinesin, and rebinding of kinesin to the microtubule have defined the mechanism for the kinesin ATPase cycle. The processivity of ATP hydrolysis is ten molecules per site at low salt concentration but is reduced to one ATP per site at higher salt concentration. Kinesin dissociates from the microtubule after ATP hydrolysis. This step is rate-limiting. The subsequent rebinding of kinesin · ADP to the microtubule is fast, so kinesin spends only a small fraction of its duty cycle in the dissociated state. These results provide an explanation for the motility differences between skeletal myosin and kinesin. PMID:7854446

  3. Enzymatic hydrolysis of poly(ethylene furanoate).

    PubMed

    Pellis, Alessandro; Haernvall, Karolina; Pichler, Christian M; Ghazaryan, Gagik; Breinbauer, Rolf; Guebitz, Georg M

    2016-10-10

    The urgency of producing new environmentally-friendly polyesters strongly enhanced the development of bio-based poly(ethylene furanoate) (PEF) as an alternative to plastics like poly(ethylene terephthalate) (PET) for applications that include food packaging, personal and home care containers and thermoforming equipment. In this study, PEF powders of various molecular weights (6, 10 and 40kDa) were synthetized and their susceptibility to enzymatic hydrolysis was investigated for the first time. According to LC/TOF-MS analysis, cutinase 1 from Thermobifida cellulosilytica liberated both 2,5-furandicarboxylic acid and oligomers of up to DP4. The enzyme preferentially hydrolyzed PEF with higher molecular weights but was active on all tested substrates. Mild enzymatic hydrolysis of PEF has a potential both for surface functionalization and monomers recycling. PMID:26854948

  4. Continuous steam hydrolysis of tulip poplar

    SciTech Connect

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

    1982-08-01

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

  5. PLA recycling by hydrolysis at high temperature

    NASA Astrophysics Data System (ADS)

    Cristina, Annesini Maria; Rosaria, Augelletti; Sara, Frattari; Fausto, Gironi

    2016-05-01

    In this work the process of PLA hydrolysis at high temperature was studied, in order to evaluate the possibility of chemical recycling of this polymer bio-based. In particular, the possibility to obtain the monomer of lactic acid from PLA degradation was investigated. The results of some preliminary tests, performed in a laboratory batch reactor at high temperature, are presented: the experimental results show that the complete degradation of PLA can be obtained in relatively low reaction times.

  6. A Dominant-negative Gα Mutant That Traps a Stable Rhodopsin-Gα-GTP-βγ Complex*

    PubMed Central

    Ramachandran, Sekar; Cerione, Richard A.

    2011-01-01

    Residues comprising the guanine nucleotide-binding sites of the α subunits of heterotrimeric (large) G-proteins (Gα subunits), as well as the Ras-related (small) G-proteins, are highly conserved. This is especially the case for the phosphate-binding loop (P-loop) where both Gα subunits and Ras-related G-proteins have a conserved serine or threonine residue. Substitutions for this residue in Ras and related (small) G-proteins yield nucleotide-depleted, dominant-negative mutants. Here we have examined the consequences of changing the conserved serine residue in the P-loop to asparagine, within a chimeric Gα subunit (designated αT*) that is mainly comprised of the α subunit of the retinal G-protein transducin and a limited region from the α subunit of Gi1. The αT*(S43N) mutant exhibits a significantly higher rate of intrinsic GDP-GTP exchange compared with wild-type αT*, with light-activated rhodopsin (R*) causing only a moderate increase in the kinetics of nucleotide exchange on αT*(S43N). The αT*(S43N) mutant, when bound to either GDP or GTP, was able to significantly slow the rate of R*-catalyzed GDP-GTP exchange on wild-type αT*. Thus, GTP-bound αT*(S43N), as well as the GDP-bound mutant, is capable of forming a stable complex with R*. αT*(S43N) activated the cGMP phosphodiesterase (PDE) with a dose-response similar to wild-type αT*. Activation of the PDE by αT*(S43N) was unaffected if either R* or β1γ1 alone was present, whereas it was inhibited when R* and the β1γ1 subunit were added together. Overall, our studies suggest that the S43N substitution on αT* stabilizes an intermediate on the G-protein activation pathway consisting of an activated G-protein-coupled receptor, a GTP-bound Gα subunit, and the β1γ1 complex. PMID:21285355

  7. Characterization of proteins that interact with the GTP-bound form of the regulatory GTPase Ran in Arabidopsis.

    PubMed

    Haizel, T; Merkle, T; Pay, A; Fejes, E; Nagy, F

    1997-01-01

    Ran, a small soluble GTP-binding protein, has been shown to be essential for the nuclear translocation of proteins and it is also thought to be involved in regulating cell cycle progression in mammalian and yeast cells. Genes encoding Ran-like proteins have been isolated from different higher plant species. Overexpression of plant Ran cDNAs, similarly to their mammalian/yeast homologues, suppresses the phenotype of the pim46-1 cell cycle mutant in yeast cells. The mammalian/yeast Ran proteins have been shown to interact with a battery of Ran-binding proteins, including the guanidine nucleotide exchange factor RCC1, the GTPase-activating Ran-GAP, nucleoporins and other Ran-binding proteins (RanBPs) specific for Ran-GTP. Here, the characterization of the first Ran-binding proteins from higher plants is reported. The yeast two-hybrid system was used to isolate cDNA clones encoding proteins of approximately 28 kDa (At-RanBP1a, At-RanBP1b) that interact with the GTP-bound forms of the Ran1, Ran2 and Ran3 proteins of Arabidopsis thaliana. The deduced amino acid sequences of the At-RanBP1s display high similarity (60%) to mammalian/yeast RanBP1 proteins and contain the characteristic Ran-binding domains. Furthermore, interaction of the plant Ran and RanBP1 proteins, is shown to require the acidic C-terminal domain (-DEDDDL) of Ran proteins in addition to the presence of an intact Ran-binding domain. In whole cell extracts, the GST-RanBP1a fusion protein binds specifically to GTP-Ran and will not interact with Rab/Ypt-type small GTP-binding proteins. Finally, in good agreement with their proposed biological function, the At-Ran and the At-RanBP genes are expressed coordinately and show the highest level of expression in meristematic tissues.

  8. Fungal secretomes enhance sugar beet pulp hydrolysis.

    PubMed

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

    2014-04-01

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

  9. Fungal secretomes enhance sugar beet pulp hydrolysis

    PubMed Central

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

    2014-01-01

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

  10. Fungal secretomes enhance sugar beet pulp hydrolysis.

    PubMed

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

    2014-04-01

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

  11. Sugarcane bagasse hydrolysis using yeast cellulolytic enzymes.

    PubMed

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

    2013-10-28

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

  12. Compact accelerator

    DOEpatents

    Caporaso, George J.; Sampayan, Stephen E.; Kirbie, Hugh C.

    2007-02-06

    A compact linear accelerator having at least one strip-shaped Blumlein module which guides a propagating wavefront between first and second ends and controls the output pulse at the second end. Each Blumlein module has first, second, and third planar conductor strips, with a first dielectric strip between the first and second conductor strips, and a second dielectric strip between the second and third conductor strips. Additionally, the compact linear accelerator includes a high voltage power supply connected to charge the second conductor strip to a high potential, and a switch for switching the high potential in the second conductor strip to at least one of the first and third conductor strips so as to initiate a propagating reverse polarity wavefront(s) in the corresponding dielectric strip(s).

  13. BICEP's acceleration

    SciTech Connect

    Contaldi, Carlo R.

    2014-10-01

    The recent Bicep2 [1] detection of, what is claimed to be primordial B-modes, opens up the possibility of constraining not only the energy scale of inflation but also the detailed acceleration history that occurred during inflation. In turn this can be used to determine the shape of the inflaton potential V(φ) for the first time — if a single, scalar inflaton is assumed to be driving the acceleration. We carry out a Monte Carlo exploration of inflationary trajectories given the current data. Using this method we obtain a posterior distribution of possible acceleration profiles ε(N) as a function of e-fold N and derived posterior distributions of the primordial power spectrum P(k) and potential V(φ). We find that the Bicep2 result, in combination with Planck measurements of total intensity Cosmic Microwave Background (CMB) anisotropies, induces a significant feature in the scalar primordial spectrum at scales k∼ 10{sup -3} Mpc {sup -1}. This is in agreement with a previous detection of a suppression in the scalar power [2].

  14. Rate of chase-promoted hydrolysis of ATP in the high affinity catalytic site of beef heart mitochondrial ATPase

    SciTech Connect

    Penefsky, H.S.

    1988-05-05

    Incubation of (..gamma..-/sup 32/P)ATP with a molar excess of the soluble, homogeneous ATPase from beef heart mitochondria (F/sub 1/) results in binding of substrate primarily in a single, very high affinity catalytic site and in a slow rate of hydrolysis characteristic of single site catalysis. Subsequent addition of millimolar concentrations of nonradioactive ATP as a cold chase, sufficient to fill catalytic sites on the enzyme, results in an acceleration of hydrolysis of bound radioactive ATP of as much as 10/sup 6/-fold, that is to V/sub max/ rates. For this reason, it was proposed that the high affinity catalytic site is a normal catalytic site on the molecule. This paper shows, in experiments with a rapid mixing-chemical quench apparatus, that hydrolysis of ATP bound in the high affinity catalytic site is accelerated to V/sub max/ rates following addition of 5 ..mu..M ATP as a cold chase. Hydrolysis of bound ATP appears to precede that of the chase. The weight of the available evidence continues to support the original suggestion that the high affinity catalytic site of beef heart F/sub 1/ is a normal catalytic site.

  15. Partial acid hydrolysis of poplar wood as a pretreatment for enzymatic hydrolysis

    SciTech Connect

    Knappert, D.; Grethlein, H.; Converse, A.

    1981-01-01

    Partial acid hydrolysis was studied as a pretreatment to enhance glucose yields from enzymatic hydrolysis of poplar. The pretreatments were carried out in a continuous flow reactor at temperatures ranging from 162 to 222/sup 0/C, acid concentrations ranging from 0 to 1.5%, and treatment times from 3.6 to 12.7 s. The pretreated slurries were hydrolyzed with Trichoderma reesei C30 cellulase at 50/sup 0/C and a pH of 4.8 for 48 h. Increased yields of glucose were achieved when poplar was pretreated at temperatures higher than 180/sup 0/C. By increasing the cellobiase activity of the cellulase with the addition of NOVO cellobiase, in some cases 100% of the potential glucose content of the substrate was converted to glucose after only 24 h of enzymatic hydrolysis.

  16. Acute Inhibition of GTP Cyclohydrolase 1 Uncouples Endothelial Nitric Oxide Synthase and Elevates Blood Pressure

    PubMed Central

    Wang, Shuangxi; Xu, Jian; Song, Ping; Wu, Yong; Zhang, Junhua; Choi, Hyoung Chul; Zou, Ming-Hui

    2012-01-01

    GTP cyclohydrolase 1 (GTPCH1) is the rate-limiting enzyme in de novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for endothelial nitric oxide synthase (eNOS) dictating at least partly, the balance of nitric oxide (NO) and superoxide (O2•−) produced by this enzyme. The aim of this study was to determine the effect of acute inhibition of GTPCH1 on BH4, eNOS function, and blood pressure in vivo. Exposure of bovine or mouse aortic endothelial cells to GTPCH1 inhibitors (DAHP or NAS) or GTPCH1- siRNA significantly reduced BH4 and NO levels, but increased superoxide (O2•−) levels. This increase was abolished by sepiapterin (BH4 precursor) or L-NAME (non-selective NOS inhibitor). Incubation of isolated murine aortas with DAHP or NAS impaired acetylcholine-induced endothelium-dependent relaxation, but not endothelium-independent relaxation. Aortas from GTPCH1 siRNA-injected mice, but not their control-siRNA injected counterparts, also exhibited impaired endothelium-dependent relaxation. BH4 reduction induced by GTPCH1 siRNA injection was associated with increased aortic levels of O2•−, 3-nitrotyrosine, and adhesion molecules (ICAM1 and VCAM1) as well as a significantly elevated systolic, diastolic, and mean blood pressure in C57BL6 mice. GTPCH1 siRNA was unable to elicit these effects in eNOS−/− mice. Sepiapterin supplementation, which had no effect on high blood pressure in eNOS−/− mice, partially reversed GTPCH1 siRNA-induced elevation of blood pressure in wild type mice. In conclusion, GTPCH1 via BH4 maintains normal blood pressure and endothelial function in vivo by preserving NO synthesis by eNOS. PMID:18645049

  17. Advanced concepts for acceleration

    SciTech Connect

    Keefe, D.

    1986-07-01

    Selected examples of advanced accelerator concepts are reviewed. Such plasma accelerators as plasma beat wave accelerator, plasma wake field accelerator, and plasma grating accelerator are discussed particularly as examples of concepts for accelerating relativistic electrons or positrons. Also covered are the pulsed electron-beam, pulsed laser accelerator, inverse Cherenkov accelerator, inverse free-electron laser, switched radial-line accelerators, and two-beam accelerator. Advanced concepts for ion acceleration discussed include the electron ring accelerator, excitation of waves on intense electron beams, and two-wave combinations. (LEW)

  18. Accelerators and the Accelerator Community

    SciTech Connect

    Malamud, Ernest; Sessler, Andrew

    2008-06-01

    In this paper, standing back--looking from afar--and adopting a historical perspective, the field of accelerator science is examined. How it grew, what are the forces that made it what it is, where it is now, and what it is likely to be in the future are the subjects explored. Clearly, a great deal of personal opinion is invoked in this process.

  19. Influence of GTP/GDP and magnesium ion on the solvated structure of the protein FtsZ: a molecular dynamics study.

    PubMed

    Jamous, Carla; Basdevant, Nathalie; Ha-Duong, Tap

    2014-01-01

    We present here a structural analysis of ten extensive all-atom molecular dynamics simulations of the monomeric protein FtsZ in various binding states. Since the polymerization and GTPase activities of FtsZ depend on the nature of a bound nucleotide as well as on the presence of a magnesium ion, we studied the structural differences between the average conformations of the following five systems: FtsZ-Apo, FtsZ-GTP, FtsZ-GDP, FtsZ-GTP-Mg, and FtsZ-GDP-Mg. The in silico solvated average structure of FtsZ-Apo significantly differs from the crystallographic structure 1W59 of FtsZ which was crystallized in a dimeric form without nucleotide and magnesium. The simulated Apo form of the protein also clearly differs from the FtsZ structures when it is bound to its ligand, the most important discrepancies being located in the loops surrounding the nucleotide binding pocket. The three average structures of FtsZ-GTP, FtsZ-GDP, and FtsZ-GTP-Mg are overall similar, except for the loop T7 located at the opposite side of the binding pocket and whose conformation in FtsZ-GDP notably differs from the one in FtsZ-GTP and FtsZ-GTP-Mg. The presence of a magnesium ion in the binding pocket has no impact on the FtsZ conformation when it is bound to GTP. In contrast, when the protein is bound to GDP, the divalent cation causes a translation of the nucleotide outwards the pocket, inducing a significant conformational change of the loop H6-H7 and the top of helix H7.

  20. Bioconversion of paper mill sludge to bioethanol in the presence of accelerants or hydrogen peroxide pretreatment.

    PubMed

    Gurram, Raghu Nandan; Al-Shannag, Mohammad; Lecher, Nicholas Joshua; Duncan, Shona M; Singsaas, Eric Lawrence; Alkasrawi, Malek

    2015-09-01

    In this study we investigated the technical feasibility of convert paper mill sludge into fuel ethanol. This involved the removal of mineral fillers by using either chemical pretreatment or mechanical fractionation to determine their effects on cellulose hydrolysis and fermentation to ethanol. In addition, we studied the effect of cationic polyelectrolyte (as accelerant) addition and hydrogen peroxide pretreatment on enzymatic hydrolysis and fermentation. We present results showing that removing the fillers content (ash and calcium carbonate) from the paper mill sludge increases the enzymatic hydrolysis performance dramatically with higher cellulose conversion at faster rates. The addition of accelerant and hydrogen peroxide pretreatment further improved the hydrolysis yields by 16% and 25% (g glucose / g cellulose), respectively with the de-ashed sludge. The fermentation process of produced sugars achieved up to 95% of the maximum theoretical ethanol yield and higher ethanol productivities within 9h of fermentation.

  1. Bioconversion of paper mill sludge to bioethanol in the presence of accelerants or hydrogen peroxide pretreatment.

    PubMed

    Gurram, Raghu Nandan; Al-Shannag, Mohammad; Lecher, Nicholas Joshua; Duncan, Shona M; Singsaas, Eric Lawrence; Alkasrawi, Malek

    2015-09-01

    In this study we investigated the technical feasibility of convert paper mill sludge into fuel ethanol. This involved the removal of mineral fillers by using either chemical pretreatment or mechanical fractionation to determine their effects on cellulose hydrolysis and fermentation to ethanol. In addition, we studied the effect of cationic polyelectrolyte (as accelerant) addition and hydrogen peroxide pretreatment on enzymatic hydrolysis and fermentation. We present results showing that removing the fillers content (ash and calcium carbonate) from the paper mill sludge increases the enzymatic hydrolysis performance dramatically with higher cellulose conversion at faster rates. The addition of accelerant and hydrogen peroxide pretreatment further improved the hydrolysis yields by 16% and 25% (g glucose / g cellulose), respectively with the de-ashed sludge. The fermentation process of produced sugars achieved up to 95% of the maximum theoretical ethanol yield and higher ethanol productivities within 9h of fermentation. PMID:26086086

  2. Protein synthesis in brine shrimp embryos. Regulation of the formation of the ternary complex (Met-tRNAf X eIF-2 X GTP) by two purified protein factors and phosphorylation of Artemia eIF-2.

    PubMed

    Woodley, C L; Roychowdhury, M; MacRae, T H; Olsen, K W; Wahba, A J

    1981-07-01

    We have purified from the ribosomal wash of dormant and developing embryos of Artemia two proteins, Co-eIF-2(A) and Co-eIF-2(B). These factors are essential for ternary complex formation and binding of [35S]-Met-tRNAf to 40-S ribosomal subunits with 15-30 microgram eIF-2/ml of reaction mixture. On polyacrylamide gel electrophoresis in dodecylsulfate, Co-eIF-2(A) is composed of a single polypeptide of Mr 65 000, whereas Co-eIF-2(B) contains polypeptides of Mr 105000 and 112000. Co-eIF-2(A) is sensitive to 4.5 microM aurintricarboxylic acid but Co-eIF-2(B) requires approximately 15 microM aurintricarboxylic acid to give 50% inhibition of ternary complex formation. The stimulatory activity of both factors is abolished by pretreatment of the proteins with N-ethylmaleimide. Artemia eIF-2 rapidly bonds [3H]GDP or [3H]GTP and at 15 degrees C the initiation factor rapidly equilibrates bound nucleotides with free GDP or GTP. Both Co-eIF-2(A) and Co-eIF-2(B) have no effect on the exchange or the amount of nucleotide bound. The small subunit (Mr 43 000) of Artemia eIF-2 is phosphorylated in the presence of the rabbit reticulocyte heme-repressible kinase. Tryptic digestion of [32P]phosphorylated eIF-2 produces a single major phosphopeptide and several minor ones. Acid hydrolysis of these phosphopeptides, as well as of [32P]phosphorylated eIF-2, demonstrates that the radioactivity is predominantly associated with phosphoserine. Phosphorylated Artemia eIF-2 is active in ternary complex formation, in AUG-dependent binding of [35S]Met-tRNAf to 40-S ribosomal subunits and in cell-free protein synthesis. Both Co-eIF-2(A) and Co-eIF-2(B) stimulate ternary complex formation with phosphorylated eIF-2. A kinase that phosphorylates the small subunit of eIF-2 is present in the post-ribosomal supernatant as well as in the ribosomal wash of developing Artemia embryos. PMID:6912815

  3. Hydrolysis of ferric chloride in solution

    SciTech Connect

    Lussiez, G.; Beckstead, L.

    1996-11-01

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

  4. Pretreatment and enzymatic hydrolysis of lignocellulosic biomass

    NASA Astrophysics Data System (ADS)

    Corredor, Deisy Y.

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

  5. Improved method for detection of starch hydrolysis

    SciTech Connect

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

    1982-09-01

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

  6. Urea hydrolysis and calcium carbonate reaction fronts

    NASA Astrophysics Data System (ADS)

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

    2010-12-01

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

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

    PubMed

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

    2015-09-01

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

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

    PubMed Central

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

    2013-01-01

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

  9. Galectin-3 Enhances Migration of Minature Pig Bone Marrow Mesenchymal Stem Cells Through Inhibition of RhoA-GTP Activity

    PubMed Central

    Gao, Qian; Xia, Ying; Liu, Lan; Huang, Lei; Liu, Yang; Zhang, Xue; Xu, Kui; Wei, Jingliang; Hu, Yanqing; Mu, Yulian; Li, Kui

    2016-01-01

    Bone marrow mesenchymal stem cells (BM-MSCs) are used in tissue engineering because of their migration characters. However, BM-MSCs have limitations in terms of reaching injuries and self-renewal. Therefore, enhancement of BM-MSC migration is important for therapeutic applications. Here, we assessed whether galectin-3 (Gal-3) increases the migration of minature pig BM-MSCs. Gal-3 was knocked down by short hairpin RNA (shRNA) or overexpressed using a lentiviral vector in Wuzhishan minature pig BM-MSCs. Proliferation and migration assays showed that knockdown of Gal-3 impaired BM-MSC proliferation and migration, whereas Gal-3 overexpression promoted these behaviors. RhoA-GTP activity was upregulated in Gal-3 shRNA-transfected BM-MSCs, while Rac-1- and Cdc42-GTP showed no changes. Western blotting indicated downregulation of p-AKT (ser473) and p-Erk1/2 after serum starvation for 12 h in Gal-3-knockdown BM-MSCs. p-AKT (ser473) expression was upregulated after serum starvation for 6 h, and p-Erk1/2 expression was unchanged in Gal-3-overexpressing BM-MSCs. Treatment with C3 transferase or Y27632 enhanced migration, whereas Gal-3 knockdown impaired migration in treated cells. These results demonstrate that Gal-3 may enhance BM-MSC migration, mainly through inhibiting RhoA-GTP activity, increasing p-AKT (ser473) expression, and regulating p-Erk1/2 levels. Our study suggests a novel function of Gal-3 in regulating minature pig BM-MSC migration, which may be beneficial for therapeutic applications. PMID:27215170

  10. The Inner Nuclear Membrane Protein Nemp1 Is a New Type of RanGTP-Binding Protein in Eukaryotes

    PubMed Central

    Shibano, Takashi; Mamada, Hiroshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Taira, Masanori

    2015-01-01

    The inner nuclear membrane (INM) protein Nemp1/TMEM194A has previously been suggested to be involved in eye development in Xenopus, and contains two evolutionarily conserved sequences in the transmembrane domains (TMs) and the C-terminal region, named region A and region B, respectively. To elucidate the molecular nature of Nemp1, we analyzed its interacting proteins through those conserved regions. First, we found that Nemp1 interacts with itself and lamin through the TMs and region A, respectively. Colocalization of Nemp1 and lamin at the INM suggests that the interaction with lamin participates in the INM localization of Nemp1. Secondly, through yeast two-hybrid screening using region B as bait, we identified the small GTPase Ran as a probable Nemp1-binding partner. GST pulldown and co-immunoprecipitation assays using region B and Ran mutants revealed that region B binds directly to the GTP-bound Ran through its effector domain. Immunostaining experiments using transfected COS-7 cells revealed that full-length Nemp1 recruits Ran near the nuclear envelope, suggesting a role for Nemp1 in the accumulation of RanGTP at the nuclear periphery. At the neurula-to-tailbud stages of Xenopus embryos, nemp1 expression overlapped with ran in several regions including the eye vesicles. Co-knockdown using antisense morpholino oligos for nemp1 and ran caused reduction of cell densities and severe eye defects more strongly than either single knockdown alone, suggesting their functional interaction. Finally we show that Arabidopsis thaliana Nemp1-orthologous proteins interact with A. thaliana Ran, suggesting their evolutionally conserved physical and functional interactions possibly in basic cellular functions including nuclear transportation. Taken together, we conclude that Nemp1 represents a new type of RanGTP-binding protein. PMID:25946333

  11. The Inner Nuclear Membrane Protein Nemp1 Is a New Type of RanGTP-Binding Protein in Eukaryotes.

    PubMed

    Shibano, Takashi; Mamada, Hiroshi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Taira, Masanori

    2015-01-01

    The inner nuclear membrane (INM) protein Nemp1/TMEM194A has previously been suggested to be involved in eye development in Xenopus, and contains two evolutionarily conserved sequences in the transmembrane domains (TMs) and the C-terminal region, named region A and region B, respectively. To elucidate the molecular nature of Nemp1, we analyzed its interacting proteins through those conserved regions. First, we found that Nemp1 interacts with itself and lamin through the TMs and region A, respectively. Colocalization of Nemp1 and lamin at the INM suggests that the interaction with lamin participates in the INM localization of Nemp1. Secondly, through yeast two-hybrid screening using region B as bait, we identified the small GTPase Ran as a probable Nemp1-binding partner. GST pulldown and co-immunoprecipitation assays using region B and Ran mutants revealed that region B binds directly to the GTP-bound Ran through its effector domain. Immunostaining experiments using transfected COS-7 cells revealed that full-length Nemp1 recruits Ran near the nuclear envelope, suggesting a role for Nemp1 in the accumulation of RanGTP at the nuclear periphery. At the neurula-to-tailbud stages of Xenopus embryos, nemp1 expression overlapped with ran in several regions including the eye vesicles. Co-knockdown using antisense morpholino oligos for nemp1 and ran caused reduction of cell densities and severe eye defects more strongly than either single knockdown alone, suggesting their functional interaction. Finally we show that Arabidopsis thaliana Nemp1-orthologous proteins interact with A. thaliana Ran, suggesting their evolutionally conserved physical and functional interactions possibly in basic cellular functions including nuclear transportation. Taken together, we conclude that Nemp1 represents a new type of RanGTP-binding protein.

  12. Galectin-3 Enhances Migration of Minature Pig Bone Marrow Mesenchymal Stem Cells Through Inhibition of RhoA-GTP Activity.

    PubMed

    Gao, Qian; Xia, Ying; Liu, Lan; Huang, Lei; Liu, Yang; Zhang, Xue; Xu, Kui; Wei, Jingliang; Hu, Yanqing; Mu, Yulian; Li, Kui

    2016-05-24

    Bone marrow mesenchymal stem cells (BM-MSCs) are used in tissue engineering because of their migration characters. However, BM-MSCs have limitations in terms of reaching injuries and self-renewal. Therefore, enhancement of BM-MSC migration is important for therapeutic applications. Here, we assessed whether galectin-3 (Gal-3) increases the migration of minature pig BM-MSCs. Gal-3 was knocked down by short hairpin RNA (shRNA) or overexpressed using a lentiviral vector in Wuzhishan minature pig BM-MSCs. Proliferation and migration assays showed that knockdown of Gal-3 impaired BM-MSC proliferation and migration, whereas Gal-3 overexpression promoted these behaviors. RhoA-GTP activity was upregulated in Gal-3 shRNA-transfected BM-MSCs, while Rac-1- and Cdc42-GTP showed no changes. Western blotting indicated downregulation of p-AKT (ser473) and p-Erk1/2 after serum starvation for 12 h in Gal-3-knockdown BM-MSCs. p-AKT (ser473) expression was upregulated after serum starvation for 6 h, and p-Erk1/2 expression was unchanged in Gal-3-overexpressing BM-MSCs. Treatment with C3 transferase or Y27632 enhanced migration, whereas Gal-3 knockdown impaired migration in treated cells. These results demonstrate that Gal-3 may enhance BM-MSC migration, mainly through inhibiting RhoA-GTP activity, increasing p-AKT (ser473) expression, and regulating p-Erk1/2 levels. Our study suggests a novel function of Gal-3 in regulating minature pig BM-MSC migration, which may be beneficial for therapeutic applications.

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

    EPA Science Inventory

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

  14. Technical bases for precipitate hydrolysis process operating parameters. Revision 1

    SciTech Connect

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

    1992-11-09

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

  15. Technical bases for precipitate hydrolysis process operating parameters

    SciTech Connect

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

    1992-11-09

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

  16. Hydrolysis of peptide esters by different enzymes.

    PubMed

    Reissmann, S; Greiner, G

    1992-08-01

    The combined use in peptide synthesis of the Fmoc-group with methyl, benzyl or p-nitro benzyl esters is not practical because of the elimination of the Fmoc-group under basic conditions and by catalytic hydrogenation. Nevertheless the solution synthesis of peptides requires those combinations in some cases. For this purpose we have investigated enzymatic hydrolysis of some tri and tetrapeptide esters. The hydrolysis were carried out under pH-control. We measured deprotection of the carboxyl group by thermitase, porcine liver esterase, carboxypeptidase A and alpha-chymotrypsin. The main problems are to suppress proteolytic degradation of the peptide bond and to bring the protected peptides into solution. To solve both problems we used dimethylformamide and dimethylsulfoxide as cosolvents. The ratios between esterolytic and proteolytic activity were estimated under various cosolvent concentrations. Advantages of this method are to avoid side reactions of alkaline instable side chains (e.g. asparagine, glutamine), cleavage of base labile protecting groups and racemization by alkaline saponification. The enzymatic deprotection was followed by HPLC, HPTLC and titration. On a preparative scale this method gives good yields and sufficiently pure products.

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

    SciTech Connect

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

    1989-09-05

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

  18. Accelerator system and method of accelerating particles

    NASA Technical Reports Server (NTRS)

    Wirz, Richard E. (Inventor)

    2010-01-01

    An accelerator system and method that utilize dust as the primary mass flux for generating thrust are provided. The accelerator system can include an accelerator capable of operating in a self-neutralizing mode and having a discharge chamber and at least one ionizer capable of charging dust particles. The system can also include a dust particle feeder that is capable of introducing the dust particles into the accelerator. By applying a pulsed positive and negative charge voltage to the accelerator, the charged dust particles can be accelerated thereby generating thrust and neutralizing the accelerator system.

  19. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

    PubMed Central

    Matsui, T; Amano, M; Yamamoto, T; Chihara, K; Nakafuku, M; Ito, M; Nakano, T; Okawa, K; Iwamatsu, A; Kaibuchi, K

    1996-01-01

    The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway. Images PMID:8641286

  20. Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade.

    PubMed Central

    Benn, J; Schneider, R J

    1994-01-01

    Hepatitis B virus produces a small (154-amino acid) transcriptional transactivating protein, HBx, which is required for viral infection and has been implicated in virus-mediated liver oncogenesis. However, the molecular mechanism for HBx activity and its possible influence on cell proliferation have remained obscure. A number of studies suggest that HBx may stimulate transcription by indirectly activating transcription factors, possibly by influencing cell signaling pathways. We now present biochemical evidence that HBx activates Ras and rapidly induces a cytoplasmic signaling cascade linking Ras, Raf, and mitogen-activated protein kinase (MAP kinase), leading to transcriptional transactivation. HBx strongly elevates levels of GTP-bound Ras, activated and phosphorylated Raf, and tyrosine-phosphorylated and activated MAP kinase. Transactivation of transcription factor AP-1 by HBx is blocked by inhibition of Ras or Raf activities but not by inhibition of Ca(2+)- and diacylglycerol-dependent protein kinase C. HBx was also found to stimulate DNA synthesis in serum-starved cells. The hepatitis B virus HBx protein therefore stimulates Ras-GTP complex formation and promotes downstream signaling through Raf and MAP kinases, and may influence cell proliferation. Images PMID:7937954

  1. Purification, crystallization and preliminary crystallographic analysis of a GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus

    SciTech Connect

    Wu, Hao; Sun, Lei; Brouns, Stan J. J.; Fu, Sheng; Akerboom, Jasper; Li, Xuemei; Oost, John van der

    2007-03-01

    A GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus has been crystallized. Combined with biochemical analyses, it is expected that the structure of this protein will give insight in the function of a relatively unknown subfamily of the GTPase superfamily. A predicted GTP-binding protein from the hyperthermophilic archaeon Sulfolobus solfataricus, termed SsGBP, has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized using the hanging-drop vapour-diffusion technique in the presence of 0.05 M cadmium sulfate and 0.8 M sodium acetate pH 7.5. A single-wavelength anomalous dispersion data set was collected to a maximum resolution of 2.0 Å using a single cadmium-incorporated crystal. The crystal form belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with approximate unit-cell parameters a = 65.0, b = 72.6, c = 95.9 Å and with a monomer in the asymmetric unit.

  2. Acid-functionalized nanoparticles for biomass hydrolysis

    NASA Astrophysics Data System (ADS)

    Pena Duque, Leidy Eugenia

    Cellulosic ethanol is a renewable source of energy. Lignocellulosic biomass is a complex material composed mainly of cellulose, hemicellulose, and lignin. Biomass pretreatment is a required step to make sugar polymers liable to hydrolysis. Mineral acids are commonly used for biomass pretreatment. Using acid catalysts that can be recovered and reused could make the process economically more attractive. The overall goal of this dissertation is the development of a recyclable nanocatalyst for the hydrolysis of biomass sugars. Cobalt iron oxide nanoparticles (CoFe2O4) were synthesized to provide a magnetic core that could be separated from reaction using a magnetic field and modified to carry acid functional groups. X-ray diffraction (XRD) confirmed the crystal structure was that of cobalt spinel ferrite. CoFe2O4 were covered with silica which served as linker for the acid functions. Silica-coated nanoparticles were functionalized with three different acid functions: perfluoropropyl-sulfonic acid, carboxylic acid, and propyl-sulfonic acid. Transmission electron microscope (TEM) images were analyzed to obtain particle size distributions of the nanoparticles. Total carbon, nitrogen, and sulfur were quantified using an elemental analyzer. Fourier transform infra-red spectra confirmed the presence of sulfonic and carboxylic acid functions and ion-exchange titrations accounted for the total amount of catalytic acid sites per nanoparticle mass. These nanoparticles were evaluated for their performance to hydrolyze the beta-1,4 glycosidic bond of the cellobiose molecule. Propyl-sulfonic (PS) and perfluoropropyl-sulfonic (PFS) acid functionalized nanoparticles catalyzed the hydrolysis of cellobiose significantly better than the control. PS and PFS were also evaluated for their capacity to solubilize wheat straw hemicelluloses and performed better than the control. Although PFS nanoparticles were stronger acid catalysts, the acid functions leached out of the nanoparticle during

  3. The Lattice As Allosteric Effector: Structural Studies of Alphabeta- And Gamma-Tubulin Clarify the Role of GTP in Microtubule Assembly

    SciTech Connect

    Rice, L.M.; Montabana, E.A.; Agard, D.A.

    2009-05-21

    GTP-dependent microtubule polymerization dynamics are required for cell division and are accompanied by domain rearrangements in the polymerizing subunit, alpha-tubulin. Two opposing models describe the role of GTP and its relationship to conformational change in alpha-tubulin. The allosteric model posits that unpolymerized alpha-tubulin adopts a more polymerization-competent conformation upon GTP binding. The lattice model posits that conformational changes occur only upon recruitment into the growing lattice. Published data support a lattice model, but are largely indirect and so the allosteric model has prevailed. We present two independent solution probes of the conformation of alpha-tubulin, the 2.3 A crystal structure of gamma-tubulin bound to GDP, and kinetic simulations to interpret the functional consequences of the structural data. These results (with our previous gamma-tubulin:GTPgammaS structure) support the lattice model by demonstrating that major domain rearrangements do not occur in eukaryotic tubulins in response to GTP binding, and that the unpolymerized conformation of alpha-tubulin differs significantly from the polymerized one. Thus, geometric constraints of lateral self-assembly must drive alpha-tubulin conformational changes, whereas GTP plays a secondary role to tune the strength of longitudinal contacts within the microtubule lattice. alpha-Tubulin behaves like a bent spring, resisting straightening until forced to do so by GTP-mediated interactions with the growing microtubule. Kinetic simulations demonstrate that resistance to straightening opposes microtubule initiation by specifically destabilizing early assembly intermediates that are especially sensitive to the strength of lateral interactions. These data provide new insights into the molecular origins of dynamic microtubule behavior.

  4. A potential link between insulin signaling and GLUT4 translocation: Association of Rab10-GTP with the exocyst subunit Exoc6/6b

    SciTech Connect

    Sano, Hiroyuki; Peck, Grantley R.; Blachon, Stephanie; Lienhard, Gustav E.

    2015-09-25

    Insulin increases glucose transport in fat and muscle cells by stimulating the exocytosis of specialized vesicles containing the glucose transporter GLUT4. This process, which is referred to as GLUT4 translocation, increases the amount of GLUT4 at the cell surface. Previous studies have provided evidence that insulin signaling increases the amount of Rab10-GTP in the GLUT4 vesicles and that GLUT4 translocation requires the exocyst, a complex that functions in the tethering of vesicles to the plasma membrane, leading to exocytosis. In the present study we show that Rab10 in its GTP form binds to Exoc6 and Exoc6b, which are the two highly homologous isotypes of an exocyst subunit, that both isotypes are found in 3T3-L1 adipocytes, and that knockdown of Exoc6, Exoc6b, or both inhibits GLUT4 translocation in 3T3-L1 adipocytes. These results suggest that the association of Rab10-GTP with Exoc6/6b is a molecular link between insulin signaling and the exocytic machinery in GLUT4 translocation. - Highlights: • Insulin stimulates the fusion of vesicles containing GLUT4 with the plasma membrane. • This requires vesicular Rab10-GTP and the exocyst plasma membrane tethering complex. • We find that Rab10-GTP associates with the Exoc6 subunit of the exocyst. • We find that knockdown of Exoc6 inhibits fusion of GLUT4 vesicles with the membrane. • The interaction of Rab10-GTP with Exoc6 potentially links signaling to exocytosis.

  5. Reactor optimization for enzymatic hydrolysis of cellulose

    SciTech Connect

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

    1982-01-01

    Enzymatic hydrolysis of cellulose furnishes sugar which can be subsequently fermented to ethanol. The production of such sugar at relatively low cost is essential for commercially viable production of ethanol. Many processes have been developed for converting cellulosic materials to sugar, and their economic feasibility has been analyzed; however, relatively little has been done to optimize such processes. A comprehensive mechanistic kinetic model for enzymatic degradation was established previously; it takes into account the structure of cellulose, mode of action of celluloytic enzyme, and mode of interaction between the enzyme and the cellulosic substrate. In the present work this model has been applied to the optimal design of cellulose hydrloysis reactors. Both batch and continously stirred reactors have been considered for this purpose. The fractional contributions of various cost parameters to the production cost have been estimated. The sensitivity of sugar cost to the important cost parameters, such as raw material and enzyme costs, have been examined. 8 figures, 7 tables.

  6. Fermentable sugars by chemical hydrolysis of biomass

    PubMed Central

    Binder, Joseph B.; Raines, Ronald T.

    2010-01-01

    Abundant plant biomass has the potential to become a sustainable source of fuels and chemicals. Realizing this potential requires the economical conversion of recalcitrant lignocellulose into useful intermediates, such as sugars. We report a high-yielding chemical process for the hydrolysis of biomass into monosaccharides. Adding water gradually to a chloride ionic liquid-containing catalytic acid leads to a nearly 90% yield of glucose from cellulose and 70–80% yield of sugars from untreated corn stover. Ion-exclusion chromatography allows recovery of the ionic liquid and delivers sugar feedstocks that support the vigorous growth of ethanologenic microbes. This simple chemical process, which requires neither an edible plant nor a cellulase, could enable crude biomass to be the sole source of carbon for a scalable biorefinery. PMID:20194793

  7. Storage oil hydrolysis during early seedling growth.

    PubMed

    Quettier, Anne-Laure; Eastmond, Peter J

    2009-06-01

    Storage oil breakdown plays an important role in the life cycle of many plants by providing the carbon skeletons that support seedling growth immediately following germination. This metabolic process is initiated by lipases (EC: 3.1.1.3), which catalyze the hydrolysis of triacylglycerols (TAGs) to release free fatty acids and glycerol. A number of lipases have been purified to near homogeneity from seed tissues and analysed for their in vitro activities. Furthermore, several genes encoding lipases have been cloned and characterised from plants. However, only recently has data been presented to establish the molecular identity of a lipase that has been shown to be required for TAG breakdown in seeds. In this review we briefly outline the processes of TAG synthesis and breakdown. We then discuss some of the biochemical literature on seed lipases and describe the cloning and characterisation of a lipase called SUGAR-DEPENDENT1, which is required for TAG breakdown in Arabidopsis thaliana seeds.

  8. Pretreatment and enzymatic hydrolysis of corn fiber

    SciTech Connect

    Grohmann, K.; Bothast, R.J.

    1996-10-01

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

  9. Enhanced functional properties of tannic acid after thermal hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. Class Projects in Physical Organic Chemistry: The Hydrolysis of Aspirin

    ERIC Educational Resources Information Center

    Marrs, Peter S.

    2004-01-01

    An exercise that provides a hands-on demonstration of the hydrolysis of aspirin is presented. The key to understanding the hydrolysis is recognizing that all six process may occur simultaneously and that the observed rate constant is the sum of the rate constants that one rate constant dominates the overall process.

  11. Bioabatement with hemicellulase supplementation to reduce enzymatic hydrolysis inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Removal of inhibitory compounds by bioabatement, combined with xylan hydrolysis, enables effective cellulose hydrolysis of pretreated corn stover, for fermentation of the sugars to fuel ethanol or other products. The fungus Coniochaeta ligniaria NRRL30616 eliminates most enzyme and fermentation inhi...

  12. Epidemic based modeling of enzymatic hydrolysis of lignocellulosic biomass.

    PubMed

    Tai, Chao; Arellano, Maria G; Keshwani, Deepak R

    2014-01-01

    An epidemic based model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, dilute sulfuric acid pretreated corn stover. The process of substrate getting adsorbed and digested by enzyme was simulated as susceptibles getting infected by viruses and becoming removed and recovered. This model simplified the dynamic enzyme "infection" process and the catalysis of cellulose into a two-parameter controlled, enzyme behavior guided mechanism. Furthermore, the model incorporates the adsorption block by lignin and inhibition effects on cellulose catalysis. The model satisfactorily predicted the enzyme adsorption and hydrolysis, negative role of lignin, and inhibition effects over hydrolysis for a broad range of substrate and enzyme loadings. Sensitivity analysis was performed to evaluate the incorporation of lignin and other inhibition effects. Our model will be a useful tool for evaluating the effects of parameters during hydrolysis and guide a design strategy for continuous hydrolysis and the associated process control.

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

    PubMed

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

    2016-03-01

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

  14. Trihalomethane hydrolysis in drinking water at elevated temperatures.

    PubMed

    Zhang, Xiao-Lu; Yang, Hong-Wei; Wang, Xiao-Mao; Karanfil, Tanju; Xie, Yuefeng F

    2015-07-01

    Hydrolysis could contribute to the loss of trihalomethanes (THMs) in the drinking water at elevated temperatures. This study was aimed at investigating THM hydrolysis pertaining to the storage of hot boiled water in enclosed containers. The water pH value was in the range of 6.1-8.2 and the water temperature was varied from 65 to 95 °C. The effects of halide ions, natural organic matter, and drinking water matrix were investigated. Results showed that the hydrolysis rates declined in the order following CHBrCl2 > CHBr2Cl > CHBr3 > CHCl3. THM hydrolysis was primarily through the alkaline pathway, except for CHCl3 in water at relatively low pH value. The activation energies for the alkaline hydrolysis of CHCl3, CHBrCl2, CHBr2Cl and CHBr3 were 109, 113, 115 and 116 kJ/mol, respectively. No hydrolysis intermediates could accumulate in the water. The natural organic matter, and probably other constituents, in drinking water could substantially decrease THM hydrolysis rates by more than 50%. When a drinking water was at 90 °C or above, the first order rate constants for THM hydrolysis were in the magnitude of 10(-2)‒10(-1) 1/h. When the boiled real tap water was stored in an enclosed container, THMs continued increasing during the first few hours and then kept decreasing later on due to the competition between hydrolysis and further formation. The removal of THMs, especially brominated THMs, by hydrolysis would greatly reduce one's exposure to disinfection by-products by consuming the boiled water stored in enclosed containers.

  15. GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes.

    PubMed

    Walker, M W; Bobak, D A; Tsai, S C; Moss, J; Vaughan, M

    1992-02-15

    ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo.

  16. Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells

    SciTech Connect

    Park, Su Hyun; Park, Tae Jun; Lim, In Kyoung

    2011-04-15

    We have previously reported that G-actin accumulation in nuclei is a universal phenomenon of cellular senescence. By employing primary culture of human diploid fibroblast (HDF) and stress-induced premature senescence (SIPS), we explored whether the failure of actin export to cytoplasm is responsible for actin accumulation in nuclei of senescent cells. Expression of exportin 6 (Exp6) and small G-protein, Ran, was significantly reduced in the replicative senescence, but not yet in SIPS, whereas nuclear import of actin by cofilin was already increased in SIPS. After treatment of young HDF cells with H{sub 2}O{sub 2}, rapid reduction of nuclear RanGTP was observed along with cytoplasmic increase of RanGDP. Furthermore, significantly reduced interaction of Exp6 with RanGTP was found by GST-Exp6 pull-down analysis. Failure of RanGTP restoration was accompanied with inhibition of ATP synthesis and NTF2 sequestration in the nuclei along with accordant change of senescence morphology. Indeed, knockdown of Exp6 expression significantly increased actin molecule in the nuclei of young HDF cells. Therefore, actin accumulation in nuclei of senescent cells is most likely due to the failure of RanGTP restoration with ATP deficiency and NTF2 accumulation in nuclei, which result in the decrease of actin export via Exp6 inactivation, in addition to actin import by cofilin activation.

  17. Reduction of exportin 6 activity leads to actin accumulation via failure of RanGTP restoration and NTF2 sequestration in the nuclei of senescent cells.

    PubMed

    Park, Su Hyun; Park, Tae Jun; Lim, In Kyoung

    2011-04-15

    We have previously reported that G-actin accumulation in nuclei is a universal phenomenon of cellular senescence. By employing primary culture of human diploid fibroblast (HDF) and stress-induced premature senescence (SIPS), we explored whether the failure of actin export to cytoplasm is responsible for actin accumulation in nuclei of senescent cells. Expression of exportin 6 (Exp6) and small G-protein, Ran, was significantly reduced in the replicative senescence, but not yet in SIPS, whereas nuclear import of actin by cofilin was already increased in SIPS. After treatment of young HDF cells with H(2)O(2), rapid reduction of nuclear RanGTP was observed along with cytoplasmic increase of RanGDP. Furthermore, significantly reduced interaction of Exp6 with RanGTP was found by GST-Exp6 pull-down analysis. Failure of RanGTP restoration was accompanied with inhibition of ATP synthesis and NTF2 sequestration in the nuclei along with accordant change of senescence morphology. Indeed, knockdown of Exp6 expression significantly increased actin molecule in the nuclei of young HDF cells. Therefore, actin accumulation in nuclei of senescent cells is most likely due to the failure of RanGTP restoration with ATP deficiency and NTF2 accumulation in nuclei, which result in the decrease of actin export via Exp6 inactivation, in addition to actin import by cofilin activation.

  18. A High-Throughput Screening Assay for the Identification of Flavivirus NS5 Capping Enzyme GTP-Binding Inhibitors: Implications for Antiviral Drug Development

    PubMed Central

    GEISS, BRIAN J.; STAHLA-BEEK, HILLARY J.; HANNAH, AMANDA M.; GARI, HAMID H.; HENDERSON, BRITTNEY R.; SAEEDI, BEJAN J.; KEENAN, SUSAN M.

    2012-01-01

    There are no effective antivirals currently available for the treatment of flavivirus infection in humans. As such, the identification and characterization of novel drug target sites are critical to developing new classes of antiviral drugs. The flavivirus NS5 N-terminal capping enzyme (CE) is vital for the formation of the viral RNA cap structure, which directs viral polyprotein translation and stabilizes the 5′ end of the viral genome. The structure of the flavivirus CE has been solved, and a detailed understanding of the CE–guanosine triphosphate (GTP) and CE–RNA cap interactions is available. Because of the essential nature of the interaction for viral replication, disrupting CE–GTP binding is an attractive approach for drug development. The authors have previously developed a robust assay for monitoring CE–GTP binding in real time. They adapted this assay for high-throughput screening and performed a pilot screen of 46 323 commercially available compounds. A number of small-molecule inhibitors capable of displacing a fluorescently labeled GTP in vitro were identified, and a second functional assay was developed to identify false positives. The results presented indicate that the flavivirus CE cap-binding site is a valuable new target site for antiviral drug discovery and should be further exploited for broad-spectrum anti-flaviviral drug development. PMID:21788392

  19. Effects of temperature and ethanol on agonist and antagonist binding to rat heart muscarinic receptors in the absence and presence of GTP.

    PubMed Central

    Waelbroeck, M; Robberecht, P; Chatelain, P; De Neef, P; Christophe, J

    1985-01-01

    The effect of temperature on the binding of four agonists and three antagonists to rat heart muscarinic receptors was studied in the absence and presence of GTP. The binding of agonists to two states (or classes) of receptors, in the absence of GTP, led to enthalpy and entropy changes that decreased sharply above 25 degrees C, suggesting that agonists induced 'isomerization' reactions (large conformational changes and/or receptor-effector association). Both temperature increase and ethanol decreased hydrophobic interactions, thereby hindering binding and/or agonist-induced 'isomerization' reactions. Addition of GTP to the incubation medium also appeared to reverse (or prevent) 'isomerization' reactions. For agonist binding to the low-affinity state, in the presence of GTP, and for antagonist binding, the thermodynamic parameters observed could be readily explained by simple receptor-ligand associations; large entropy increases and small enthalpy increases, provoked by hydrophobic and ionic interactions, were partly neutralized by entropy and enthalpy decreases, due to hydrogen bonds and van der Waals interactions. The muscarinic antagonists used (atropine, n-methylscopolamine and dexetimide), being more hydrophobic molecules than the agonists tested (carbamylcholine, oxotremorine and pilocarpine), induced larger entropy changes or more negative enthalpy changes. PMID:4062907

  20. Effects of temperature and ethanol on agonist and antagonist binding to rat heart muscarinic receptors in the absence and presence of GTP.

    PubMed

    Waelbroeck, M; Robberecht, P; Chatelain, P; De Neef, P; Christophe, J

    1985-10-15

    The effect of temperature on the binding of four agonists and three antagonists to rat heart muscarinic receptors was studied in the absence and presence of GTP. The binding of agonists to two states (or classes) of receptors, in the absence of GTP, led to enthalpy and entropy changes that decreased sharply above 25 degrees C, suggesting that agonists induced 'isomerization' reactions (large conformational changes and/or receptor-effector association). Both temperature increase and ethanol decreased hydrophobic interactions, thereby hindering binding and/or agonist-induced 'isomerization' reactions. Addition of GTP to the incubation medium also appeared to reverse (or prevent) 'isomerization' reactions. For agonist binding to the low-affinity state, in the presence of GTP, and for antagonist binding, the thermodynamic parameters observed could be readily explained by simple receptor-ligand associations; large entropy increases and small enthalpy increases, provoked by hydrophobic and ionic interactions, were partly neutralized by entropy and enthalpy decreases, due to hydrogen bonds and van der Waals interactions. The muscarinic antagonists used (atropine, n-methylscopolamine and dexetimide), being more hydrophobic molecules than the agonists tested (carbamylcholine, oxotremorine and pilocarpine), induced larger entropy changes or more negative enthalpy changes. PMID:4062907

  1. The amino acid sequence of GTP:AMP phosphotransferase from beef-heart mitochondria. Extensive homology with cytosolic adenylate kinase.

    PubMed

    Wieland, B; Tomasselli, A G; Noda, L H; Frank, R; Schulz, G E

    1984-09-01

    The amino acid sequence of GTP:AMP phosphotransferase (AK3) from beef-heart mitochondria has been determined, except for one segment of about 33 residues in the middle of the polypeptide chain. The established sequence has been unambiguously aligned to the sequence of cytosolic ATP:AMP phosphotransferase (AK1) from pig muscle, allowing for six insertions and deletions. With 30% of all aligned residues being identical, the homology between AK3 and AK1 is well established. As derived from the known three-dimensional structure of AK1, the missing segment is localized at a small surface area of the molecule, far apart from the active center. The pattern of conserved residues demonstrates that earlier views on substrate binding have to be modified. The observation of three different consecutive N-termini indicates enzyme processing.

  2. Hydrolysis of milk triglycerides by human gastric lipase.

    PubMed

    Jaśkiewicz, J; Szafran, Z; Popiela, T; Szafran, H

    1980-01-01

    The concentrations of myristic, palmitic, palmitoleic, stearic and oleic acids were determined in the products of hydrolysis of lipids of cow milk incubated with human gastric juice using thin-layer chromatography for the separation of lipid fractions, and gas liquid chromatography for the determination of fatty acids. It was found that the percentage ratio of the above fatty acids in hydrolysis products was similar to that in milk triglycerides. It was concluded that triglycerides containing higher fatty acids present in milk are hydrolysed by the lipase appearing in human gastric juice, the rate of hydrolysis of the individual acids being roughly proportional to the concentration of these acids in triglyceride substrate.

  3. Adsorption and recovery of cellulases during hydrolysis of newspaper

    SciTech Connect

    Castanon, M.; Wilke, C.R.

    1980-01-01

    The adsorption of cellulases from Trichoderma viride was studied during the hydrolysis of newspaper. By measuring individual enzyme activities it was found that in the early stage of hydrolysis enzyme components showing C/sub x/A were adsorbed preferentially to those showing C/sub 1/A; afterwards this situation was inverted. Electrophoretic resolution of proteins in hydrolysates showed a continuous decrease of enzyme proteins in solution, and furthermore suggested that the enzymes once adsorbed remained immobilized on the substrate (even after extensive digestion). Experiments to recover the enzymes that had remained in solution after typical hydrolysis showed a potential saving of enzyme of up to 40%.

  4. Isolation of the alpha subunits of GTP-binding regulatory proteins by affinity chromatography with immobilized beta gamma subunits.

    PubMed Central

    Pang, I H; Sternweis, P C

    1989-01-01

    Immobilized beta gamma subunits of GTP-binding regulatory proteins (G proteins) were used to isolate alpha subunits from solubilized membranes of bovine tissues and to separate specific alpha subunits based on their differential affinities for beta gamma subunits. The beta gamma subunits were cross-linked to omega-aminobutyl agarose. Up to 7 nmol of alpha subunit could bind to each milliliter of beta gamma-agarose and be recovered by elution with AIF4-. This affinity resin effectively separated the alpha subunits of Gi1 and Gi2 from "contaminating" alpha subunits of Go, the most abundant G protein in bovine brain, by taking advantage of the apparent lower affinity of the alpha subunits of Go for beta gamma subunits. The beta gamma-agarose was also used to isolate mixtures of alpha subunits from cholate extracts of membranes from different bovine tissues. alpha subunits of 39-41 kDa (in various ratios) as well as the alpha subunits of Gs were purified. The yields from extracts exceeded 60% for all alpha subunits examined and apparently represented the relative content of alpha subunits in the tissues. This technique can rapidly isolate and identify, from a small amount of sample, the endogenous G proteins in various tissues and cells. So far, only polypeptides in the range of 39-52 kDa have been detected with this approach. If other GTP-binding proteins interact with these beta gamma subunits, the interaction is either of low affinity or mechanistically unique from the alpha subunits isolated in this study. Images PMID:2510152

  5. Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein.

    PubMed

    Ohtsuka, T; Shimizu, K; Yamamori, B; Kuroda, S; Takai, Y

    1996-01-19

    Rap1 small GTP-binding protein has the same amino acid sequence at its effector domain as that of Ras. Rap1 has been shown to antagonize the Ras functions, such as the Ras-induced transformation of NIH 3T3 cells and the Ras-induced activation of the c-Raf-1 protein kinase-dependent mitogen-activated protein (MAP) kinase cascade in Rat-1 cells, whereas we have shown that Rap1 as well as Ras stimulates DNA synthesis in Swiss 3T3 cells. We have established a cell-free assay system in which Ras activates bovine brain B-Raf protein kinase. Here we have used this assay system and examined the effect of Rap1 on the B-Raf activity to phosphorylate recombinant MAP kinase kinase (MEK). Recombinant Rap1B stimulated the activity of B-Raf, which was partially purified from bovine brain and immunoprecipitated by an anti-B-Raf antibody. The GTP-bound form was active, but the GDP-bound form was inactive. The fully post-translationally lipid-modified form was active, but the unmodified form was nearly inactive. The maximum B-Raf activity stimulated by Rap1B was nearly the same as that stimulated by Ki-Ras. Rap1B enhanced the Ki-Ras-stimulated B-Raf activity in an additive manner. These results indicate that not only Ras but also Rap1 is involved in the activation of the B-Raf-dependent MAP kinase cascade.

  6. Enzymatic hydrolysis of biomass from wood.

    PubMed

    Álvarez, Consolación; Reyes-Sosa, Francisco Manuel; Díez, Bruno

    2016-03-01

    Current research and development in cellulosic ethanol production has been focused mainly on agricultural residues and dedicated energy crops such as corn stover and switchgrass; however, woody biomass remains a very important feedstock for ethanol production. The precise composition of hemicellulose in the wood is strongly dependent on the plant species, therefore different types of enzymes are needed based on hemicellulose complexity and type of pretreatment. In general, hardwood species have much lower recalcitrance to enzymes than softwood. For hardwood, xylanases, beta-xylosidases and xyloglucanases are the main hemicellulases involved in degradation of the hemicellulose backbone, while for softwood the effect of mannanases and beta-mannosidases is more relevant. Furthermore, there are different key accessory enzymes involved in removing the hemicellulosic fraction and increasing accessibility of cellulases to the cellulose fibres improving the hydrolysis process. A diversity of enzymatic cocktails has been tested using from low to high densities of biomass (2-20% total solids) and a broad range of results has been obtained. The performance of recently developed commercial cocktails on hardwoods and softwoods will enable a further step for the commercialization of fuel ethanol from wood. PMID:26833542

  7. Synthesis, hydrolysis and stability of psilocin glucuronide.

    PubMed

    Martin, Rafaela; Schürenkamp, Jennifer; Pfeiffer, Heidi; Lehr, Matthias; Köhler, Helga

    2014-04-01

    A two-step synthesis of psilocin glucuronide (PCG), the main metabolite of psilocin, with methyl 2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-α-d-glucopyranuronate is reported. With the synthesized PCG, hydrolysis conditions in serum and urine were optimized. Escherichia coli proved to be a better enzyme source for β-glucuronidase than Helix pomatia. It was essential to add ascorbic acid to serum samples to protect psilocin during incubation. Furthermore the stability of PCG and psilocin was compared as stability data are the basis for forensic interpretation of measurements. PCG showed a greater long-term stability after six months in deep frozen serum and urine samples than psilocin. The short-term stability of PCG for one week in whole blood at room temperature and in deep frozen samples was also better than that of psilocin. Therefore, PCG can be considered to be more stable than the labile psilocin and should always be included if psilocin is analyzed in samples. PMID:24513688

  8. Synthesis, hydrolysis and stability of psilocin glucuronide.

    PubMed

    Martin, Rafaela; Schürenkamp, Jennifer; Pfeiffer, Heidi; Lehr, Matthias; Köhler, Helga

    2014-04-01

    A two-step synthesis of psilocin glucuronide (PCG), the main metabolite of psilocin, with methyl 2,3,4-tri-O-isobutyryl-1-O-trichloroacetimidoyl-α-d-glucopyranuronate is reported. With the synthesized PCG, hydrolysis conditions in serum and urine were optimized. Escherichia coli proved to be a better enzyme source for β-glucuronidase than Helix pomatia. It was essential to add ascorbic acid to serum samples to protect psilocin during incubation. Furthermore the stability of PCG and psilocin was compared as stability data are the basis for forensic interpretation of measurements. PCG showed a greater long-term stability after six months in deep frozen serum and urine samples than psilocin. The short-term stability of PCG for one week in whole blood at room temperature and in deep frozen samples was also better than that of psilocin. Therefore, PCG can be considered to be more stable than the labile psilocin and should always be included if psilocin is analyzed in samples.

  9. Preparation of highly charged cellulose nanofibrils using high-pressure homogenization coupled with strong acid hydrolysis pretreatments.

    PubMed

    Tian, Cuihua; Yi, Jianan; Wu, Yiqiang; Wu, Qinglin; Qing, Yan; Wang, Lijun

    2016-01-20

    Cellulose nanofibrils (CNFs) are attracting much attention for the advantages of excellent mechanical strength, good optical transparency, and high surface area. An eco-friendly and energy-saving method was created in this work to produce highly negative charged CNFs using high-pressure mechanical defibrillation coupled with strong acid hydrolysis pretreatments. The morphological development, zeta potential, crystal structure, chemical composition and thermal degradation behavior of the resultant materials were evaluated by transmission electron microscopy (TEM), zeta potential analysis, X-ray diffraction (XRD), Fourier transform infrared spectrometry (FTIR), and thermogravimetric analysis (TGA). These CNFs were fully separated, surface-charged, and highly entangled. They showed a large fiber aspect ratio compared to traditional cellulose nanocrystrals that are produced by strong acid hydrolysis. Compared to hydrochloric acid hydrolysis, the CNFs produced by sulfuric acid pretreatments were completely defibrillated and presented stable suspensions (or gels) even at low fiber content. On the other hand, CNFs pretreated by hydrochloric acid hydrolysis trended to aggregate because of the absence of surface charge. The crystallinity index (CI) of CNFs decreased because of mechanical defibrillation, and then increased dramatically with increased sulfuric acid concentration and reaction time. FTIR analysis showed that the C-O-SO3 group was introduced on the surfaces of CNFs during sulfuric acid hydrolysis. These sulfate groups accelerated the thermal degradation of CNFs, which occurred at lower temperature than wood pulp, indicating that the thermal stability of sulfuric acid hydrolyzed CNFs was decreased. The temperature of the maximum decomposition rate (Tmax) and the maximum weight-loss rates (MWLRmax) were much lower than for wood pulp because of the retardant effect of sulfuric acid during the combustion of CNFs. By contrast, the CNFs treated with hydrochloric acid

  10. Laser driven ion accelerator

    DOEpatents

    Tajima, Toshiki

    2006-04-18

    A system and method of accelerating ions in an accelerator to optimize the energy produced by a light source. Several parameters may be controlled in constructing a target used in the accelerator system to adjust performance of the accelerator system. These parameters include the material, thickness, geometry and surface of the target.

  11. Evaluation of hydrolysis-esterification biodiesel production from wet microalgae.

    PubMed

    Song, Chunfeng; Liu, Qingling; Ji, Na; Deng, Shuai; Zhao, Jun; Li, Shuhong; Kitamura, Yutaka

    2016-08-01

    Wet microalgae hydrolysis-esterification route has the advantage to avoid the energy-intensive units (e.g. drying and lipid extraction) in the biodiesel production process. In this study, techno-economic evaluation of hydrolysis-esterification biodiesel production process was carried out and compared with conventional (usually including drying, lipid extraction, esterification and transesterification) biodiesel production process. Energy and material balance of the conventional and hydrolysis-esterification processes was evaluated by Aspen Plus. The simulation results indicated that drying (2.36MJ/L biodiesel) and triolein transesterification (1.89MJ/L biodiesel) are the dominant energy-intensive stages in the conventional route (5.42MJ/L biodiesel). By contrast, the total energy consumption of hydrolysis-esterification route can be reduced to 1.81MJ/L biodiesel, and approximately 3.61MJ can be saved to produce per liter biodiesel.

  12. A General Approach for Teaching Hydrolysis of Salts.

    ERIC Educational Resources Information Center

    Aguirre-Ode, Fernando

    1987-01-01

    Presented is a general approach and equation for teaching the hydrolysis of salts. This general equation covers many more sets of conditions than those currently in textbooks. The simplifying assumptions leading to the known limiting equations are straightforward. (RH)

  13. Kinetics of the hydrolysis of guanosine 5'-phospho-2-methylimidazolide

    NASA Technical Reports Server (NTRS)

    Kanavarioti, Anastassia

    1986-01-01

    The hydrolysis kinetics of guanosine 5'-phospho-2-methylimidazolide (2-MeImpG) in aqueous buffered solutions of various pH's was studied at 75 and 37 C, using spectrophotometric and HPLC techniques. The hydrolysis was found to be very slow even at low pH. At 75 C and pH at or below l.0, two kinetic processes were observed: the more rapid one was attributed to the hydrolysis of the phosphoimidazolide P-N bond; the second, much slower one, was attributed to the cleavage of the glycosidic bond. It is noted that the P-N hydrolysis in phosphoimidazolides is very slow compared to other phosphoramidates, and that this might be one of the reasons why the phosphoimidazolides showed an extraordinary ability to form long oligomers under template-directed conditions.

  14. Hydrolysis of Al3+ from constrained molecular dynamics

    NASA Astrophysics Data System (ADS)

    Ikeda, Takashi; Hirata, Masaru; Kimura, Takaumi

    2006-02-01

    We investigated the hydrolysis reactions of Al3+ in AlCl3 aqueous solution using the constrained molecular dynamics based on the Car-Parrinello molecular-dynamics method. By employing the proton-aluminum coordination number as a reaction coordinate in the constrained molecular dynamics the deprotonation as well as dehydration processes are successfully realized. From our free-energy difference of ΔG0≃8.0kcalmol-1 the hydrolysis constant pKa1 is roughly estimated as 5.8, comparable to the literature value of 5.07. We show that the free-energy difference for the hydrolysis of Al3+ in acidic conditions is at least 4kcalmol-1 higher than that in neutral condition, indicating that the hydrolysis reaction is inhibited by the presence of excess protons located around the hydrated ion, in agreement with the change of the predominant species by pH.

  15. Hydrolysis of whey lactose using CTAB-permeabilized yeast cells.

    PubMed

    Kaur, Gurpreet; Panesar, Parmjit S; Bera, Manav B; Kumar, Harish

    2009-01-01

    Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. The enzymatic hydrolysis of whey lactose to glucose and galactose by beta-galactosidase constitutes the basis of the most biotechnological processes currently developed to exploit the sugar content of whey. Keeping this in view, lactose hydrolysis in whey was performed using CTAB permeabilized Kluyveromyces marxianus cells. Permeabilization of K. marxianus cells in relation to beta-galactosidase activity was carried out using cetyltrimethyl ammonium bromide (CTAB) to avoid the problem of enzyme extraction. Different process parameters (biomass load, pH, temperature, and incubation time) were optimized to enhance the lactose hydrolysis in whey. Maximum hydrolysis (90.5%) of whey lactose was observed with 200 mg DW yeast biomass after 90 min of incubation period at optimum pH of 6.5 and temperature of 40 degrees C.

  16. Energetic approach of biomass hydrolysis in supercritical water.

    PubMed

    Cantero, Danilo A; Vaquerizo, Luis; Mato, Fidel; Bermejo, M Dolores; Cocero, M José

    2015-03-01

    Cellulose hydrolysis can be performed in supercritical water with a high selectivity of soluble sugars. The process produces high-pressure steam that can be integrated, from an energy point of view, with the whole biomass treating process. This work investigates the integration of biomass hydrolysis reactors with commercial combined heat and power (CHP) schemes, with special attention to reactor outlet streams. The innovation developed in this work allows adequate energy integration possibilities for heating and compression by using high temperature of the flue gases and direct shaft work from the turbine. The integration of biomass hydrolysis with a CHP process allows the selective conversion of biomass into sugars with low heat requirements. Integrating these two processes, the CHP scheme yield is enhanced around 10% by injecting water in the gas turbine. Furthermore, the hydrolysis reactor can be held at 400°C and 23 MPa using only the gas turbine outlet streams.

  17. Energetic approach of biomass hydrolysis in supercritical water.

    PubMed

    Cantero, Danilo A; Vaquerizo, Luis; Mato, Fidel; Bermejo, M Dolores; Cocero, M José

    2015-03-01

    Cellulose hydrolysis can be performed in supercritical water with a high selectivity of soluble sugars. The process produces high-pressure steam that can be integrated, from an energy point of view, with the whole biomass treating process. This work investigates the integration of biomass hydrolysis reactors with commercial combined heat and power (CHP) schemes, with special attention to reactor outlet streams. The innovation developed in this work allows adequate energy integration possibilities for heating and compression by using high temperature of the flue gases and direct shaft work from the turbine. The integration of biomass hydrolysis with a CHP process allows the selective conversion of biomass into sugars with low heat requirements. Integrating these two processes, the CHP scheme yield is enhanced around 10% by injecting water in the gas turbine. Furthermore, the hydrolysis reactor can be held at 400°C and 23 MPa using only the gas turbine outlet streams. PMID:25536511

  18. Evaluation of hydrolysis-esterification biodiesel production from wet microalgae.

    PubMed

    Song, Chunfeng; Liu, Qingling; Ji, Na; Deng, Shuai; Zhao, Jun; Li, Shuhong; Kitamura, Yutaka

    2016-08-01

    Wet microalgae hydrolysis-esterification route has the advantage to avoid the energy-intensive units (e.g. drying and lipid extraction) in the biodiesel production process. In this study, techno-economic evaluation of hydrolysis-esterification biodiesel production process was carried out and compared with conventional (usually including drying, lipid extraction, esterification and transesterification) biodiesel production process. Energy and material balance of the conventional and hydrolysis-esterification processes was evaluated by Aspen Plus. The simulation results indicated that drying (2.36MJ/L biodiesel) and triolein transesterification (1.89MJ/L biodiesel) are the dominant energy-intensive stages in the conventional route (5.42MJ/L biodiesel). By contrast, the total energy consumption of hydrolysis-esterification route can be reduced to 1.81MJ/L biodiesel, and approximately 3.61MJ can be saved to produce per liter biodiesel. PMID:27209457

  19. ESTIMATION OF CARBOXYLIC ACID ESTER HYDROLYSIS RATE CONSTANTS

    EPA Science Inventory

    SPARC chemical reactivity models were extended to calculate hydrolysis rate constants for carboxylic acid esters from molecular structure. The energy differences between the initial state and the transition state for a molecule of interest are factored into internal and external...

  20. Mathematical modeling of maltose hydrolysis in different types of reactor.

    PubMed

    Findrik, Zvjezdana; Presecki, Ana Vrsalović; Vasić-Racki, Durda

    2010-03-01

    A commercial enzyme Dextrozyme was tested as catalyst for maltose hydrolysis at two different temperatures: 40 and 65 degrees C at pH 5.5. Its operational stability was studied in different reactor types: batch, repetitive batch, fed-batch and continuously operated enzyme membrane reactor. Dextrozyme was more active at 65 degrees C, but operational stability decay was observed during the prolonged use in the reactor at this temperature. The reactor efficiencies were compared according to the volumetric productivity, biocatalyst productivity and enzyme consumption. The best reactor type according to the volumetric productivity for maltose hydrolysis is batch and the best reactor type according to the biocatalyst productivity and enzyme consumption is continuously operated enzyme membrane reactor. The mathematical model developed for the maltose hydrolysis in the different reactors was validated by the experiments at both temperatures. The Michaelis-Menten kinetics describing maltose hydrolysis was used.

  1. Sub-Equimolar Hydrolysis and Condensation of Organophosphates

    DOE PAGES

    Alam, Todd M.; Kinnan, Mark K.; Wilson, Brendan W.; Wheeler, David R.

    2016-07-16

    We characterized the in-situ hydrolysis and subsequent condensation reaction of the chemical agent simulant diethyl chlorophosphate (DECP) by high-resolution 31P NMR spectroscopy following the addition of water in sub-equimolar concentrations. Moreover, the identification and quantification of the multiple pyrophosphate and larger polyphosphate chemical species formed through a series of self-condensation reactions are reported. Finally, the DECP hydrolysis kinetics and distribution of breakdown species was strongly influenced by the water concentration and reaction temperature.

  2. Protein hydrolysis by immobilized and stabilized trypsin.

    PubMed

    Marques, Daniela; Pessela, Benavides C; Betancor, Lorena; Monti, Rubens; Carrascosa, Alfonso V; Rocha-Martin, Javier; Guisán, Jose M; Fernandez-Lorente, Gloria

    2011-01-01

    The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates [benzoyl-arginine p-nitroanilide (BAPNA)] and were 50,000-fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was immobilized on highly activated glyoxyl-Sepharose following a two-step immobilization strategy: (a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pK(a) amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed and (b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pK(a) amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a nonstabilized derivative prepared on CNBr-activated Sepharose. In fact, all the proteins contained a cheese whey extract had been completely proteolyzed after 6 h at pH 9 and 50°C, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the three-dimensional (3D) structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and five lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high-molecular weight proteins.

  3. Acceleration of the rate of ethanol fermentation by addition of nitrogen in high tannin grain sorghum

    SciTech Connect

    Mullins, J.T.; NeSmith, C.C.

    1987-01-01

    In this communication, the authors show that accelerated rates of ethanol production, comparable to sorghum varieties containing low levels of tannins and to corn, can occur without the removal of the tannins. The basis of the inhibition appears to be a lack of sufficient nitrogen in the mash for protein synthesis required to support an accelerated fermentative metabolism in Saccharomyces. No inhibition of the enzymes used for starch hydrolysis was found.

  4. Development of complete hydrolysis of pectins from apple pomace.

    PubMed

    Wikiera, Agnieszka; Mika, Magdalena; Starzyńska-Janiszewska, Anna; Stodolak, Bożena

    2015-04-01

    Enzymatically extracted pectins have a more complex structure than those obtained by conventional methods. As a result, they are less susceptible to hydrolysis, which makes the precise determination of their composition difficult. The aim of the study was to develop a method of complete hydrolysis of enzymatically extracted apple pectins. Substrates were pectins isolated from apple pomace by the use of xylanase and multicatalytic preparation Celluclast and apple pomace. Hydrolysis was performed by a chemical method with 2M TFA at 100 °C and 120 °C and a combined acidic/enzymatic method. After hydrolysis, the contents of galacturonic acid and neutral sugars were measured by HPLC. Complete hydrolysis of polygalacturonic acid occurred after 2.5h incubation with 2M TFA at 120 °C. The efficient hydrolysis of neutral sugars in pectins was performed with 2M TFA at 100 °C for 2.5h. Monomers most susceptible to concentrated acid were rhamnose, mannose and arabinose.

  5. Development of complete hydrolysis of pectins from apple pomace.

    PubMed

    Wikiera, Agnieszka; Mika, Magdalena; Starzyńska-Janiszewska, Anna; Stodolak, Bożena

    2015-04-01

    Enzymatically extracted pectins have a more complex structure than those obtained by conventional methods. As a result, they are less susceptible to hydrolysis, which makes the precise determination of their composition difficult. The aim of the study was to develop a method of complete hydrolysis of enzymatically extracted apple pectins. Substrates were pectins isolated from apple pomace by the use of xylanase and multicatalytic preparation Celluclast and apple pomace. Hydrolysis was performed by a chemical method with 2M TFA at 100 °C and 120 °C and a combined acidic/enzymatic method. After hydrolysis, the contents of galacturonic acid and neutral sugars were measured by HPLC. Complete hydrolysis of polygalacturonic acid occurred after 2.5h incubation with 2M TFA at 120 °C. The efficient hydrolysis of neutral sugars in pectins was performed with 2M TFA at 100 °C for 2.5h. Monomers most susceptible to concentrated acid were rhamnose, mannose and arabinose. PMID:25442606

  6. Study of enzyme adsorption and reaction kinetics for cellulose hydrolysis

    SciTech Connect

    Gilbert, I.G.

    1982-01-01

    Enzymatic hydrolysis of cellulose occurs due to the combined catalytic action of two types of cellulase components commonly referred to as C/sub 1/ and C/sub x/. However, before the hydrolysis reaction can begin, it is necessary for these enzymes to first adsorb onto the accessible surfaces of the insoluble cellulose substrate. The objective of the study was to gain a better understanding of the relationships between the adsorption of these enzyme components, the hydrolysis kinetics, the cellulosic surface area accessible to the enzymes, and the cellulose crystallinity. These relationships were investigated by passing a Trichoderma viride cellulase solution through columns of cellulose powder having different accessibility and crystallinity, and then analyzing the quantities of the different enzyme components and the hydrolysis product in the effluent. The amounts of the different cellulase components were analyzed using high-performance anion-exchange chromatography. Additional adsorption and hydrolysis experiments were done using columns of cellulose beads specially developed to provide amodel substrate for this analysis. A mathematical model has been formulated to describe the kinetics of enzyme adsorption and the resultant, initial hydrolysis rate in cellulose column. The analytical solutions obtained have been linearized into a convenient form so that the kinetic parameters of the model can be readily determined from experimental breakthrough curves.

  7. How efficacious are 5-HT1B/D receptor ligands: an answer from GTP gamma S binding studies with stably transfected C6-glial cell lines.

    PubMed

    Pauwels, P J; Tardif, S; Palmier, C; Wurch, T; Colpaert, F C

    1997-01-01

    The intrinsic activity of a series of 5-hydroxytryptamine (serotonin, 5-HT) receptor ligands was analysed at recombinant h5-HT1B and h5-HT1D receptor sites using a [35S]GTP gamma S binding assay and membrane preparations of stably transfected C6-glial cell lines. Compounds either stimulated or inhibited [35S]GTP gamma S binding to a membrane preparation containing either h5-HT1B or h5-HT1D receptors. The potencies observed for most of the compounds at the h5-HT1B receptor subtype correlated with their potencies measured by inhibition of stimulated cAMP formation on intact cells. Apparent agonist potencies in the [35S]GTP gamma S binding assay to C6-glial/h5-HT1D membranes were, with the exception of 2-[5-[3-(4-methylsulphonylamino)benzyl-1 2,4-oxadiazol-5-yl]-1H-indol-3-yl] ethanamine (L694247), 5- to 13-times lower than in the cAMP assay on intact cells. This suggests that receptor coupling in the h5-HT1D membrane preparation is less efficient than that in the intact cell. It further appeared that 6-times more h5-HT1D than h5-HT1B binding sites were required to attain a similar, maximal (73%), 5-HT-stimulated [35S]GTP gamma S binding response: Hence, the h5-HT1B receptor in C6-glial cell membranes could be more efficiently coupled, even though some compounds more readily displayed intrinsic activity at h5-HT1D receptor sites [e.g. dihydroergotamine and (2'-methyl-4'-(5-methyl[1,2,4]oxadiazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]amide (GR127935)]. Efficacy differences were apparent for most of the compounds (sumatriptan, zolmitriptan, rizatriptan, N-methyl-3-[pyrrolidin-2(R)-ylmethyl]-1H-indol-5-ylmethyl sulfonamide (CP122638), dihydroergotamine, naratriptan and GR127935) that stimulated [35S]GTP gamma S binding compared to the native agonist 5-HT. The observed maximal responses were different for the h5-HT1B and h5-HT1D receptor subtypes. Few compounds behaved as full agonists: L694247, zolmitriptan and sumatriptan did so at

  8. Amine-Promoted Organosilicate Hydrolysis Mechanism at Near-Neutral pH

    NASA Astrophysics Data System (ADS)

    Delak, K. M.; Sahai, N.

    2006-12-01

    Proteins bearing polylysine moeities and histidine and serine amino-aicd residues, isolated from diatoms and sponges, are known to promote biological nanoporous silica formation [1, 2]. Using 29Si NMR, we have shown quantitatively that monoamines and small polyamines can chemically accelerate the hydrolysis and condensation rates of organosilicate starting materials, in biomimetic silica synthesis pathways, at circum- neutral pHs and room temperature [3, 4]. The present study is focused on understanding the mechanistic role of these amines in catalyzing the hydrolysis step that precedes condensation [5]. We conducted 29Si NMR experimental studies over a range of temperature and pHs for the hydrolysis rates of trimethylethoxysilane (TMES), a model compound with only one hydrolyzable bond. Experimental results were combined with quantum mechanical hybrid Density Functional Theory calculations of putative intermediate and transition state structures for TMES and tetramethylorthosilicate (TMOS) which has four hydrolyzable bonds. Comparison of calculated energies with experimentally-determined activation energies indicated that amines promote TMES hydrolysis mainly due to the amine's acidity at neutral pH. The proton released by the amine is transferred to the organosilicate, producing a protonated, ethoxy leaving group that can be displaced by water in an SN2 reaction. For TMOS, the activation energy of proton-transfer coupled with SN2 substitution is comparable to that for Corriu's nucleophile-activated nucleophilic displacement mechanism [6], such that the mechanism of amine-catalyzed hydrolysis is mostly dependent on the ambient pH conditions as well as the type of amine. The molecular mechanisms of hydrolysis and aggregation are reflected, ultimately, on the larger scale in the silica morphology where amines promoting faster hydrolysis result in glassy products compared to slower hydrolyzing amines forming particulate silica [7, 8]. REFERENCES [1] Kroger N

  9. Effect of D-amino acid substitutions on Ni(II)-assisted peptide bond hydrolysis.

    PubMed

    Ariani, Hanieh H; Polkowska-Nowakowska, Agnieszka; Bal, Wojciech

    2013-03-01

    Previously we demonstrated the sequence-specific hydrolysis of the R1-(Ser/Thr)-peptide bond in Ni(II) complexes of peptides with a general R1-(Ser/Thr)-Xaa-His-Zaa-R2 sequence (R1 and R2 being any sequences) (Kopera, E.; Krezel, A.; Protas, A. M.; Belczyk, A.; Bonna, A.; Wyslouch-Cieszynska, A.; Poznanski, J.; Bal, W. Inorg. Chem. 2010, 49, 6636). In order to refine our understanding of the mechanism of this reaction and to find ways to accelerate it, we undertook a systematic study of effects of d-amino acid substitutions in the template Ac-Gly-Ala-Ser-Arg-His-Trp-Lys-Phe-Leu-NH2 peptide on the hydrolysis rate constants. We found that all stereochemical alterations made around the Ni(II) chelate plane resulted in the decrease of the reaction rate. However, the Ni(II) coordination, a prerequisite to the reaction, was not compromised by these substitutions. We demonstrated that the reaction is only possible when either the side chain of the crucial Ser (or Thr) residue is on the same part of the chelate plane as the next residue in the sequence (Arg), or the side chain of the residue following His (Trp) resides on the opposite side of the plane. The rate of reaction is the fastest when both these conditions are fulfilled. Another novel effect is the strong dependence of the rate of the acyl shift step on the character of the leaving group. PMID:23427909

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

    PubMed

    Ometto, Francesco; Quiroga, Gerardo; Pšenička, Pavel; Whitton, Rachel; Jefferson, Bruce; Villa, Raffaella

    2014-11-15

    Anaerobic digestion (AD) of microalgae is primarily inhibited by the chemical composition of their cell walls containing biopolymers able to resist bacterial degradation. Adoption of pre-treatments such as thermal, thermal hydrolysis, ultrasound and enzymatic hydrolysis have the potential to remove these inhibitory compounds and enhance biogas yields by degrading the cell wall, and releasing the intracellular algogenic organic matter (AOM). This work investigated the effect of four pre-treatments on three microalgae species, and their impact on the quantity of soluble biomass released in the media and thus on the digestion process yields. The analysis of the composition of the soluble COD released and of the TEM images of the cells showed two main degradation actions associated with the processes: (1) cell wall damage with the release of intracellular AOM (thermal, thermal hydrolysis and ultrasound) and (2) degradation of the cell wall constituents with the release of intracellular AOM and the solubilisation of the cell wall biopolymers (enzymatic hydrolysis). As a result of this, enzymatic hydrolysis showed the greatest biogas yield increments (>270%) followed by thermal hydrolysis (60-100%) and ultrasounds (30-60%).

  11. Hydrolysis and fractionation of lignocellulosic biomass

    DOEpatents

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

    2000-01-01

    A multi-function process is described for the hydrolysis and fractionation of lignocellulosic biomass to separate hemicellulosic sugars from other biomass components such as extractives and proteins; a portion of the solubilized lignin; cellulose; glucose derived from cellulose; and insoluble lignin from said biomass comprising one or more of the following: optionally, as function 1, introducing a dilute acid of pH 1.0-5.0 into a continual shrinking bed reactor containing a lignocellulosic biomass material at a temperature of about 94 to about 160.degree. C. for a period of about 10 to about 120 minutes at a volumetric flow rate of about 1 to about 5 reactor volumes to effect solubilization of extractives, lignin, and protein by keeping the solid to liquid ratio constant throughout the solubilization process; as function 2, introducing a dilute acid of pH 1.0-5.0, either as virgin acid or an acidic stream from another function, into a continual shrinking bed reactor containing either fresh biomass or the partially fractionated lignocellulosic biomass material from function 1 at a temperature of about 94-220.degree. C. for a period of about 10 to about 60 minutes at a volumetric flow rate of about 1 to about 5 reactor volumes to effect solubilization of hemicellulosic sugars, semisoluble sugars and other compounds, and amorphous glucans by keeping the solid to liquid ratio constant throughout the solubilization process; as function 3, optionally, introducing a dilute acid of pH 1.0-5.0 either as virgin acid or an acidic stream from another function, into a continual shrinking bed reactor containing the partially fractionated lignocellulosic biomass material from function 2 at a temperature of about 180-280.degree. C. for a period of about 10 to about 60 minutes at a volumetric flow rate of 1 to about 5 reactor volumes to effect solubilization of cellulosic sugars by keeping the solid to liquid ratio constant throughout the solubilization process; and as function 4

  12. TURBULENT SHEAR ACCELERATION

    SciTech Connect

    Ohira, Yutaka

    2013-04-10

    We consider particle acceleration by large-scale incompressible turbulence with a length scale larger than the particle mean free path. We derive an ensemble-averaged transport equation of energetic charged particles from an extended transport equation that contains the shear acceleration. The ensemble-averaged transport equation describes particle acceleration by incompressible turbulence (turbulent shear acceleration). We find that for Kolmogorov turbulence, the turbulent shear acceleration becomes important on small scales. Moreover, using Monte Carlo simulations, we confirm that the ensemble-averaged transport equation describes the turbulent shear acceleration.

  13. The direction of acceleration

    NASA Astrophysics Data System (ADS)

    Wilhelm, Thomas; Burde, Jan-Philipp; Lück, Stephan

    2015-11-01

    Acceleration is a physical quantity that is difficult to understand and hence its complexity is often erroneously simplified. Many students think of acceleration as equivalent to velocity, a ˜ v. For others, acceleration is a scalar quantity, which describes the change in speed Δ|v| or Δ|v|/Δt (as opposed to the change in velocity). The main difficulty with the concept of acceleration therefore lies in developing a correct understanding of its direction. The free iOS app AccelVisu supports students in acquiring a correct conception of acceleration by showing acceleration arrows directly at moving objects.

  14. Thermal synthesis and hydrolysis of polyglyceric acid. [in orgin of life studying

    NASA Technical Reports Server (NTRS)

    Weber, Arthur L.

    1989-01-01

    Polyglyceric acid was synthesized by thermal condensation of glyceric acid at 80 C in the presence and absence of two mole percent of sulfuric acid catalyst. The acid catalyst accelerated the polymerization over 100-fold and made possible the synthesis of insoluble polymers of both L- and DL-glyceric acid by heating for less than 1 day. Racemization of L-glyceric acid yielded less than 1 percent D-glyceric acid in condensations carried out at 80 C with catalyst for 1 day and without catalyst for 12 days. The condensation of L-glyceric acid yielded an insoluble polymer much more readily than condensation of DL-glyceric acid. Studies of the hydrolysis of poly-DL-glyceric acid revealed that it was considerably more stable under mild acidic conditions compared to neutral pH. The relationship of this study to the origin of life is discussed.

  15. Identification of Carboxylesterase-Dependent Dabigatran Etexilate Hydrolysis

    PubMed Central

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

    2014-01-01

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

  16. Accelerating Particles with Plasma

    SciTech Connect

    Litos, Michael; Hogan, Mark

    2014-11-05

    Researchers at SLAC explain how they use plasma wakefields to accelerate bunches of electrons to very high energies over only a short distance. Their experiments offer a possible path for the future of particle accelerators.

  17. Accelerator Technology Division

    NASA Astrophysics Data System (ADS)

    1992-04-01

    In fiscal year (FY) 1991, the Accelerator Technology (AT) division continued fulfilling its mission to pursue accelerator science and technology and to develop new accelerator concepts for application to research, defense, energy, industry, and other areas of national interest. This report discusses the following programs: The Ground Test Accelerator Program; APLE Free-Electron Laser Program; Accelerator Transmutation of Waste; JAERI, OMEGA Project, and Intense Neutron Source for Materials Testing; Advanced Free-Electron Laser Initiative; Superconducting Super Collider; The High-Power Microwave Program; (Phi) Factory Collaboration; Neutral Particle Beam Power System Highlights; Accelerator Physics and Special Projects; Magnetic Optics and Beam Diagnostics; Accelerator Design and Engineering; Radio-Frequency Technology; Free-Electron Laser Technology; Accelerator Controls and Automation; Very High-Power Microwave Sources and Effects; and GTA Installation, Commissioning, and Operations.

  18. Linear accelerator: A concept

    NASA Technical Reports Server (NTRS)

    Mutzberg, J.

    1972-01-01

    Design is proposed for inexpensive accelerometer which would work by applying pressure to fluid during acceleration. Pressure is used to move shuttle, and shuttle movement is sensed and calibrated to give acceleration readings.

  19. Improved plasma accelerator

    NASA Technical Reports Server (NTRS)

    Cheng, D. Y.

    1971-01-01

    Converging, coaxial accelerator electrode configuration operates in vacuum as plasma gun. Plasma forms by periodic injections of high pressure gas that is ionized by electrical discharges. Deflagration mode of discharge provides acceleration, and converging contours of plasma gun provide focusing.

  20. Enzymatic hydrolysis of fructans in the tequila production process.

    PubMed

    Avila-Fernández, Angela; Rendón-Poujol, Xóchitl; Olvera, Clarita; González, Fernando; Capella, Santiago; Peña-Alvarez, Araceli; López-Munguía, Agustín

    2009-06-24

    In contrast to the hydrolysis of reserve carbohydrates in most plant-derived alcoholic beverage processes carried out with enzymes, agave fructans in tequila production have traditionally been transformed to fermentable sugars through acid thermal hydrolysis. Experiments at the bench scale demonstrated that the extraction and hydrolysis of agave fructans can be carried out continuously using commercial inulinases in a countercurrent extraction process with shredded agave fibers. Difficulties in the temperature control of large extraction diffusers did not allow the scaling up of this procedure. Nevertheless, batch enzymatic hydrolysis of agave extracts obtained in diffusers operating at 60 and 90 degrees C was studied at the laboratory and industrial levels. The effects of the enzymatic process on some tequila congeners were studied, demonstrating that although a short thermal treatment is essential for the development of tequila's organoleptic characteristics, the fructan hydrolysis can be performed with enzymes without major modifications in the flavor or aroma, as determined by a plant sensory panel and corroborated by the analysis of tequila congeners.

  1. Hydrolysis of aluminum dross material to achieve zero hazardous waste.

    PubMed

    David, E; Kopac, J

    2012-03-30

    A simple method with high efficiency for generating high pure hydrogen by hydrolysis in tap water of highly activated aluminum dross is established. Aluminum dross is activated by mechanically milling to particles of about 45 μm. This leads to removal of surface layer of the aluminum particles and creation of a fresh chemically active metal surface. In contact with water the hydrolysis reaction takes place and hydrogen is released. In this process a Zero Waste concept is achieved because the other product of reaction is aluminum oxide hydroxide (AlOOH), which is nature-friendly and can be used to make high quality refractory or calcium aluminate cement. For comparison we also used pure aluminum powder and alkaline tap water solution (NaOH, KOH) at a ratio similar to that of aluminum dross content. The rates of hydrogen generated in hydrolysis reaction of pure aluminum and aluminum dross have been found to be similar. As a result of the experimental setup, a hydrogen generator was designed and assembled. Hydrogen volume generated by hydrolysis reaction was measured. The experimental results obtained reveal that aluminum dross could be economically recycled by hydrolysis process with achieving zero hazardous aluminum dross waste and hydrogen generation. PMID:22326245

  2. Enzymatic hydrolysis of cellulose and various pretreated wood fractions

    SciTech Connect

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

    1982-06-01

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

  3. Enzymatic hydrolysis of fractionated products from oil thermally oxidated

    SciTech Connect

    Yashida, H.; Alexander, J.C.

    1983-01-01

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

  4. Fluoride incorporation into apatite crystals delays amelogenin hydrolysis

    PubMed Central

    DenBesten, Pamela; Zhu, Li; Li, Wu; Tanimoto, Kotaro; Liu, Haichuan; Witkowska, Halina Ewa

    2012-01-01

    Enamel fluorosis has been related to an increase in the amount of amelogenin in fluorosed enamel as compared to normal enamel in the maturation stage. In this study we tested the hypothesis that fluoride incorporated into carbonated apatite alters amelogenin hydrolysis. Recombinant human amelogenin (rh174) was allowed to bind to 0.15 mg of carbonated hydroxyapatite (CAP) or fluoride-containing carbonated hydroxyapatite (F-CAP) synthesized to contain 100, 1000 or 4000 ppm F-. After 3 h digestion with recombinant human MMP20 or KLK4, bound protein was characterized by reverse-phase HPLC. Proteolytic fragments formed after 24 h digestion of amelogenin, were identified by LC tandem mass spectrometry (LCMS/MS). The hydrolysis of amelogenin bound to F100-CAP by both MMP20 and KLK4 was significantly reduced in a dose dependent manner as compared to CAP. After 24 h hydrolysis, the number of cleavage sites in bound amelogenin by MMP20 were similar in CAP and F100-CAP, whereas there were 24 fewer cleavage sites identified for the KLK4 hydrolysis on F100-CAP as compared to CAP. These results suggest that the reduced hydrolysis of amelogenins in fluorosed enamel may be partially due to the increased fluoride content in fluoride containing apatite, contributing to the hypomineralized enamel matrix phenotype observed in fluorosed enamel. PMID:22243219

  5. MEQALAC rf accelerating structure

    SciTech Connect

    Keane, J.; Brodowski, J.

    1981-01-01

    A prototype MEQALAC capable of replacing the Cockcroft Walton pre-injector at BNL is being fabricated. Ten milliamperes of H/sup -/ beam supplied from a source sitting at a potential of -40 kilovolt is to be accelerated to 750 keV. This energy gain is provided by a 200 Megahertz accelerating system rather than the normal dc acceleration. Substantial size and cost reduction would be realized by such a system over conventional pre-accelerator systems.

  6. /sup 19/F nuclear magnetic resonance measurement of the distance between the E-site GTP and the high-affinity Mg/sup 2 +/ in tubulin

    SciTech Connect

    Monasterio, O.

    1987-09-22

    The distance separating the divalent metal ion high-affinity binding site and the exchangeable nucleotide binding site on tubulin was evaluated by using high-resolution /sup 19/F NMR. The /sup 31/P and /sup 19/F NMR spectra of guanosine 5'-(..gamma..-fluorotriphosphate) (GTP(..gamma..F)) were studied. Both the fluorine and the ..gamma..-phosphate were split into a doublet with a coupling constant of 936 Hz. Tubulin purified according to the method of Weisenberg was incubated with 1 mM Mn/sup 2 +/. After one cycle of assembly, Mn/sup 2 +/ only partially, i.e., 60% at the high-affinity binding site. After colchicine treatment of tubulin to stabilize it, GTP(..gamma..F) was added, and the 254-MHz fluorine-19 relaxation rates were measured within the first 4 h. Longitudinal and transversal relaxation rates were determined at two concentrations of GTP(..gamma..F) and variable concentrations of colchicine-tubulin-Mn(II) (paramagnetic complex) or the ternary complex with magnesium diamagnetic complex). The analysis of the relaxation data indicates that the rate of exchange of GTP(..gamma..F) from the exchangeable nucleotide site has a lower limit of 8.7 x 10/sup 4/ s/sup -1/ and the metal and exchangeable nucleotide binding sites are separated by an upper distance between 6 and 8 A. These data confirm that the high-affinity divalent cation site is situated in the same locus as that of the exchangeable nucleotide, forming a metal-nucleotide complex.

  7. Acceleration gradient of a plasma wakefield accelerator

    SciTech Connect

    Uhm, Han S.

    2008-02-25

    The phase velocity of the wakefield waves is identical to the electron beam velocity. A theoretical analysis indicates that the acceleration gradient of the wakefield accelerator normalized by the wave breaking amplitude is K{sub 0}({xi})/K{sub 1}({xi}), where K{sub 0}({xi}) and K{sub 1}({xi}) are the modified Bessel functions of the second kind of order zero and one, respectively and {xi} is the beam parameter representing the beam intensity. It is also shown that the beam density must be considerably higher than the diffuse plasma density for the large radial velocity of plasma electrons that are required for a high acceleration gradient.

  8. Volatile organic acid adsorption and cation dissociation by porphyritic andesite for enhancing hydrolysis and acidogenesis of solid food wastes.

    PubMed

    Cheng, Fan; Li, Ming; Li, Dawei; Chen, Ling; Jiang, Weizhong; Kitamura, Yutaka; Li, Baoming

    2010-07-01

    Volatile organic acid adsorption, cation dissociation by porphyritic andesite, and their effects on the hydrolysis and acidogenesis of solid food wastes were evaluated through batch experiments. The acetic acid adsorption experiments show that pH was mainly regulated by H(+) adsorption. The mono-layer and multi-layer adsorption were found under the low (8.3-83.2 mmol/L) and high (133.22-532.89 mmol/L) initial acetic acid concentration, respectively. The dissociated cations concentration in acidic solution showed the predominance of Ca(2+). Porphyritic andesite addition elevated the pH levels and accelerated hydrolysis and acidogenesis in the batch fermentation experiment. Leachate of porphyritic andesite addition achieved the highest hydrolysis constant of 22.1 x 10(-3)kgm(-2)d(-1) and VS degradation rates of 3.9 g L(-1)d(-1). The highest activity of microorganisms represented by specific growth rate of ATP, 0.16d(-1), and specific consumption rate of Ca(2+), 0.18d(-1), was obtained by adding leachate of porphyritic andesite.

  9. Volatile organic acid adsorption and cation dissociation by porphyritic andesite for enhancing hydrolysis and acidogenesis of solid food wastes.

    PubMed

    Cheng, Fan; Li, Ming; Li, Dawei; Chen, Ling; Jiang, Weizhong; Kitamura, Yutaka; Li, Baoming

    2010-07-01

    Volatile organic acid adsorption, cation dissociation by porphyritic andesite, and their effects on the hydrolysis and acidogenesis of solid food wastes were evaluated through batch experiments. The acetic acid adsorption experiments show that pH was mainly regulated by H(+) adsorption. The mono-layer and multi-layer adsorption were found under the low (8.3-83.2 mmol/L) and high (133.22-532.89 mmol/L) initial acetic acid concentration, respectively. The dissociated cations concentration in acidic solution showed the predominance of Ca(2+). Porphyritic andesite addition elevated the pH levels and accelerated hydrolysis and acidogenesis in the batch fermentation experiment. Leachate of porphyritic andesite addition achieved the highest hydrolysis constant of 22.1 x 10(-3)kgm(-2)d(-1) and VS degradation rates of 3.9 g L(-1)d(-1). The highest activity of microorganisms represented by specific growth rate of ATP, 0.16d(-1), and specific consumption rate of Ca(2+), 0.18d(-1), was obtained by adding leachate of porphyritic andesite. PMID:20156676

  10. Acceleration: It's Elementary

    ERIC Educational Resources Information Center

    Willis, Mariam

    2012-01-01

    Acceleration is one tool for providing high-ability students the opportunity to learn something new every day. Some people talk about acceleration as taking a student out of step. In actuality, what one is doing is putting a student in step with the right curriculum. Whole-grade acceleration, also called grade-skipping, usually happens between…

  11. Far field acceleration

    SciTech Connect

    Fernow, R.C.

    1995-07-01

    Far fields are propagating electromagnetic waves far from their source, boundary surfaces, and free charges. The general principles governing the acceleration of charged particles by far fields are reviewed. A survey of proposed field configurations is given. The two most important schemes, Inverse Cerenkov acceleration and Inverse free electron laser acceleration, are discussed in detail.

  12. Angular Acceleration without Torque?

    ERIC Educational Resources Information Center

    Kaufman, Richard D.

    2012-01-01

    Hardly. Just as Robert Johns qualitatively describes angular acceleration by an internal force in his article "Acceleration Without Force?" here we will extend the discussion to consider angular acceleration by an internal torque. As we will see, this internal torque is due to an internal force acting at a distance from an instantaneous center.

  13. Granular starch hydrolysis for fuel ethanol production

    NASA Astrophysics Data System (ADS)

    Wang, Ping

    addition were evaluated in the dry grind process using GSHE (GSH process). Addition of proteases resulted in higher ethanol concentrations (15.2 to 18.0% v/v) and lower (DDGS) yields (32.9 to 45.8% db) compared to the control (no protease addition). As level of proteases and GSHE increased, ethanol concentrations increased and DDGS yields decreased. Proteases addition reduced required GSHE dose. Ethanol concentrations with protease addition alone were higher than with urea or with addition of both protease and urea. Corn endosperm consists of soft and hard endosperm. More exposed starch granules and rough surfaces produced from soft endosperm compared to hard endosperm will create more surface area which will benefit the solid phase hydrolysis as used in GSH process. In this study, the effects of protease, urea, endosperm hardness and GSHE levels on the GSH process were evaluated. Soft and hard endosperm materials were obtained by grinding and sifting flaking grits from dry milling pilot plant. Soft endosperm resulted in higher ethanol concentrations (at 72 hr) compared to ground corn or hard endosperm. Addition of urea increased ethanol concentrations (at 72 hr) for soft and hard endosperm. The effect of protease addition on increasing ethanol concentrations and fermentation rates was more predominant for soft endosperm, less for hard endosperm and least for ground corn. The GSH process with protease resulted in higher ethanol concentration than that with urea. For fermentation of soft endosperm, GSHE dose can be reduced. Ground corn fermented faster at the beginning than hard and soft endosperm due to the presence of inherent nutrients which enhanced yeast growth.

  14. Association of the N-formyl-Met-Leu-Phe receptor in human neutrophils with a GTP-binding protein sensitive to pertussis toxin.

    PubMed Central

    Lad, P M; Olson, C V; Smiley, P A

    1985-01-01

    Pertussis toxin inhibits the N-formyl-Met-Leu-Phe (fMet-Leu-Phe) mediated human neutrophil functions of enzyme release, superoxide generation, aggregation, and chemotaxis. As pertussis toxin modifies the GTP binding receptor-regulatory protein "Ni," the association of the fMet-Leu-Phe receptor with such a protein was further examined in purified neutrophil plasma membranes. Both fMet-Leu-Phe-mediated guanine nucleotide exchange and nucleotide-mediated regulation of the fMet-Leu-Phe receptor are inhibited by pertussis toxin. In addition, membrane pretreatment with pertussis toxin abolishes the fMet-Leu-Phe-mediated inhibition of adenylate cyclase. Actions of pertussis toxin are due to the ADP-ribosylation of a single subunit at 41 kDa in the neutrophil plasma membrane, which comigrates on NaDodSO4 gels with the Ni GTP-binding protein in the platelet plasma membrane. Our results suggest that (i) the fMet-Leu-Phe receptor is associated with a Ni GTP regulatory protein, and (ii) a fMet-Leu-Phe-Ni complex is important in the control of several neutrophil functions, probably involving multiple transduction systems, including adenylate cyclase. Images PMID:2983319

  15. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase.

    PubMed Central

    Ishizaki, T; Maekawa, M; Fujisawa, K; Okawa, K; Iwamatsu, A; Fujita, A; Watanabe, N; Saito, Y; Kakizuka, A; Morii, N; Narumiya, S

    1996-01-01

    The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP-bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N-terminus, followed by a coiled-coil structure approximately 600 amino acids long, and a cysteine-rich zinc finger-like motif and a pleckstrin homology region in the C-terminus. The N-terminus region including a kinase domain and a part of coiled-coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 was co-precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo. Images PMID:8617235

  16. Tandem duplications of a degenerated GTP-binding domain at the origin of GTPase receptors Toc159 and thylakoidal SRP

    SciTech Connect

    Hernandez Torres, Jorge Maldonado, Monica Alexandra Arias; Chomilier, Jacques

    2007-12-14

    The evolutionary origin of some nuclear encoded proteins that translocate proteins across the chloroplast envelope remains unknown. Therefore, sequences of GTPase proteins constituting the Arabidopsis thaliana translocon at the outer membrane of chloroplast (atToc) complexes were analyzed by means of HCA. In particular, atToc159 and related proteins (atToc132, atToc120, and atToc90) do not have proven homologues of prokaryotic or eukaryotic ancestry. We established that the three domains commonly referred to as A, G, and M originate from the GTPase G domain, tandemly repeated, and probably evolving toward an unstructured conformation in the case of the A domain. It resulted from this study a putative common ancestor for these proteins and a new domain definition, in particular the splitting of A into three domains (A1, A2, and A3), has been proposed. The family of Toc159, previously containing A. thaliana and Pisum sativum, has been extended to Medicago truncatula and Populus trichocarpa and it has been revised for Oryza sativa. They have also been compared to GTPase subunits involved in the cpSRP system. A distant homology has been revealed among Toc and cpSRP GTP-hydrolyzing proteins of A. thaliana, and repetitions of a GTPase domain were also found in cpSRP protein receptors, by means of HCA analysis.

  17. Overexpression of GTP cyclohydrolase 1 feedback regulatory protein is protective in a murine model of septic shock.

    PubMed

    Starr, Anna; Sand, Claire A; Heikal, Lamia; Kelly, Peter D; Spina, Domenico; Crabtree, Mark; Channon, Keith M; Leiper, James M; Nandi, Manasi

    2014-11-01

    Overproduction of nitric oxide (NO) by inducible NO synthase contributes toward refractory hypotension, impaired microvascular perfusion, and end-organ damage in septic shock patients. Tetrahydrobiopterin (BH4) is an essential NOS cofactor. GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme for BH4 biosynthesis. Under inflammatory conditions, GCH1 activity and hence BH4 levels are increased, supporting pathological NOS activity. GCH1 activity can be controlled through allosteric interactions with GCH1 feedback regulatory protein (GFRP). We investigated whether overexpression of GFRP can regulate BH4 and NO production and attenuate cardiovascular dysfunction in sepsis. Sepsis was induced in mice conditionally overexpressing GFRP and wild-type littermates by cecal ligation and puncture. Blood pressure was monitored by radiotelemetry, and mesenteric blood flow was quantified by laser speckle contrast imaging. Blood biochemistry data were obtained using an iSTAT analyzer, and BH4 levels were measured in plasma and tissues by high-performance liquid chromatography. Increased BH4 and NO production and hypotension were observed in all mice, but the extents of these pathophysiological changes were attenuated in GFRP OE mice. Perturbations in blood biochemistry were similarly attenuated in GFRP OE compared with wild-type controls. These results suggest that GFRP overexpression regulates GCH1 activity during septic shock, which in turn limits BH4 bioavailability for iNOS. We conclude that the GCH1-GFRP axis is a critical regulator of BH4 and NO production and the cardiovascular derangements that occur in septic shock.

  18. The TITAN5 gene of Arabidopsis encodes a protein related to the ADP ribosylation factor family of GTP binding proteins.

    PubMed

    McElver, J; Patton, D; Rumbaugh, M; Liu, C; Yang, L J; Meinke, D

    2000-08-01

    The titan (ttn) mutants of Arabidopsis exhibit dramatic alterations in mitosis and cell cycle control during seed development. Endosperm development in these mutants is characterized by the formation of giant polyploid nuclei with enlarged nucleoli. Embryo development is accompanied by significant cell enlargement in some mutants (ttn1 and ttn5) but not others (ttn2 and ttn3). We describe here the molecular cloning of TTN5 using a T-DNA-tagged allele. A second allele with a similar phenotype contains a nonsense mutation in the same coding region. The predicted protein is related to ADP ribosylation factors (ARFs), members of the RAS family of small GTP binding proteins that regulate various cellular functions in eukaryotes. TTN5 is most closely related in sequence to the ARL2 class of ARF-like proteins isolated from humans, rats, and mice. Although the cellular functions of ARL proteins remain unclear, the ttn5 phenotype is consistent with the known roles of ARFs in the regulation of intracellular vesicle transport.

  19. Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement

    PubMed Central

    Zick, Michael; Wickner, William

    2016-01-01

    In vitro reconstitution of homotypic yeast vacuole fusion from purified components enables detailed study of membrane fusion mechanisms. Current reconstitutions have yet to faithfully replicate the fusion process in at least three respects: 1) The density of SNARE proteins required for fusion in vitro is substantially higher than on the organelle. 2) Substantial lysis accompanies reconstituted fusion. 3) The Rab GTPase Ypt7 is essential in vivo but often dispensable in vitro. Here we report that changes in fatty acyl chain composition dramatically lower the density of SNAREs that are required for fusion. By providing more physiological lipids with a lower phase transition temperature, we achieved efficient fusion with SNARE concentrations as low as on the native organelle. Although fused proteoliposomes became unstable at elevated SNARE concentrations, releasing their content after fusion had occurred, reconstituted proteoliposomes with substantially reduced SNARE concentrations fused without concomitant lysis. The Rab GTPase Ypt7 is essential on both membranes for proteoliposome fusion to occur at these SNARE concentrations. Strikingly, it was only critical for Ypt7 to be GTP loaded on membranes bearing the R-SNARE Nyv1, whereas the bound nucleotide of Ypt7 was irrelevant on membranes bearing the Q-SNAREs Vam3 and Vti1. PMID:27385334

  20. A presynaptic role for the ADP ribosylation factor (ARF)-specific GDP/GTP exchange factor msec7-1.

    PubMed

    Ashery, U; Koch, H; Scheuss, V; Brose, N; Rettig, J

    1999-02-01

    ADP ribosylation factors (ARFs) represent a family of small monomeric G proteins that switch from an inactive, GDP-bound state to an active, GTP-bound state. One member of this family, ARF6, translocates on activation from intracellular compartments to the plasma membrane and has been implicated in regulated exocytosis in neuroendocrine cells. Because GDP release in vivo is rather slow, ARF activation is facilitated by specific guanine nucleotide exchange factors like cytohesin-1 or ARNO. Here we show that msec7-1, a rat homologue of cytohesin-1, translocates ARF6 to the plasma membrane in living cells. Overexpression of msec7-1 leads to an increase in basal synaptic transmission at the Xenopus neuromuscular junction. msec7-1-containing synapses have a 5-fold higher frequency of spontaneous synaptic currents than control synapses. On stimulation, the amplitudes of the resulting evoked postsynaptic currents of msec7-1-overexpressing neurons are increased as well. However, further stimulation leads to a decline in amplitudes approaching the values of control synapses. This transient effect on amplitude is strongly reduced on overexpression of msec7-1E157K, a mutant incapable of translocating ARFs. Our results provide evidence that small G proteins of the ARF family and activating factors like msec7-1 play an important role in synaptic transmission, most likely by making more vesicles available for fusion at the plasma membrane.

  1. Enzymatic hydrolysis of plant polysaccharides: substrates for fermentation.

    PubMed

    Dekker, R F

    1989-01-01

    The enzymatic hydrolysis of plant carbohydrate polymers is discussed with particular emphasis on lignocellulose. The polysaccharides include starch, inulin, cellulose and the hemicelluloses, i.e., the heteroxylans and glucomannans. Commercial operations exist for the enzymatic hydrolysis of starch and its fermentation into chemicals such as ethanol. Enzymatic hydrolysis of lignocellulose is more complex and the enzymes are rather expensive to produce, which currently precludes the commercial processing of lignocellulosic materials. The bioconversion of lignocellulose consists of 4 process steps: pretreatment, enzyme production, enzymatic saccharification and fermentation. Except for the last step, each of these process steps is discussed. The discussion is highlighted with examples of lignocellulosic waste materials (e.g., sugar cane and a hardwood and softwood sawdust) which are of potential use in a bioconversion process for providing sugar hydrolysates that can serve as fermentation substrates.

  2. Monoolein production by triglycerides hydrolysis using immobilized Rhizopus oryzae lipase.

    PubMed

    Ghattas, Nesrine; Abidi, Ferid; Galai, Said; Marzouki, M Nejib; Salah, Abderraouf Ben

    2014-07-01

    Lipase extracted from Rhizopus oryzae was immobilized in alginate gel beads. The effects of the immobilization conditions, such as, alginate concentration, CaCl2 concentration and amount of initial enzyme on retained activity (specific activity ratio of entrapped active lipase to free lipase) were investigated. The optimal conditions for lipase entrapment were determined: 2% (w/v) alginate concentration, 100mM CaCl2 and enzyme ratio of 2000IU/mL.In such conditions, immobilized lipase by inclusion in alginate showed a highest stability and activity, on olive oil hydrolysis reaction where it could be reused for 10 cycles. After 15min of hydrolysis reaction, the mass composition of monoolein, diolein and triolein were about 78%, 10% and 12%. Hydrolysis' products purification by column chromatography lead to a successful separation of reaction compounds and provide a pure fraction of monoolein which is considered as the widest used emulsifier in food and pharmaceutical industries. PMID:24755261

  3. Complex enzyme hydrolysis releases antioxidative phenolics from rice bran.

    PubMed

    Liu, Lei; Wen, Wei; Zhang, Ruifen; Wei, Zhencheng; Deng, Yuanyuan; Xiao, Juan; Zhang, Mingwei

    2017-01-01

    In this study, phenolic profiles and antioxidant activity of rice bran were analyzed following successive treatment by gelatinization, liquefaction and complex enzyme hydrolysis. Compared with gelatinization alone, liquefaction slightly increased the total amount of phenolics and antioxidant activity as measured by ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. Complex enzyme hydrolysis significantly increased the total phenolics, flavonoids, FRAP and ORAC by 46.24%, 79.13%, 159.14% and 41.98%, respectively, compared to gelatinization alone. Furthermore, ten individual phenolics present in free or soluble conjugate forms were also analyzed following enzymatic processing. Ferulic acid experienced the largest release, followed by protocatechuic acid and then quercetin. Interestingly, a major proportion of phenolics existed as soluble conjugates, rather than free form. Overall, complex enzyme hydrolysis releases phenolics, thus increasing the antioxidant activity of rice bran extract. This study provides useful information for processing rice bran into functional beverage rich in phenolics. PMID:27507440

  4. Monoolein production by triglycerides hydrolysis using immobilized Rhizopus oryzae lipase.

    PubMed

    Ghattas, Nesrine; Abidi, Ferid; Galai, Said; Marzouki, M Nejib; Salah, Abderraouf Ben

    2014-07-01

    Lipase extracted from Rhizopus oryzae was immobilized in alginate gel beads. The effects of the immobilization conditions, such as, alginate concentration, CaCl2 concentration and amount of initial enzyme on retained activity (specific activity ratio of entrapped active lipase to free lipase) were investigated. The optimal conditions for lipase entrapment were determined: 2% (w/v) alginate concentration, 100mM CaCl2 and enzyme ratio of 2000IU/mL.In such conditions, immobilized lipase by inclusion in alginate showed a highest stability and activity, on olive oil hydrolysis reaction where it could be reused for 10 cycles. After 15min of hydrolysis reaction, the mass composition of monoolein, diolein and triolein were about 78%, 10% and 12%. Hydrolysis' products purification by column chromatography lead to a successful separation of reaction compounds and provide a pure fraction of monoolein which is considered as the widest used emulsifier in food and pharmaceutical industries.

  5. Homogeneous catalysis of valeronitrile hydrolysis under supercritical conditions.

    PubMed

    Sarlea, Michael; Kohl, Sabine; Blickhan, Nina; Vogel, Herbert

    2012-01-01

    Supercritical nitrile hydrolysis can be used for both, amide and acid production as well as waste water treatment, as the hydrolysis products show good biodegradability. The conventional process at ambient conditions requires large amounts of mineral acid or base. Approaches that use supercritical water as a green solvent without a catalyst have been investigated over recent years. Findings for valeronitrile hydrolysis presented recently showed promising reaction rates and valeric acid yields. In an attempt to further maximize product yield and to better understand the impact of the pH, reactions in dilute sulfuric acid (0.01 mol L(-1)) were performed in a continuous high-pressure laboratory-scale apparatus at 400-500 °C, 30 MPa, and a maximum residence time of 100 s. Results from both reaction media were compared with regard to productivity and sustainability.

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

    PubMed

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

    2009-05-01

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

  7. Comparative hydrolysis and fermentation of sugarcane and agave bagasse.

    PubMed

    Hernández-Salas, J M; Villa-Ramírez, M S; Veloz-Rendón, J S; Rivera-Hernández, K N; González-César, R A; Plascencia-Espinosa, M A; Trejo-Estrada, S R

    2009-02-01

    Sugarcane and agave bagasse samples were hydrolyzed with either mineral acids (HCl), commercial glucanases or a combined treatment consisting of alkaline delignification followed by enzymatic hydrolysis. Acid hydrolysis of sugar cane bagasse yielded a higher level of reducing sugars (37.21% for depithed bagasse and 35.37% for pith bagasse), when compared to metzal or metzontete (agave pinecone and leaves, 5.02% and 9.91%, respectively). An optimized enzyme formulation was used to process sugar cane bagasse, which contained Celluclast, Novozyme and Viscozyme L. From alkaline-enzymatic hydrolysis of sugarcane bagasse samples, a reduced level of reducing sugar yield was obtained (11-20%) compared to agave bagasse (12-58%). Selected hydrolyzates were fermented with a non-recombinant strain of Saccharomyces cerevisiae. Maximum alcohol yield by fermentation (32.6%) was obtained from the hydrolyzate of sugarcane depithed bagasse. Hydrolyzed agave waste residues provide an increased glucose decreased xylose product useful for biotechnological conversion.

  8. Compact Plasma Accelerator

    NASA Technical Reports Server (NTRS)

    Foster, John E.

    2004-01-01

    A plasma accelerator has been conceived for both material-processing and spacecraft-propulsion applications. This accelerator generates and accelerates ions within a very small volume. Because of its compactness, this accelerator could be nearly ideal for primary or station-keeping propulsion for spacecraft having masses between 1 and 20 kg. Because this accelerator is designed to generate beams of ions having energies between 50 and 200 eV, it could also be used for surface modification or activation of thin films.

  9. A kinetic study on sesame cake protein hydrolysis by Alcalase.

    PubMed

    Demirhan, Elçin; Apar, Dilek Kılıç; Özbek, Belma

    2011-01-01

    In the present study, the hydrolysis of sesame cake protein was performed by Alcalase, a bacterial protease produced by Bacillus licheniformis, to investigate the reaction kinetics of sesame cake hydrolysis and to determine decay and product inhibition effects for Alcalase. The reactions were carried out for 10 min in 0.1 L of aqueous solutions containing 10, 15, 20, 25, and 30 g protein/L at various temperature and pH values. To determine decay and product inhibition effects for Alcalase, a series of inhibition experiments were conducted with the addition of various amounts of hydrolysate. The reaction kinetics was investigated by initial rate approach. The initial reaction rates were determined from the slopes of the linear models that fitted to the experimental data. The kinetic parameters, K(m) and V(max), were estimated as 41.17 g/L and 9.24 meqv/L x min. The Lineweaver-Burk plots showed that the type of inhibition for Alcalase determined as uncompetitive, and the inhibition constant, K(i), was estimated as 38.24% (hydrolysate/substrate mixture). Practical Application: Plant proteins are increasingly being used as an alternative to proteins from animal sources to perform functional roles in food formulation. Knowledge of the kinetics of the hydrolysis reaction is essential for the optimization of enzymatic protein hydrolysis and for increasing the utilization of plant proteins in food products. Therefore, in the present study, the hydrolysis of sesame cake protein was performed by Alcalase, a bacterial protease produced by B. licheniformis, to investigate the reaction kinetics of sesame cake hydrolysis and to determine decay and product inhibition effects for Alcalase.

  10. High brightness electron accelerator

    DOEpatents

    Sheffield, Richard L.; Carlsten, Bruce E.; Young, Lloyd M.

    1994-01-01

    A compact high brightness linear accelerator is provided for use, e.g., in a free electron laser. The accelerator has a first plurality of acclerating cavities having end walls with four coupling slots for accelerating electrons to high velocities in the absence of quadrupole fields. A second plurality of cavities receives the high velocity electrons for further acceleration, where each of the second cavities has end walls with two coupling slots for acceleration in the absence of dipole fields. The accelerator also includes a first cavity with an extended length to provide for phase matching the electron beam along the accelerating cavities. A solenoid is provided about the photocathode that emits the electons, where the solenoid is configured to provide a substantially uniform magnetic field over the photocathode surface to minimize emittance of the electons as the electrons enter the first cavity.

  11. Fiber Accelerating Structures

    SciTech Connect

    Hammond, Andrew P.; /Reed Coll. /SLAC

    2010-08-25

    One of the options for future particle accelerators are photonic band gap (PBG) fiber accelerators. PBG fibers are specially designed optical fibers that use lasers to excite an electric field that is used to accelerate electrons. To improve PBG accelerators, the basic parameters of the fiber were tested to maximize defect size and acceleration. Using the program CUDOS, several accelerating modes were found that maximized these parameters for several wavelengths. The design of multiple defects, similar to having closely bound fibers, was studied to find possible coupling or the change of modes. The amount of coupling was found to be dependent on distance separated. For certain distances accelerating coupled modes were found and examined. In addition, several non-periodic fiber structures were examined using CUDOS. The non-periodic fibers produced several interesting results and promised more modes given time to study them in more detail.

  12. Enzymatic hydrolysis of ammonia-treated sugar beet pulp.

    PubMed

    Foster, B L; Dale, B E; Doran-Peterson, J B

    2001-01-01

    Sugar beet pulp is a carbohydrate-rich coproduct generated by the table sugar industry. Beet pulp has shown promise as a feedstock for ethanol production using enzymes to hydrolyze polymeric carbohydrates and engineered bacteria to ferment sugars to ethanol. In this study, sugar beet pulp underwent an ammonia pressurization depressurization (APD) pretreatment in which the pulp was exploded by the sudden evaporation of ammonia in a reactor vessel. APD was found to substantially increase hydrolysis efficiency of the cellulose component, but when hemicellulose- and pectin-degrading enzymes were added, treated pulp hydrolysis was no better than the untreated control.

  13. Benzene/nitrous oxide flammability in the precipitate hydrolysis process

    SciTech Connect

    Jacobs, R A

    1989-09-18

    The HAN (hydroxylamine nitrate) process for destruction of nitrite in precipitate hydrolysis produces nitrous oxide (N2O) gas as one of the products. N2O can form flammable mixtures with benzene which is also present due to radiolysis and hydrolysis of tetraphenylborate. Extensive flame modeling and explosion testing was undertaken to define the minimum oxidant for combustion of N2O/benzene using both nitrogen and carbon dioxide as diluents. The attached memorandum interprets and documents the results of the studies.

  14. Hydrolysis of xylan by an immobilized xylanase from Aureobasidium pullulans

    SciTech Connect

    Allenza, P.; Scherl, D.S.; Detroy, R.W.; Leathers, T.D.; Scott, C.D. .

    1986-01-01

    The beta-(1,4)-linked xylose residues that comprise the backbone of the abundant plant polymer xylan can be released by enzymic hydrolysis. Xylanase, which is produced in exceptionally high levels by the color-variant strain Y-2311-1 of A. pullulans, was immobilized onto a macroporous ceramic carrier. Despite a low coupling efficiency, it was possible to run the reactor under a wide range of conditions with flow rates of 3-10 bed volumes/minute of 1% soluble xylan with no detectable leaching of enzyme. The size distribution of products and rate of xylan hydrolysis were very similar for the immobilized and soluble enzymes. (Refs. 13).

  15. Hydrolysis of xylan by an immobilized xylanase from Aureobasidium pullanans

    SciTech Connect

    Allenza, P.; Scherl, D.S.; Detroy, R.W.; Leathers, T.D.; Scott, C.D.

    1986-01-01

    The beta-(1,4)-linked xylose residues that comprise the backbone of the abundant plant polymer xylan can be released by enzymic hydrolysis. Xylanase, which is produced in exceptionally high levels by the color-variant strain of A. pullulans, was immobilized onto a macroporous ceramic carrier. Despite a low coupling efficiency, it was possible to run the reactor under a wide range of conditions with flow rates of 3-10 bed volumes/minute of 1% soluble xylan with no detectable leaching of enzyme. The size distribution of products and rate of xylan hydrolysis were very similar for the immobilized and soluble enzymes. (Refs. 13).

  16. Acceleration in astrophysics

    SciTech Connect

    Colgate, S.A.

    1993-12-31

    The origin of cosmic rays and applicable laboratory experiments are discussed. Some of the problems of shock acceleration for the production of cosmic rays are discussed in the context of astrophysical conditions. These are: The presumed unique explanation of the power law spectrum is shown instead to be a universal property of all lossy accelerators; the extraordinary isotropy of cosmic rays and the limited diffusion distances implied by supernova induced shock acceleration requires a more frequent and space-filling source than supernovae; the near perfect adiabaticity of strong hydromagnetic turbulence necessary for reflecting the accelerated particles each doubling in energy roughly 10{sup 5} to {sup 6} scatterings with negligible energy loss seems most unlikely; the evidence for acceleration due to quasi-parallel heliosphere shocks is weak. There is small evidence for the expected strong hydromagnetic turbulence, and instead, only a small number of particles accelerate after only a few shock traversals; the acceleration of electrons in the same collisionless shock that accelerates ions is difficult to reconcile with the theoretical picture of strong hydromagnetic turbulence that reflects the ions. The hydromagnetic turbulence will appear adiabatic to the electrons at their much higher Larmor frequency and so the electrons should not be scattered incoherently as they must be for acceleration. Therefore the electrons must be accelerated by a different mechanism. This is unsatisfactory, because wherever electrons are accelerated these sites, observed in radio emission, may accelerate ions more favorably. The acceleration is coherent provided the reconnection is coherent, in which case the total flux, as for example of collimated radio sources, predicts single charge accelerated energies much greater than observed.

  17. Results of the hydrolysis of fusinitic brown coals

    SciTech Connect

    Perednikova, Z.M.; Garstman, B.B.; Rakitina, E.V.; Rumyantseva, Z.A.

    1984-01-01

    The products of the alkaline hydrolysis of debituminized fusinitic brown coals have been separated into relatively homogeneous groups of substances with the aid of extraction, chromatography, and alkaline saponification. The group compositions of the substances isolated have been studied by IR spectroscopy.

  18. Small peptides hydrolysis in dry-cured meats.

    PubMed

    Mora, Leticia; Gallego, Marta; Escudero, Elizabeth; Reig, Milagro; Aristoy, M-Concepción; Toldrá, Fidel

    2015-11-01

    Large amounts of different peptides are naturally generated in dry-cured meats as a consequence of the intense proteolysis mechanisms which take place during their processing. In fact, meat proteins are extensively hydrolysed by muscle endo-peptidases (mainly calpains and cathepsins) followed by exo-peptidases (mainly, tri- and di-peptidyl peptidases, dipeptidases, aminopeptidases and carboxypeptidases). The result is a large amount of released free amino acids and a pool of numerous peptides with different sequences and lengths, some of them with interesting sequences for bioactivity. This manuscript is presenting the proteomic identification of small peptides resulting from the hydrolysis of four target proteins (glyceraldehyde-3-phosphate dehydrogenase, beta-enolase, myozenin-1 and troponin T) and discusses the enzymatic routes for their generation during the dry-curing process. The results indicate that the hydrolysis of peptides follows similar exo-peptidase mechanisms. In the case of dry-fermented sausages, most of the observed hydrolysis is the result of the combined action of muscle and microbial exo-peptidases except for the hydrolysis of di- and tri-peptides, mostly due to microbial di- and tri-peptidases, and the release of amino acids at the C-terminal that appears to be mostly due to muscle carboxypeptidases.

  19. Effects of hydrolysis and carbonization reactions on hydrochar production.

    PubMed

    Fakkaew, K; Koottatep, T; Polprasert, C

    2015-09-01

    Hydrothermal carbonization (HTC) is a thermal conversion process which converts wet biomass into hydrochar. In this study, a low-energy HTC process named "Two-stage HTC" comprising of hydrolysis and carbonization stages using faecal sludge as feedstock was developed and optimized. The experimental results indicated the optimum conditions of the two-stage HTC to be; hydrolysis temperature of 170 °C, hydrolysis reaction time of 155 min, carbonization temperature of 215 °C, and carbonization reaction time of 100 min. The hydrolysis reaction time and carbonization temperature had a statistically significant effect on energy content of the produced hydrochar. Energy input of the two-stage HTC was about 25% less than conventional HTC. Energy efficiency of the two-stage HTC for treating faecal sludge was higher than that of conventional HTC and other thermal conversion processes such as pyrolysis and gasification. The two-stage HTC could be considered as a potential technology for treating FS and producing hydrochar.

  20. Bioabatement with xylanase supplementation to reduce enzymatic hydrolysis inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bioabatement, using the fungus Coniochaeta ligniaria NRRL30616 can effectively eliminate enzyme inhibitors from pretreated biomass hydrolysis. However, our recent research suggested that bioabatement had no beneficial effect on removing xylo-oligomers which were identified as strong inhibitors to ce...

  1. Designer xylanosomes: protein nanostructures for enhanced xylan hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This work is the first report of the successful design, construction, and application of multi-functional, self-assembling biocatalysts for targeted xylan hydrolysis, termed xylanosomes. Using the architecture of cellulosomes found in some anaerobic cellulolytic microbes, four different xylanosomes...

  2. Ethanol production with dilute acid hydrolysis using partially dried lignocellulosics

    DOEpatents

    Nguyen, Quang A.; Keller, Fred A.; Tucker, Melvin P.

    2003-12-09

    A process of converting lignocellulosic biomass to ethanol, comprising hydrolyzing lignocellulosic materials by subjecting dried lignocellulosic material in a reactor to a catalyst comprised of a dilute solution of a strong acid and a metal salt to lower the activation energy (i.e., the temperature) of cellulose hydrolysis and ultimately obtain higher sugar yields.

  3. Validation of lignocellulosic biomass carbohydrates determination via acid hydrolysis.

    PubMed

    Zhou, Shengfei; Runge, Troy M

    2014-11-01

    This work studied the two-step acid hydrolysis for determining carbohydrates in lignocellulosic biomass. Estimation of sugar loss based on acid hydrolyzed sugar standards or analysis of sugar derivatives was investigated. Four model substrates (starch, holocellulose, filter paper and cotton) and three levels of acid/material ratios (7.8, 10.3 and 15.4, v/w) were studied to demonstrate the range of test artifacts. The method for carbohydrates estimation based on acid hydrolyzed sugar standards having the most satisfactory carbohydrate recovery and relative standard deviation. Raw material and the acid/material ratio both had significant effect on carbohydrate hydrolysis, suggesting the acid to have impacts beyond a catalyst in the hydrolysis. Following optimal procedures, we were able to reach a carbohydrate recovery of 96% with a relative standard deviation less than 3%. The carbohydrates recovery lower than 100% was likely due to the incomplete hydrolysis of substrates, which was supported by scanning electron microscope (SEM) images.

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

    ERIC Educational Resources Information Center

    Sanford, Elizabeth M.; Smith, Traci L.

    2008-01-01

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

  5. Radioactive demonstration of the ``late wash`` Precipitate Hydrolysis Process

    SciTech Connect

    Bibler, N.E.; Ferrara, D.M.; Ha, B.C.

    1992-06-30

    This report presents results of the radioactive demonstration of the DWPF Precipitate Hydrolysis Process as it would occur in the ``late wash`` flowsheet in the absence of hydroxylamine nitrate. Radioactive precipitate containing Cs-137 from the April, 1983, in-tank precipitation demonstration in Tank 48 was used for these tests.

  6. Radioactive demonstration of the late wash'' Precipitate Hydrolysis Process

    SciTech Connect

    Bibler, N.E.; Ferrara, D.M.; Ha, B.C.

    1992-06-30

    This report presents results of the radioactive demonstration of the DWPF Precipitate Hydrolysis Process as it would occur in the late wash'' flowsheet in the absence of hydroxylamine nitrate. Radioactive precipitate containing Cs-137 from the April, 1983, in-tank precipitation demonstration in Tank 48 was used for these tests.

  7. Acid hydrolysis of Jerusalem artichoke for ethanol fermentation

    SciTech Connect

    Kim, K.; Hamdy, M.K.

    1986-01-01

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

  8. DFT STUDY OF THE HYDROLYSIS OF SOME S-TRIAZINES

    EPA Science Inventory

    The acid-catalyzed hydrolysis of atrazine and related 2-chloro-s-triazines to the corresponding 2-hydroxy-s-triazines was investigated using the B3LYP hybrid density functional theory method. Gas-phase calculations were performed at the B3LYP/6-311++G(d,p)//B3LYP/6-31G* level of ...

  9. Penicillin Hydrolysis: A Kinetic Study of a Multistep, Multiproduct Reaction.

    ERIC Educational Resources Information Center

    McCarrick, Thomas A.; McLafferty, Fred W.

    1984-01-01

    Background, procedures used, and typical results are provided for an experiment in which students carry out the necessary measurements on the acid-catalysis of penicillin in two hours. By applying kinetic theory to the data obtained, the reaction pathways for the hydrolysis of potassium benzyl penicillin are elucidated. (JN)

  10. Destruction of waste energetic materials using base hydrolysis

    SciTech Connect

    Benziger, T.M.; Buntain, G.A.; Sanchez, J.A.; Spontarelli, T.

    1993-01-01

    In dismantling weapons from stockpile reduction, environmentally acceptable degradation of the associated high explosive (HE) waste to non-energetic forms is a critical objective. Base hydrolysis appears to be a simple, inexpensive method for converting propellants, explosives, and pyrotechnics (PEPS) into non-energetic materials that can be released directly or, if necessary, treated further. We have demonstrated that many PEPs can be hydrolyzed with aqueous sodium hydroxide or ammonia at temperatures ranging from 60 to 150[degree]C. Hydrolysis experiments have been performed on pure compounds as well as DOE and DoD formulations, such as plastic-bonded explosive (PBX) 9404, tritonal, and rocket motor propellant. Small particle size of the energetic material is desirable, but not necessary. We have decomposed molding powder, pressed charges up to two pounds in weight, and partially exposed, metal-encased pieces. The products formed are dependent on the starting material composition, but usually consist of organic and inorganic salts, e.g., sodium formate, acetate, nitrite and nitrate. The major gaseous product from the base hydrolysis of PEPs is nitrous oxide. The time required for complete destruction varies with the material being hydrolyzed, and is dependent on solubility and mass transfer. Hydrolysis rates can be increased by particle size reduction, efficient stirring, and addition of organic solvent to the alkaline solution. Rate enhancement by ultrasonic agitation is a possibility that we have just begun to study.

  11. Release of bound procyanidins from cranberry pomace by alkaline hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Procyanidins in plant products are present as extractable or unextractable/bound forms. We optimized alkaline hydrolysis conditions to liberate bound procyanidins from dried cranberry pomace. Five mL of sodium hydroxide (2, 4, or 6N) was added to 0.5 g of cranberry pomace in screw top glass tubes,...

  12. Single Molecule Study of Cellulase Hydrolysis of Crystalline Cellulose

    SciTech Connect

    Liu, Y.-S.; Luo, Y.; Baker, J. O.; Zeng, Y.; Himmel, M. E.; Smith, S.; Ding, S.-Y.

    2009-12-01

    This report seeks to elucidate the role of cellobiohydrolase-I (CBH I) in the hydrolysis of crystalline cellulose. A single-molecule approach uses various imaging techniques to investigate the surface structure of crystalline cellulose and changes made in the structure by CBH I.

  13. Solvent effects and alkali metal ion catalysis in phosphodiester hydrolysis.

    PubMed

    Gomez-Tagle, Paola; Vargas-Zúñiga, Idania; Taran, Olga; Yatsimirsky, Anatoly K

    2006-12-22

    The kinetics of the alkaline hydrolysis of bis(p-nitrophenyl) phosphate (BNPP) have been studied in aqueous DMSO, dioxane, and MeCN. In all solvent mixtures the reaction rate steadily decreases to half of its value in pure water in the range of 0-70 vol % of organic cosolvent and sharply increases in mixtures with lower water content. Correlations based on different scales of solvent empirical parameters failed to describe the solvent effect in this system, but it can be satisfactorily treated in terms of a simplified stepwise solvent-exchange model. Alkali metal ions catalyze the BNPP hydrolysis but do not affect the rate of hydrolysis of neutral phosphotriester p-nitrophenyl diphenyl phosphate in DMSO-rich mixtures. The catalytic activity decreases in the order Li+ > Na+ > K+ > Rb+ > Cs+. For all cations except Na+, the reaction rate is first-order in metal ion. With Na+, both first- and second-order kinetics in metal ions are observed. Binding constants of cations to the dianionic transition state of BNPP alkaline hydrolysis are of the same order of magnitude and show a similar trend as their binding constants to p-nitrophenyl phosphate dianion employed as a transition-state model. The appearance of alkali metal ion catalysis in a medium, which solvates metal ions stronger than water, is attributed to the increased affinity of cations to dianions, which undergo a strong destabilization in the presence of an aprotic dipolar cosolvent.

  14. Acid hydrolysis of sweet potato for ethanol production

    SciTech Connect

    Kim, K.; Hamdy, M.K.

    1985-01-01

    Studies were conducted to establish optimal conditions for the acid hydrolysis of sweet potato for maximal ethanol yield. The starch contents of two sweet potato cultivars (Georgia Red and TG-4), based on fresh weight, were 21.1 +/- 0.6% and 27.5 +/- 1.6%, respectively. The results of acid hydrolysis experiments showed the following: (1) both hydrolysis rate and hydroxymethylfurfural (HMF) concentration were a function of HCL concentration, temperature, and time; (2) the reducing sugars were rapidly formed with elevated concentrations of HCl and temperature, but also destroyed quickly; and (3) HMF concentration increased significantly with the concentration of HCl, temperature, and hydrolysis time. Maximum reducing sugar value of 84.2 DE and 0.056% HMF (based on wet weight) was achieved after heating 8% SPS for 15 min in 1N HCl at 110/sup 0/C. Degraded 8% SPS (1N HCl, 97/sup 0/C for 20 min or 110/sup 0/C for 10 min) was utilized as substrate for ethanol fermentation and 3.8% ethanol (v/v) was produced from 1400 mL fermented wort. This is equal to 41.6 g ethanol (200 proof) from 400 g of fresh sweet potato tuber (Georgia Red) or an ethanol yield potential of 431 gal of 200-proof ethanol/acre (from 500 bushel tubers/acre).

  15. Evaluation of Cation Hydrolysis Schemes with a Pocket Calculator.

    ERIC Educational Resources Information Center

    Clare, Brian W.

    1979-01-01

    Described is the use of two models of pocket calculators. The Hewlett-Packard HP67 and the Texas Instruments TI59, to solve problems arising in connection with ionic equilibria in solution. A three-parameter regression program is described and listed as a specific example, the hydrolysis of hexavalent uranium, is provided. (BT)

  16. Structural modifications of lignocellulosics by pretreatments to enhance enzymatic hydrolysis

    SciTech Connect

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

    1983-01-01

    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 the mechanism of the enhancement in the hyrolysis rate upon pretreatment. It has been found that, in general, multiple pretreatments were not promising, since the hydrolysis rates rarely exceeded those achieved by single pretreatments. Ball-milling pretreatment was found to be effective in increasing the specific surface area and decreasing the crystallinity index. Treatment with ethylene glycol was highly effective in increasing the specific surface area, in addition to a high degree of delignification. Peracetic acid pretreatment was highly effective in delignifying substrate. Among multiple pretreatments, those involving peracetic acid treatment generally had lower crystallinity indices and lignin content values. The relationship between the hydrolysis rate and the set of structural features indicated that an increase in surface area and a decrease in the crystallinity and lignin content enhance the hydrolysis; the specific surface area is the most influential of the structural features, followed by the lignin content. (Refs. 23).

  17. 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...

  18. Effects of hydrolysis and carbonization reactions on hydrochar production.

    PubMed

    Fakkaew, K; Koottatep, T; Polprasert, C

    2015-09-01

    Hydrothermal carbonization (HTC) is a thermal conversion process which converts wet biomass into hydrochar. In this study, a low-energy HTC process named "Two-stage HTC" comprising of hydrolysis and carbonization stages using faecal sludge as feedstock was developed and optimized. The experimental results indicated the optimum conditions of the two-stage HTC to be; hydrolysis temperature of 170 °C, hydrolysis reaction time of 155 min, carbonization temperature of 215 °C, and carbonization reaction time of 100 min. The hydrolysis reaction time and carbonization temperature had a statistically significant effect on energy content of the produced hydrochar. Energy input of the two-stage HTC was about 25% less than conventional HTC. Energy efficiency of the two-stage HTC for treating faecal sludge was higher than that of conventional HTC and other thermal conversion processes such as pyrolysis and gasification. The two-stage HTC could be considered as a potential technology for treating FS and producing hydrochar. PMID:26051497

  19. Arabidopsis ARF-GTP exchange factor, GNOM, mediates transport required for innate immunity and focal accumulation of syntaxin PEN1.

    PubMed

    Nielsen, Mads Eggert; Feechan, Angela; Böhlenius, Henrik; Ueda, Takashi; Thordal-Christensen, Hans

    2012-07-10

    Penetration resistance to powdery mildew fungi, conferred by localized cell wall appositions (papillae), is one of the best-studied processes in plant innate immunity. The syntaxin PENETRATION (PEN)1 is required for timely appearance of papillae, which contain callose and extracellular membrane material, as well as PEN1 itself. Appearance of membrane material in papillae suggests secretion of exosomes. These are potentially derived from multivesicular bodies (MVBs), supported by our observation that ARA6-labeled organelles assemble at the fungal attack site. However, the trafficking components that mediate delivery of extracellular membrane material are unknown. Here, we show that the delivery is independent of PEN1 function. Instead, we find that application of brefeldin (BF)A blocks the papillary accumulation of GFP-PEN1-labeled extracellular membrane and callose, while impeding penetration resistance. We subsequently provide evidence indicating that the responsible BFA-sensitive ADP ribosylation factor-GTP exchange factor (ARF-GEF) is GNOM. Firstly, analysis of the transheterozygote gnom(B4049/emb30-1) (gnom(B)(/E)) mutant revealed a delay in papilla formation and reduced penetration resistance. Furthermore, a BFA-resistant version of GNOM restored the BFA-sensitive papillary accumulation of GFP-PEN1 and callose. Our data, therefore, provide a link between GNOM and disease resistance. We suggest that papilla formation requires rapid reorganization of material from the plasma membrane mediated by GNOM. The papilla material is subsequently presumed to be sorted into MVBs and directed to the site of fungal attack, rendering the epidermal plant cell inaccessible for the invading powdery mildew fungus.

  20. Regulation of β-adrenergic control of heart rate by GTP-cyclohydrolase 1 (GCH1) and tetrahydrobiopterin

    PubMed Central

    Adlam, David; Herring, Neil; Douglas, Gillian; De Bono, Joseph P.; Li, Dan; Danson, Edward J.; Tatham, Amy; Lu, Cheih-Ju; Jennings, Katie A.; Cragg, Stephanie J.; Casadei, Barbara; Paterson, David J.; Channon, Keith M.

    2012-01-01

    Aims Clinical markers of cardiac autonomic function, such as heart rate and response to exercise, are important predictors of cardiovascular risk. Tetrahydrobiopterin (BH4) is a required cofactor for enzymes with roles in cardiac autonomic function, including tyrosine hydroxylase and nitric oxide synthase. Synthesis of BH4 is regulated by GTP cyclohydrolase I (GTPCH), encoded by GCH1. Recent clinical studies report associations between GCH1 variants and increased heart rate, but the mechanistic importance of GCH1 and BH4 in autonomic function remains unclear. We investigate the effect of BH4 deficiency on the autonomic regulation of heart rate in the hph-1 mouse model of BH4 deficiency. Methods and results In the hph-1 mouse, reduced cardiac GCH1 expression, GTPCH enzymatic activity, and BH4 were associated with increased resting heart rate; blood pressure was not different. Exercise training decreased resting heart rate, but hph-1 mice retained a relative tachycardia. Vagal nerve stimulation in vitro induced bradycardia equally in hph-1 and wild-type mice both before and after exercise training. Direct atrial responses to carbamylcholine were equal. In contrast, propranolol treatment normalized the resting tachycardia in vivo. Stellate ganglion stimulation and isoproterenol but not forskolin application in vitro induced a greater tachycardic response in hph-1 mice. β1-adrenoceptor protein was increased as was the cAMP response to isoproterenol stimulation. Conclusion Reduced GCH1 expression and BH4 deficiency cause tachycardia through enhanced β-adrenergic sensitivity, with no effect on vagal function. GCH1 expression and BH4 are novel determinants of cardiac autonomic regulation that may have important roles in cardiovascular pathophysiology. PMID:22241166

  1. Induction of Cytoplasmic Rods and Rings Structures by Inhibition of the CTP and GTP Synthetic Pathway in Mammalian Cells

    PubMed Central

    Carcamo, Wendy C.; Satoh, Minoru; Kasahara, Hideko; Terada, Naohiro; Hamazaki, Takashi; Chan, Jason Y. F.; Yao, Bing; Tamayo, Stephanie; Covini, Giovanni; von Mühlen, Carlos A.; Chan, Edward K. L.

    2011-01-01

    Background Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined. Methodology/Principal Findings Distinct cytoplasmic rods (∼3–10 µm in length) and rings (∼2–5 µm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin. Conclusions/Significance RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases. PMID:22220215

  2. GTP cyclohydrolase I deficiency, a new enzyme defect causing hyperphenylalaninemia with neopterin, biopterin, dopamine, and serotonin deficiencies and muscular hypotonia.

    PubMed

    Niederwieser, A; Blau, N; Wang, M; Joller, P; Atarés, M; Cardesa-Garcia, J

    1984-02-01

    A 4-year-old patient is described with hyperphenylalaninemia, severe retardation in development, severe muscular hypotonia of the trunk and hypertonia of the extremities, convulsions, and frequent episodes of hyperthermia without infections. Urinary excretion of neopterin, biopterin, pterin, isoxanthopterin, dopamine, and serotonin was very low, although the relative proportions of pterins were normal. In lumbar cerebrospinal fluid, homovanillic acid, 5-hydroxyindoleacetic acid, neopterin and biopterin were low. Oral administration of L-erythro tetrahydrobiopterin normalized the elevated serum phenylalanine within 4 h, serum tyrosine was increased briefly and serum alanine and glutamic acid for a longer time. Urinary dopamine and serotonin excretion were also increased. Administration of an equivalent dose of D-erythro tetrahydroneopterin was ineffective and demonstrated that this compound is not a cofactor in vivo and cannot be transformed into an active cofactor. GTP cyclohydrolase I activity was not detectable in liver biopsies from the patient. The presence of an endogenous inhibitor in the patient's liver was excluded. This is the first case of a new variant of hyperphenylalaninemia in which the formation of dihydroneopterin triphosphate and its pterin metabolites in liver is markedly diminished. Normal activities of xanthine oxidase and sulfite oxidase were apparent since uric acid levels were normal and no increase in hypoxanthine, xanthine, and S-sulfocysteine concentrations could be observed in urine. It is concluded that the molybdenum cofactor of these enzymes may not be derived from dihydroneopterin triphosphate in man. Also, since no gross abnormalities in the patient's immune system could be found, it seems unlikely that dihydroneopterin triphosphate metabolites, such as neopterin, participate actively in immunological processes, as postulated by others. See Note added in proof. PMID:6734669

  3. Structure of an archaeal heterotrimeric initiation factor 2 reveals a nucleotide state between the GTP and the GDP states

    PubMed Central

    Yatime, Laure; Mechulam, Yves; Blanquet, Sylvain; Schmitt, Emmanuelle

    2007-01-01

    Initiation of translation in eukaryotes and in archaea involves eukaryotic/archaeal initiation factor (e/aIF)1 and the heterotrimeric initiation factor e/aIF2. In its GTP-bound form, e/aIF2 provides the initiation complex with Met–tRNAiMet. After recognition of the start codon by initiator tRNA, e/aIF1 leaves the complex. Finally, e/aIF2, now in a GDP-bound form, loses affinity for Met–tRNAiMet and dissociates from the ribosome. Here, we report a 3D structure of an aIF2 heterotrimer from the archeon Sulfolobus solfataricus obtained in the presence of GDP. Our report highlights how the two-switch regions involved in formation of the tRNA-binding site on subunit γ exchange conformational information with α and β. The zinc-binding domain of β lies close to the guanine nucleotide and directly contacts the switch 1 region. As a result, switch 1 adopts a not yet described conformation. Moreover, unexpectedly for a GDP-bound state, switch 2 has the “ON” conformation. The stability of these conformations is accounted for by a ligand, most probably a phosphate ion, bound near the nucleotide binding site. The structure suggests that this GDP–inorganic phosphate (Pi) bound state of aIF2 may be proficient for tRNA binding. Recently, it has been proposed that dissociation of eIF2 from the initiation complex is closely coupled to that of Pi from eIF2γ upon start codon recognition. The nucleotide state of aIF2 shown here is indicative of a similar mechanism in archaea. Finally, we consider the possibility that release of Pi takes place after e/aIF2γ has been informed of e/aIF1 dissociation by e/aIF2β. PMID:18000047

  4. The Dielectric Wall Accelerator

    SciTech Connect

    Caporaso, George J.; Chen, Yu-Jiuan; Sampayan, Stephen E.

    2009-01-01

    The Dielectric Wall Accelerator (DWA), a class of induction accelerators, employs a novel insulating beam tube to impress a longitudinal electric field on a bunch of charged particles. The surface flashover characteristics of this tube may permit the attainment of accelerating gradients on the order of 100 MV/m for accelerating pulses on the order of a nanosecond in duration. A virtual traveling wave of excitation along the tube is produced at any desired speed by controlling the timing of pulse generating modules that supply a tangential electric field to the tube wall. Because of the ability to control the speed of this virtual wave, the accelerator is capable of handling any charge to mass ratio particle; hence it can be used for electrons, protons and any ion. The accelerator architectures, key technologies and development challenges will be described.

  5. Switched matrix accelerator

    SciTech Connect

    Whittum, David H.; Tantawi, Sami G.

    2001-01-01

    We describe a new concept for a microwave circuit functioning as a charged-particle accelerator at mm wavelengths, permitting an accelerating gradient higher than conventional passive circuits can withstand consistent with cyclic fatigue. The device provides acceleration for multiple bunches in parallel channels, and permits a short exposure time for the conducting surface of the accelerating cavities. Our analysis includes scalings based on a smooth transmission line model and a complementary treatment with a coupled-cavity simulation. We also provide an electromagnetic design for the accelerating structure, arriving at rough dimensions for a seven-cell accelerator matched to standard waveguide and suitable for bench tests at low power in air at 91.392 GHz. A critical element in the concept is a fast mm-wave switch suitable for operation at high power, and we present the considerations for implementation in an H-plane tee. We discuss the use of diamond as the photoconductor switch medium.

  6. Switched Matrix Accelerator

    SciTech Connect

    Whittum, David H

    2000-10-04

    We describe a new concept for a microwave circuit functioning as a charged-particle accelerator at mm-wavelengths, permitting an accelerating gradient higher than conventional passive circuits can withstand consistent with cyclic fatigue. The device provides acceleration for multiple bunches in parallel channels, and permits a short exposure time for the conducting surface of the accelerating cavities. Our analysis includes scalings based on a smooth transmission line model and a complementary treatment with a coupled-cavity simulation. We provide also an electromagnetic design for the accelerating structure, arriving at rough dimensions for a seven-cell accelerator matched to standard waveguide and suitable for bench tests at low power in air at 91.392. GHz. A critical element in the concept is a fast mm-wave switch suitable for operation at high-power, and we present the considerations for implementation in an H-plane tee. We discuss the use of diamond as the photoconductor switch medium.

  7. Wake field accelerators

    SciTech Connect

    Wilson, P.B.

    1986-02-01

    In a wake field accelerator a high current driving bunch injected into a structure or plasma produces intense induced fields, which are in turn used to accelerate a trailing charge or bunch. The basic concepts of wake field acceleration are described. Wake potentials for closed cavities and periodic structures are derived, as are wake potentials on a collinear path with a charge distribution. Cylindrically symmetric structures excited by a beam in the form of a ring are considered. (LEW)

  8. ACCELERATION RESPONSIVE SWITCH

    DOEpatents

    Chabrek, A.F.; Maxwell, R.L.

    1963-07-01

    An acceleration-responsive device with dual channel capabilities whereby a first circuit is actuated upon attainment of a predetermined maximum acceleration level and when the acceleration drops to a predetermined minimum acceleriltion level another circuit is actuated is described. A fluid-damped sensing mass slidably mounted in a relatively frictionless manner on a shaft through the intermediation of a ball bushing and biased by an adjustable compression spring provides inertially operated means for actuating the circuits. (AEC)

  9. Optically pulsed electron accelerator

    DOEpatents

    Fraser, John S.; Sheffield, Richard L.

    1987-01-01

    An optically pulsed electron accelerator can be used as an injector for a free electron laser and comprises a pulsed light source, such as a laser, for providing discrete incident light pulses. A photoemissive electron source emits electron bursts having the same duration as the incident light pulses when impinged upon by same. The photoemissive electron source is located on an inside wall of a radio frequency powered accelerator cell which accelerates the electron burst emitted by the photoemissive electron source.

  10. Optically pulsed electron accelerator

    DOEpatents

    Fraser, J.S.; Sheffield, R.L.

    1985-05-20

    An optically pulsed electron accelerator can be used as an injector for a free electron laser and comprises a pulsed light source, such as a laser, for providing discrete incident light pulses. A photoemissive electron source emits electron bursts having the same duration as the incident light pulses when impinged upon by same. The photoemissive electron source is located on an inside wall of a radiofrequency-powered accelerator cell which accelerates the electron burst emitted by the photoemissive electron source.

  11. Generation of group B soyasaponins I and III by hydrolysis.

    PubMed

    Zhang, Wei; Teng, Su Ping; Popovich, David G

    2009-05-13

    Soyasaponins are a group of oleanane triterpenoids found in soy and other legumes that have been associated with some of the benefits achieved by consuming plant-based diets. However, these groups of compounds are diverse and structurally complicated to chemically characterize, separate from the isoflavones, and isolate in sufficient quantities for bioactive testing. Therefore, the aim of this study was to maximize the extraction of soyasaponins from soy flour, remove isoflavones, separate group B soyasaponins from group A, and produce an extract that contained a majority of non-DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one)-conjugated group B soyasaponins I and III. Room temperature extraction in methanol for 24 or 48 h resulted in the maximum recovery of soyasaponins, and Soxhlet extraction resulted in the least. A solid-phase extraction using methanol (45%) was found to virtually eliminate the interfering isoflavones as compared to butanol-water liquid-liquid extraction and ammonium sulfate precipitation, while maximizing saponin recovery. Alkaline hydrolysis in anhydrous methanol produced the maximum amount of soyasaponins I and III as compared to aqueous methanol and acid hydrolysis in both aqueous and anhydrous methanol. The soyasaponin I amount was increased by 175%, and soyasaponin III was increased by 211% after alkaline hydrolysis. Furthermore, after alkaline hydrolysis, a majority of DDMP-conjugated group B soyasaponins such as betag, betaa, gammag, and gammaa transformed into the non-DDMP-conjugated soyasaponins I and III without affecting the glycosidic bond at position C-3 of the ring structure. Therefore, we have developed a method that maximizes the recovery of DDMP-conjugated saponins and uses alkaline hydrolysis to produce an extract containing mainly soyasaponins I and III. PMID:19338335

  12. Hydrolysis of thorium(iv) at variable temperatures.

    PubMed

    Zanonato, P L; Di Bernardo, P; Zhang, Z; Gong, Y; Tian, G; Gibson, J K; Rao, L

    2016-08-01

    Hydrolysis of Th(iv) was studied in tetraethylammonium perchlorate (0.10 mol kg(-1)) at variable temperatures (283-358 K) by potentiometry and microcalorimetry. Three hydrolysis reactions, mTh(4+) + nH2O = Thm(OH)n((4m-n)+) + nH(+), in which (n,m) = (2,2), (8,4), and (15,6), were invoked to describe the potentiometric and calorimetric data for solutions with the [hydroxide]/[Th(iv)] ratio ≤ 2. At higher ratios, the formation of (16,5) cannot be excluded. The hydrolysis constants, *β2,2, *β8,4, and *β15,6, increased by 3, 7, and 11 orders of magnitude, respectively, as the temperature was increased from 283 to 358 K. The enhancement is mainly due to the significant increase of the degree of ionization of water as the temperature rises. All three hydrolysis reactions are endothermic at 298 K, with enthalpies of (118 ± 4) kJ mol(-1), (236 ± 7) kJ mol(-1), and (554 ± 4) kJ mol(-1) for ΔH2,2, ΔH8,4, and ΔH15,6 respectively. The hydrolysis constants at infinite dilution have been obtained with the specific ion interaction approach. The applicability of three approaches for estimating the equilibrium constants at different temperatures, including the constant enthalpy approach, the constant heat capacity approach and the DQUANT equation was evaluated with the data from this work. PMID:27460458

  13. Lipase-catalyzed hydrolysis of TG containing acetylenic FA.

    PubMed

    Jie, Marcel S F Lie Ken; Fua, Xun; Lau, Maureen M L; Chye, M L

    2002-10-01

    Hydrolysis of symmetrical acetylenic TG of type AAA [viz., glycerol tri-(4-decynoate), glycerol tri-(6-octadecynoate), glycerol tri-(9-octadecynoate), glycerol tri-(10-undecynoate), and glycerol tri-(13-docosynoate)] in the presence of eight microbial lipases was studied. Novozyme 435 (Candida antarctica), an efficient enzyme for esterification, showed a significant resistance in the hydrolysis of glycerol tri-(9-octadecynoate) and glycerol tri-(13-docosynoate). Hydrolysis of acetylenic TG with Lipolase 100T (Humicola lanuginosa) was rapidly accomplished. Lipase PS-D (Pseudomonas cepacia) showed a fair resistance toward the hydrolysis of glycerol tri-(6-octadecynoate) only, which reflected its ability to recognize the delta6 positional isomer of 18:1. Lipase CCL (Candida cylindracea, syn. C. rugosa) and AY-30 (C. rugosa) were able to catalyze the release of 10-undecynoic acid and 9-octadecynoic acid from the corresponding TG, but less readily the 13-docosynoic acid in the case of glycerol tri-(13-docosynoate). The two lipases CCL and AY-30 were able to distinguish the small difference in structure of fatty acyl moieties in the TG substrate. To confirm this trend, three regioisomers of mixed acetylenic TG of type ABC (containing one each of delta6, delta9, and delta13 acetylenic FA in various positions) were prepared and hydrolyzed with CCL and AY-40. The results reconfirmed the observation that AY-30 and CCL were able to distinguish the slight differences in the molecular structure (position of the acetylenic bond and chain length) of the acyl groups in the TG during the hydrolysis of such TG substrates.

  14. Acceleration of polarized protons in circular accelerators

    SciTech Connect

    Courant, E.D.; Ruth, R.D.

    1980-09-12

    The theory of depolarization in circular accelerators is presented. The spin equation is first expressed in terms of the particle orbit and then converted to the equivalent spinor equation. The spinor equation is then solved for three different situations: (1) a beam on a flat top near a resonance, (2) uniform acceleration through an isolated resonance, and (3) a model of a fast resonance jump. Finally, the depolarization coefficient, epsilon, is calculated in terms of properties of the particle orbit and the results are applied to a calculation of depolarization in the AGS.

  15. Charged particle accelerator grating

    DOEpatents

    Palmer, Robert B.

    1986-01-01

    A readily disposable and replaceable accelerator grating for a relativistic particle accelerator. The grating is formed for a plurality of liquid droplets that are directed in precisely positioned jet streams to periodically dispose rows of droplets along the borders of a predetermined particle beam path. A plurality of lasers are used to direct laser beams into the droplets, at predetermined angles, thereby to excite the droplets to support electromagnetic accelerating resonances on their surfaces. Those resonances operate to accelerate and focus particles moving along the beam path. As the droplets are distorted or destroyed by the incoming radiation, they are replaced at a predetermined frequency by other droplets supplied through the jet streams.

  16. Particle acceleration in flares

    NASA Technical Reports Server (NTRS)

    Benz, Arnold O.; Kosugi, Takeo; Aschwanden, Markus J.; Benka, Steve G.; Chupp, Edward L.; Enome, Shinzo; Garcia, Howard; Holman, Gordon D.; Kurt, Victoria G.; Sakao, Taro

    1994-01-01

    Particle acceleration is intrinsic to the primary energy release in the impulsive phase of solar flares, and we cannot understand flares without understanding acceleration. New observations in soft and hard X-rays, gamma-rays and coherent radio emissions are presented, suggesting flare fragmentation in time and space. X-ray and radio measurements exhibit at least five different time scales in flares. In addition, some new observations of delayed acceleration signatures are also presented. The theory of acceleration by parallel electric fields is used to model the spectral shape and evolution of hard X-rays. The possibility of the appearance of double layers is further investigated.

  17. Accelerator-based BNCT.

    PubMed

    Kreiner, A J; Baldo, M; Bergueiro, J R; Cartelli, D; Castell, W; Thatar Vento, V; Gomez Asoia, J; Mercuri, D; Padulo, J; Suarez Sandin, J C; Erhardt, J; Kesque, J M; Valda, A A; Debray, M E; Somacal, H R; Igarzabal, M; Minsky, D M; Herrera, M S; Capoulat, M E; Gonzalez, S J; del Grosso, M F; Gagetti, L; Suarez Anzorena, M; Gun, M; Carranza, O

    2014-06-01

    The activity in accelerator development for accelerator-based BNCT (AB-BNCT) both worldwide and in Argentina is described. Projects in Russia, UK, Italy, Japan, Israel, and Argentina to develop AB-BNCT around different types of accelerators are briefly presented. In particular, the present status and recent progress of the Argentine project will be reviewed. The topics will cover: intense ion sources, accelerator tubes, transport of intense beams, beam diagnostics, the (9)Be(d,n) reaction as a possible neutron source, Beam Shaping Assemblies (BSA), a treatment room, and treatment planning in realistic cases.

  18. Accelerator-based BNCT.

    PubMed

    Kreiner, A J; Baldo, M; Bergueiro, J R; Cartelli, D; Castell, W; Thatar Vento, V; Gomez Asoia, J; Mercuri, D; Padulo, J; Suarez Sandin, J C; Erhardt, J; Kesque, J M; Valda, A A; Debray, M E; Somacal, H R; Igarzabal, M; Minsky, D M; Herrera, M S; Capoulat, M E; Gonzalez, S J; del Grosso, M F; Gagetti, L; Suarez Anzorena, M; Gun, M; Carranza, O

    2014-06-01

    The activity in accelerator development for accelerator-based BNCT (AB-BNCT) both worldwide and in Argentina is described. Projects in Russia, UK, Italy, Japan, Israel, and Argentina to develop AB-BNCT around different types of accelerators are briefly presented. In particular, the present status and recent progress of the Argentine project will be reviewed. The topics will cover: intense ion sources, accelerator tubes, transport of intense beams, beam diagnostics, the (9)Be(d,n) reaction as a possible neutron source, Beam Shaping Assemblies (BSA), a treatment room, and treatment planning in realistic cases. PMID:24365468

  19. Charged particle accelerator grating

    DOEpatents

    Palmer, Robert B.

    1986-09-02

    A readily disposable and replaceable accelerator grating for a relativistic particle accelerator. The grating is formed for a plurality of liquid droplets that are directed in precisely positioned jet streams to periodically dispose rows of droplets along the borders of a predetermined particle beam path. A plurality of lasers are used to direct laser beams into the droplets, at predetermined angles, thereby to excite the droplets to support electromagnetic accelerating resonances on their surfaces. Those resonances operate to accelerate and focus particles moving along the beam path. As the droplets are distorted or destroyed by the incoming radiation, they are replaced at a predetermined frequency by other droplets supplied through the jet streams.

  20. Non-ionic surfactants do not consistently improve the enzymatic hydrolysis of pure cellulose.

    PubMed

    Zhou, Yan; Chen, Hongmei; Qi, Feng; Zhao, Xuebing; Liu, Dehua

    2015-04-01

    Non-ionic surfactants have been frequently reported to improve the enzymatic hydrolysis of pretreated lignocellulosic biomass and pure cellulose. However, how the hydrolysis condition, substrate structure and cellulase formulation affect the beneficial action of surfactants has not been well elucidated. In this work, it was found that the enzymatic hydrolysis of pure cellulose was not consistently improved by surfactants. Contrarily, high surfactant concentration, e.g. 5 g/L, which greatly improved the hydrolysis of dilute acid pretreated substrates, actually showed notable inhibition to pure cellulose conversion in the late phase of hydrolysis. Under an optimal hydrolysis condition, the improvement by surfactant was limited, but under harsh conditions surfactant indeed could enhance cellulose conversion. It was proposed that non-ionic surfactants could interact with substrates and cellulases to impact the adsorption behaviors of cellulases. Therefore, the beneficial action of surfactants on pure cellulose hydrolysis is influenced by hydrolysis condition, cellulose structural features and cellulase formulation.

  1. A kinetic study of hydrolysis of polyester elastomer in magnetic tape

    NASA Technical Reports Server (NTRS)

    Yamamoto, K.; Watanabe, H.

    1994-01-01

    A useful method for kinetic study of the hydrolysis of polyester elastomer is established which uses the number-average molecular weight. The reasonableness of this method is confirmed and the effect of magnetic particles on hydrolysis is considered.

  2. Reactivity of Dimeric Tetrazirconium(IV) Wells-Dawson Polyoxometalate toward Dipeptide Hydrolysis Studied by a Combined Experimental and Density Functional Theory Approach.

    PubMed

    Ly, Hong Giang T; Mihaylov, Tzvetan; Absillis, Gregory; Pierloot, Kristine; Parac-Vogt, Tatjana N

    2015-12-01

    Detailed kinetic studies on the hydrolysis of glycylglycine (Gly-Gly) in the presence of the dimeric tetrazirconium(IV)-substituted Wells-Dawson-type polyoxometalate Na14[Zr4(P2W16O59)2(μ3-O)2(OH)2(H2O)4] · 57H2O (1) were performed by a combination of (1)H, (13)C, and (31)P NMR spectroscopies. The catalyst was shown to be stable under a broad range of reaction conditions. The effect of pD on the hydrolysis of Gly-Gly showed a bell-shaped profile with the fastest hydrolysis observed at pD 7.4. The observed rate constant for the hydrolysis of Gly-Gly at pD 7.4 and 60 °C was 4.67 × 10(-7) s(-1), representing a significant acceleration as compared to the uncatalyzed reaction. (13)C NMR data were indicative for coordination of Gly-Gly to 1 via its amide oxygen and amine nitrogen atoms, resulting in a hydrolytically active complex. Importantly, the effective hydrolysis of a series of Gly-X dipeptides with different X side chain amino acids in the presence of 1 was achieved, and the observed rate constant was shown to be dependent on the volume, chemical nature, and charge of the X amino acid side chain. To give a mechanistic explanation of the observed catalytic hydrolysis of Gly-Gly, a detailed quantum-chemical study was performed. The theoretical results confirmed the nature of the experimentally suggested binding mode in the hydrolytically active complex formed between Gly-Gly and 1. To elucidate the role of 1 in the hydrolytic process, both the uncatalyzed and the polyoxometalate-catalyzed reactions were examined. In the rate-determining step of the uncatalyzed Gly-Gly hydrolysis, a carboxylic oxygen atom abstracts a proton from a solvent water molecule and the nascent OH nucleophile attacks the peptide carbon atom. Analogous general-base activity of the free carboxylic group was found to take place also in the case of polyoxometalate-catalyzed hydrolysis as the main catalytic effect originates from the -C═O···Zr(IV) binding.

  3. Effect of the extent of the hydrolysis of tetrabutoxytitanium on catalysis efficiency in the esterification reaction

    SciTech Connect

    Chervina, S.I.; Maksimenko, E.G.; Barshtein, R.S.; Shabanova, N.V.; Bulai, A.K.; Kotov, Yu.I.; Slonim, I.Ya.

    1988-03-01

    A study was carried out on the products of the hydrolysis of tetrabutoxytitanium and their catalytic activity in the esterification reaction. A maximum is observed in the dependence of the reaction rate constant on the extent of the hydrolysis of tetrabutoxytitanium. The maximum effective esterification rate constant corresponds to 60% hydrolysis of tetrabutoxytitanium. The hydrolysis products in this case have largely linear structure. The relationship between the catalytic activity of linear polytitanates and their stability in the reaction medium is discussed.

  4. Angular velocities, angular accelerations, and coriolis accelerations

    NASA Technical Reports Server (NTRS)

    Graybiel, A.

    1975-01-01

    Weightlessness, rotating environment, and mathematical analysis of Coriolis acceleration is described for man's biological effective force environments. Effects on the vestibular system are summarized, including the end organs, functional neurology, and input-output relations. Ground-based studies in preparation for space missions are examined, including functional tests, provocative tests, adaptive capacity tests, simulation studies, and antimotion sickness.

  5. 40 CFR 721.10498 - Substituted alkyl ester, hydrolysis products with silica (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Substituted alkyl ester, hydrolysis... Significant New Uses for Specific Chemical Substances § 721.10498 Substituted alkyl ester, hydrolysis products... chemical substances identified generically as substituted alkyl ester, hydrolysis products with...

  6. 40 CFR 721.10498 - Substituted alkyl ester, hydrolysis products with silica (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Substituted alkyl ester, hydrolysis... Significant New Uses for Specific Chemical Substances § 721.10498 Substituted alkyl ester, hydrolysis products... chemical substances identified generically as substituted alkyl ester, hydrolysis products with...

  7. Comparison of Enzymatic Hydrolysis and Acid Hydrolysis of Sterol Glycosides from Foods Rich in Δ(7)-Sterols.

    PubMed

    Münger, Linda H; Jutzi, Sabrina; Lampi, Anna-Maija; Nyström, Laura

    2015-08-01

    In this study, we present the difference in sterol composition of extracted steryl glycosides (SG) hydrolyzed by either enzymatic or acid hydrolysis. SG were analyzed from foods belonging to the plant families Cucurbitaceae (melon and pumpkin seeds) and Amaranthaceae (amaranth and beetroot), both of which are dominated by Δ(7)-sterols. Released sterols were quantified by gas chromatography with a flame ionization detector (GC-FID) and identified using gas chromatography/mass spectrometry (GC-MS). All Δ(7)-sterols identified (Δ(7)-stigmastenyl, spinasteryl, Δ(7)-campesteryl, Δ(7)-avenasteryl, poriferasta-7,25-dienyl and poriferasta-7,22,25-trienyl glucoside) underwent isomerization under acidic conditions and high temperature. Sterols with an ethylidene or methylidene side chain were found to form multiple artifacts. The artifact sterols coeluted with residues of incompletely isomerized Δ(7)-sterols, or Δ(5)-sterols if present, and could be identified as Δ(8(14))-sterols on the basis of relative retention time, and their MS spectra as trimethylsilyl (TMS) and acetate derivatives. For instance, SG from melon were composed of 66% Δ(7)-stigmastenol when enzymatic hydrolysis was performed, whereas with acid hydrolysis only 8% of Δ(7)-stigmastenol was determined. The artifact of Δ(7)-stigmastenol coeluted with residual non-isomerized spinasterol, demonstrating the high risk of misinterpretation of compositional data obtained after acid hydrolysis. Therefore, the accurate composition of SG from foods containing sterols with a double bond at C-7 can only be obtained by enzymatic hydrolysis or by direct analysis of the intact SG.

  8. Feasibility of pressurization to speed up enzymatic hydrolysis of biological materials for multielement determinations.

    PubMed

    Moreda-Piñeiro, Antonio; Bermejo-Barrera, Adela; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Jorge; Alonso-Rodríguez, Elia; Muniategui-Lorenzo, Soledad; López-Mahía, Purificación; Prada-Rodríguez, Darío

    2007-03-01

    The feasibility of pressurized solvents (liquids at a high pressure and/or high temperature without the subcritical point being reached) has been newly investigated to accelerate enzymatic hydrolysis processes of mussel tissue for multielement determinations. The target elements (Al, As, Cd, Co, Cu, Fe, Hg, Li, Mn, Pb, Se, Sr, V, and Zn) were released from dried mussel tissue by action of two proteases (pepsin and pancreatin), and they have been evaluated by inductively coupled plasma optical emission spectrometry (ICP-OES). Variables inherent to the enzymatic activity (pH, ionic strength, temperature, and enzyme mass) and factors affecting pressurization (static time, pressure, and number of cycles) were simultaneously studied by applying a Plackett-Burman design (PBD) as the screening method. Results showed that pH, ionic strength, and temperature were the most statistically significant factors (confidence interval of 95%) under pressurized conditions for pepsin, while pH and ionic strength affected pancreatin activity. This means that metal extraction is mostly attributed to enzymatic activity. The static time (enzymatic hydrolysis time) was found statistically nonsignificant for most of the elements, meaning that the hydrolysis procedure can be finished within a 2-15 min range. For pepsin, optimized conditions (pH 1.0, temperature 40 degrees C, pressure 1500 psi, static time 2 min, and number of cycles 3) gave quantitative extractions for As, Cd, Co, Cu, Hg, Li, Mn, Pb, Se, Sr, V, and Zn. The pepsin mass was 0.05 g, and the solution was Milli-Q water at pH 1.0 (adjusted with hydrochloric acid). For pancreatin, quantitative recoveries were only reached for As, Cd, Cu, Li, Pb, and Sr at room temperature, at a pressure of 1500 psi, for a static time of 2 min and a number of cycles of 3. The extraction solution was a 0.3 M potassium dihydrogen phosphate/potassium hydrogen phosphate buffer at a pH of 7.5 working at room temperature. Around 0.5 g of diatomaceous

  9. Accelerators Beyond The Tevatron?

    SciTech Connect

    Lach, Joseph; /Fermilab

    2010-07-01

    Following the successful operation of the Fermilab superconducting accelerator three new higher energy accelerators were planned. They were the UNK in the Soviet Union, the LHC in Europe, and the SSC in the United States. All were expected to start producing physics about 1995. They did not. Why?

  10. Accelerators Beyond The Tevatron?

    SciTech Connect

    Lach, Joseph

    2010-07-29

    Following the successful operation of the Fermilab superconducting accelerator three new higher energy accelerators were planned. They were the UNK in the Soviet Union, the LHC in Europe, and the SSC in the United States. All were expected to start producing physics about 1995. They did not. Why?.

  11. Accelerators (4/5)

    ScienceCinema

    None

    2016-07-12

    1a) Introduction and motivation 1b) History and accelerator types 2) Transverse beam dynamics 3a) Longitudinal beam dynamics 3b) Figure of merit of a synchrotron/collider 3c) Beam control 4) Main limiting factors 5) Technical challenges Prerequisite knowledge: Previous knowledge of accelerators is not required.

  12. Induction linear accelerators

    NASA Astrophysics Data System (ADS)

    Birx, Daniel

    1992-03-01

    Among the family of particle accelerators, the Induction Linear Accelerator is the best suited for the acceleration of high current electron beams. Because the electromagnetic radiation used to accelerate the electron beam is not stored in the cavities but is supplied by transmission lines during the beam pulse it is possible to utilize very low Q (typically<10) structures and very large beam pipes. This combination increases the beam breakup limited maximum currents to of order kiloamperes. The micropulse lengths of these machines are measured in 10's of nanoseconds and duty factors as high as 10-4 have been achieved. Until recently the major problem with these machines has been associated with the pulse power drive. Beam currents of kiloamperes and accelerating potentials of megavolts require peak power drives of gigawatts since no energy is stored in the structure. The marriage of liner accelerator technology and nonlinear magnetic compressors has produced some unique capabilities. It now appears possible to produce electron beams with average currents measured in amperes, peak currents in kiloamperes and gradients exceeding 1 MeV/meter, with power efficiencies approaching 50%. The nonlinear magnetic compression technology has replaced the spark gap drivers used on earlier accelerators with state-of-the-art all-solid-state SCR commutated compression chains. The reliability of these machines is now approaching 1010 shot MTBF. In the following paper we will briefly review the historical development of induction linear accelerators and then discuss the design considerations.

  13. Accelerators (3/5)

    ScienceCinema

    None

    2016-07-12

    1a) Introduction and motivation 1b) History and accelerator types 2) Transverse beam dynamics 3a) Longitudinal beam dynamics 3b) Figure of merit of a synchrotron/collider 3c) Beam control 4) Main limiting factors 5) Technical challenges Prerequisite knowledge: Previous knowledge of accelerators is not required.

  14. Accelerators (5/5)

    ScienceCinema

    None

    2016-07-12

    1a) Introduction and motivation 1b) History and accelerator types 2) Transverse beam dynamics 3a) Longitudinal beam dynamics 3b) Figure of merit of a synchrotron/collider 3c) Beam control 4) Main limiting factors 5) Technical challenges Prerequisite knowledge: Previous knowledge of accelerators is not required.

  15. Accelerators (5/5)

    SciTech Connect

    2009-07-09

    1a) Introduction and motivation 1b) History and accelerator types 2) Transverse beam dynamics 3a) Longitudinal beam dynamics 3b) Figure of merit of a synchrotron/collider 3c) Beam control 4) Main limiting factors 5) Technical challenges Prerequisite knowledge: Previous knowledge of accelerators is not required.

  16. Accelerators (4/5)

    SciTech Connect

    2009-07-08

    1a) Introduction and motivation 1b) History and accelerator types 2) Transverse beam dynamics 3a) Longitudinal beam dynamics 3b) Figure of merit of a synchrotron/collider 3c) Beam control 4) Main limiting factors 5) Technical challenges Prerequisite knowledge: Previous knowledge of accelerators is not required.

  17. Accelerators (3/5)

    SciTech Connect

    2009-07-07

    1a) Introduction and motivation 1b) History and accelerator types 2) Transverse beam dynamics 3a) Longitudinal beam dynamics 3b) Figure of merit of a synchrotron/collider 3c) Beam control 4) Main limiting factors 5) Technical challenges Prerequisite knowledge: Previous knowledge of accelerators is not required.

  18. Ion Induction Accelerators

    NASA Astrophysics Data System (ADS)

    Barnard, John J.; Horioka, Kazuhiko

    The description of beams in RF and induction accelerators share many common features. Likewise, there is considerable commonality between electron induction accelerators (see Chap. 7) and ion induction accelerators. However, in contrast to electron induction accelerators, there are fewer ion induction accelerators that have been operated as application-driven user facilities. Ion induction accelerators are envisioned for applications (see Chap. 10) such as Heavy Ion Fusion (HIF), High Energy Density Physics (HEDP), and spallation neutron sources. Most ion induction accelerators constructed to date have been limited scale facilities built for feasibility studies for HIF and HEDP where a large numbers of ions are required on target in short pulses. Because ions are typically non-relativistic or weakly relativistic in much of the machine, space-charge effects can be of crucial importance. This contrasts the situation with electron machines, which are usually strongly relativistic leading to weaker transverse space-charge effects and simplified longitudinal dynamics. Similarly, the bunch structure of ion induction accelerators relative to RF machines results in significant differences in the longitudinal physics.

  19. Particle Acceleration in Jets

    NASA Technical Reports Server (NTRS)

    Nishikawa, Ken-Ichi

    2005-01-01

    Nonthermal radiation observed from astrophysical systems containing relativistic jets and shocks, e.g., active galactic nuclei (AGNs), gamma ray burst (GRBs), and Galactic microquasar systems usually have power-law emission spectra. Fermi acceleration is the mechanism usually assumed for the acceleration of particles in astrophysical environments.

  20. Microscale acceleration history discriminators

    DOEpatents

    Polosky, Marc A.; Plummer, David W.

    2002-01-01

    A new class of micromechanical acceleration history discriminators is claimed. These discriminators allow the precise differentiation of a wide range of acceleration-time histories, thereby allowing adaptive events to be triggered in response to the severity (or lack thereof) of an external environment. Such devices have applications in airbag activation, and other safety and surety applications.

  1. Diagnostics for induction accelerators

    SciTech Connect

    Fessenden, T.J.

    1996-04-01

    The induction accelerator was conceived by N. C. Christofilos and first realized as the Astron accelerator that operated at LLNL from the early 1960`s to the end of 1975. This accelerator generated electron beams at energies near 6 MeV with typical currents of 600 Amperes in 400 ns pulses. The Advanced Test Accelerator (ATA) built at Livermore`s Site 300 produced 10,000 Ampere beams with pulse widths of 70 ns at energies approaching 50 MeV. Several other electron and ion induction accelerators have been fabricated at LLNL and LBNL. This paper reviews the principal diagnostics developed through efforts by scientists at both laboratories for measuring the current, position, energy, and emittance of beams generated by these high current, short pulse accelerators. Many of these diagnostics are closely related to those developed for other accelerators. However, the very fast and intense current pulses often require special diagnostic techniques and considerations. The physics and design of the more unique diagnostics developed for electron induction accelerators are presented and discussed in detail.

  2. KEK digital accelerator

    NASA Astrophysics Data System (ADS)

    Iwashita, T.; Adachi, T.; Takayama, K.; Leo, K. W.; Arai, T.; Arakida, Y.; Hashimoto, M.; Kadokura, E.; Kawai, M.; Kawakubo, T.; Kubo, Tomio; Koyama, K.; Nakanishi, H.; Okazaki, K.; Okamura, K.; Someya, H.; Takagi, A.; Tokuchi, A.; Wake, M.

    2011-07-01

    The High Energy Accelerator Research Organization KEK digital accelerator (KEK-DA) is a renovation of the KEK 500 MeV booster proton synchrotron, which was shut down in 2006. The existing 40 MeV drift tube linac and rf cavities have been replaced by an electron cyclotron resonance (ECR) ion source embedded in a 200 kV high-voltage terminal and induction acceleration cells, respectively. A DA is, in principle, capable of accelerating any species of ion in all possible charge states. The KEK-DA is characterized by specific accelerator components such as a permanent magnet X-band ECR ion source, a low-energy transport line, an electrostatic injection kicker, an extraction septum magnet operated in air, combined-function main magnets, and an induction acceleration system. The induction acceleration method, integrating modern pulse power technology and state-of-art digital control, is crucial for the rapid-cycle KEK-DA. The key issues of beam dynamics associated with low-energy injection of heavy ions are beam loss caused by electron capture and stripping as results of the interaction with residual gas molecules and the closed orbit distortion resulting from relatively high remanent fields in the bending magnets. Attractive applications of this accelerator in materials and biological sciences are discussed.

  3. The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle

    DOE PAGES

    Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

    2015-02-27

    Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNAmore » transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.« less

  4. The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle

    SciTech Connect

    Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

    2015-02-27

    Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.

  5. GTP dysregulation in Bacillus subtilis cells lacking (p)ppGpp results in phenotypic amino acid auxotrophy and failure to adapt to nutrient downshift and regulate biosynthesis genes.

    PubMed

    Kriel, Allison; Brinsmade, Shaun R; Tse, Jessica L; Tehranchi, Ashley K; Bittner, Alycia N; Sonenshein, Abraham L; Wang, Jue D

    2014-01-01

    The nucleotide (p)ppGpp inhibits GTP biosynthesis in the Gram-positive bacterium Bacillus subtilis. Here we examined how this regulation allows cells to grow in the absence of amino acids. We showed that B. subtilis cells lacking (p)ppGpp, due to either deletions or point mutations in all three (p)ppGpp synthetase genes, yjbM, ywaC, and relA, strongly require supplementation of leucine, isoleucine, valine, methionine, and threonine and modestly require three additional amino acids. This polyauxotrophy is rescued by reducing GTP levels. Reduction of GTP levels activates transcription of genes responsible for the biosynthesis of the five strongly required amino acids by inactivating the transcription factor CodY, which represses the ybgE, ilvD, ilvBHC-leuABCD, ilvA, ywaA, and hom-thrCB operons, and by a CodY-independent activation of transcription of the ilvA, ywaA, hom-thrCB, and metE operons. Interestingly, providing the eight required amino acids does not allow for colony formation of (p)ppGpp(0) cells when transitioning from amino acid-replete medium to amino acid-limiting medium, and we found that this is due to an additional role that (p)ppGpp plays in protecting cells during nutrient downshifts. We conclude that (p)ppGpp allows adaptation to amino acid limitation by a combined effect of preventing death during metabolic transitions and sustaining growth by activating amino acid biosynthesis. This ability of (p)ppGpp to integrate a general stress response with a targeted reprogramming of gene regulation allows appropriate adaptation and is likely conserved among diverse bacteria.

  6. The cysteine-rich region of raf-1 kinase contains zinc, translocates to liposomes, and is adjacent to a segment that binds GTP-ras.

    PubMed

    Ghosh, S; Xie, W Q; Quest, A F; Mabrouk, G M; Strum, J C; Bell, R M

    1994-04-01

    Different domains of the serine/threonine kinase, raf-1, were expressed as fusion proteins with glutathione S-transferase (GST) in Escherichia coli and purified to near homogeneity by affinity chromatography. A cysteine-rich domain of raf-1 was found to contain 2 mol of zinc (molar basis), similar to analogous cysteine-rich domains of protein kinase C. GST-fusion proteins, containing the cysteine-rich domain of raf-1, bound to liposomes in a phosphatidylserine-dependent manner. In contrast to protein kinase C, the translocation of raf-1 was not dependent upon diacylglycerol, phorbol ester, or calcium, nor did raf-1 bind phorbol esters. A GST-fusion protein encoding residues 1-147 of raf-1 bound to normal GTP-ras with high affinity, but not to mutant GTP-Ala35 ras; no binding was detected to GDP-ras. The binding of a smaller fusion protein (residues 1-130 of raf-1) was about 10-fold weaker, inferring that a 17-amino acid sequence represents a critical binding determinant in intact raf-1. These residues are adjacent to the amino-terminal end of, and partially extend into, the cysteine-rich domain (amino acids 139-184). A synthetic peptide corresponding to this 17-amino acid sequence blocked the interaction of raf-1 with ras. The function of the cysteine-rich region of raf-1 homologous to protein kinase C is to promote translocation of raf-1 kinase to membranes and to form part of the high affinity binding site for GTP-ras.

  7. GTP-dependent association of Raf-1 with Ha-Ras: identification of Raf as a target downstream of Ras in mammalian cells.

    PubMed Central

    Koide, H; Satoh, T; Nakafuku, M; Kaziro, Y

    1993-01-01

    Ras is involved in signal transduction of various factors for growth, differentiation, and oncogenesis. Recent studies have revealed several proteins that function upstream and downstream of the Ras signaling pathway. However, its immediate downstream target molecular has not yet been identified. In an effort to identify the Ras-associated downstream proteins, we added recombinant Ha-Ras in a GTP-bound form to cell-free lysates and used several antibodies against Ras to immunoprecipitate Ras complexes. We found that a serine/threonine kinase, Raf-1, was coimmunoprecipitated with Ha-Ras by two anti-Ras antibodies (LA069 and Y13-238), whereas a neutralizing antibody against Ras (Y13-259) could not precipitate Raf-1. The coimmunoprecipitation was observed with a complex of Ras and guanosine 5'-[gamma- thio]triphosphate but not with a complex of Ras and guanosine 5'-[beta-thio]diphosphate. The GTP-dependent association of Ha-Ras with Raf-1 was observed with lysates of various types of cultured cells, including NIH 3T3, pheochromocytoma (PC) 12, Ba/F3, and Jurkat T cells, and also with crude extracts from rat brain. Furthermore, Raf-1 was precipitated with a transforming Ha-Ras mutant ([Val12]Ras) and wild-type Ha-Ras but not with an effector-region mutant ([Leu35,ARg37]Ras) that lacks transforming activity. These results indicate that Ras.GTP physically associates with Raf either directly or through other component(s) and strongly suggest that Raf functions in close downstream proximity to Ras in mammalian cells. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8378348

  8. NADPH oxidase-mediated Rac1 GTP activity is necessary for nongenomic actions of the mineralocorticoid receptor in the CA1 region of the rat hippocampus.

    PubMed

    Kawakami-Mori, Fumiko; Shimosawa, Tatsuo; Mu, Shengyu; Wang, Hong; Ogura, Sayoko; Yatomi, Yutaka; Fujita, Toshiro

    2012-02-15

    Mineralocorticoid receptors (MRs) in the central nervous system play important roles in spatial memory, fear memory, salt sensitivity, and hypertension. Corticosterone binds to MRs to induce presynaptic vesicle release and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor aggregation, which are necessary for induction of long-term potentiation under psychological stress. On the other hand, cognitive dysfunction is an important problem clinically in patients with hypertension, diabetes, and cerebral infarction, and all of these conditions are associated with an increase in reactive oxygen species (ROS) generation. Oxidative stress has been shown to modify the genomic actions of MRs in the peripheral organs; however, there have been no reports until now about the relation between the nongenomic actions of MRs and ROS in the central nervous system. In this study, we investigated the relationship between ROS and the nongenomic actions of MR. We examined the nongenomic actions of MR by measuring the slope of the field excitatory postsynaptic potentials and found that ROS induced an additive increase of these potentials, which was accompanied by Rac1 GTP activation and ERK1/2 phosphorylation. An NADPH oxidase inhibitor, apocynin, blocked the nongenomic actions of MRs. A Rac1 inhibitor, NSC23766, was also found to block synaptic enhancement and ERK1/2 phosphorylation induced by NADPH and corticosterone. We concluded that NADPH oxidase activity and Rac1 GTP activity are indispensable for the nongenomic actions of MRs and that Rac1 GTP activation induces ERK1/2 phosphorylation in the brain.

  9. The Interaction of RNA Helicase DDX3 with HIV-1 Rev-CRM1-RanGTP Complex during the HIV Replication Cycle

    PubMed Central

    Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

    2015-01-01

    Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP. PMID:25723178

  10. Reaction kinetics of cellulose hydrolysis in subcritical and supercritical water

    NASA Astrophysics Data System (ADS)

    Olanrewaju, Kazeem Bode

    The uncertainties in the continuous supply of fossil fuels from the crisis-ridden oil-rich region of the world is fast shifting focus on the need to utilize cellulosic biomass and develop more efficient technologies for its conversion to fuels and chemicals. One such technology is the rapid degradation of cellulose in supercritical water without the need for an enzyme or inorganic catalyst such as acid. This project focused on the study of reaction kinetics of cellulose hydrolysis in subcritical and supercritical water. Cellulose reactions at hydrothermal conditions can proceed via the homogeneous route involving dissolution and hydrolysis or the heterogeneous path of surface hydrolysis. The work is divided into three main parts. First, the detailed kinetic analysis of cellulose reactions in micro- and tubular reactors was conducted. Reaction kinetics models were applied, and kinetics parameters at both subcritical and supercritical conditions were evaluated. The second major task was the evaluation of yields of water soluble hydrolysates obtained from the hydrolysis of cellulose and starch in hydrothermal reactors. Lastly, changes in molecular weight distribution due to hydrothermolytic degradation of cellulose were investigated. These changes were also simulated based on different modes of scission, and the pattern generated from simulation was compared with the distribution pattern from experiments. For a better understanding of the reaction kinetics of cellulose in subcritical and supercritical water, a series of reactions was conducted in the microreactor. Hydrolysis of cellulose was performed at subcritical temperatures ranging from 270 to 340 °C (tau = 0.40--0.88 s). For the dissolution of cellulose, the reaction was conducted at supercritical temperatures ranging from 375 to 395 °C (tau = 0.27--0.44 s). The operating pressure for the reactions at both subcritical and supercritical conditions was 5000 psig. The results show that the rate-limiting step in

  11. Endotoxemia alters nucleotide hydrolysis in platelets of rats.

    PubMed

    Vuaden, Fernanda Cenci; Furstenau, Cristina Ribas; Savio, Luiz Eduardo Baggio; Sarkis, João José Freitas; Bonan, Carla Denise

    2009-03-01

    Platelets play a critical role in homeostasis and blood clotting at sites of vascular injury, and also in various ways in innate immunity and inflammation. Platelets are one of the first cells to accumulate at an injured site, and local release of their secretome at some point initiate an inflammatory cascade that attracts leukocytes, activates target cells, stimulates vessel growth and repair. The level of exogenous ATP in the body may be increased in various inflammatory and shock conditions, primarily as a consequence of nucleotide release from platelets, endothelium and blood vessel cells. An increase of ATP release has been described during inflammation and this compound presents proinflammatory properties. ADP is a nucleotide known to induce changes in platelets shape and aggregation, to promote the exposure of fibrinogen-binding sites and to inhibit the stimulation of adenylate cyclase. Adenosine, the final product of the nucleotide hydrolysis, is a vasodilator and an inhibitor of platelet aggregation. There is a group of ecto-enzymes responsible for extracellular nucleotide hydrolysis named ectonucleotidases, which includes the NTPDase (nucleoside triphosphate diphosphohydrolase) family, the NPP (nucleoside pyrophosphatase/phosphodiesterase) family and an ecto-5'-nucleotidase. Therefore, we have aimed to investigate the effect of lipopolysaccharide endotoxin from Escherichia coli on ectonucleotidases in platelets from adult rats in order to better understand the role of extracellular adenine nucleotides and nucleosides in the maintenance of blood homeostasis in inflammatory processes. LPS administered in vitro was not able to alter the ATP, ADP, AMP and rho-Nph-5'-TMP hydrolysis of platelets from untreated rats in all concentrations tested (25-100 microg/ml). There was a significant decrease in ATP, ADP, AMP and rho-Nph-5'-TMP hydrolysis in rat platelets after 48 hours of LPS exposure (2 mg/Kg, i.p.). ATP and ADP hydrolysis has been reduced about 28

  12. Switching Catalysis from Hydrolysis to Perhydrolysis in Pseudomonas fluorescens Esterase

    SciTech Connect

    Yin, D.; Bernhardt, P; Morley, K; Jiang, Y; Cheeseman, J; Purpero, V; Schrag, J; Kazlauskas, R

    2010-01-01

    Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of {var_epsilon}-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen peroxide, occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed 2-fold higher k{sub cat}, but K{sub m} also increased so the specificity constant, k{sub cat}/K{sub m}, remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate) but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of {var_epsilon}-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the

  13. Controllable Laser Ion Acceleration

    NASA Astrophysics Data System (ADS)

    Kawata, S.; Kamiyama, D.; Ohtake, Y.; Takano, M.; Barada, D.; Kong, Q.; Wang, P. X.; Gu, Y. J.; Wang, W. M.; Limpouch, J.; Andreev, A.; Bulanov, S. V.; Sheng, Z. M.; Klimo, O.; Psikal, J.; Ma, Y. Y.; Li, X. F.; Yu, Q. S.

    2016-02-01

    In this paper a future laser ion accelerator is discussed to make the laser-based ion accelerator compact and controllable. Especially a collimation device is focused in this paper. The future laser ion accelerator should have an ion source, ion collimators, ion beam bunchers, and ion post acceleration devices [Laser Therapy 22, 103(2013)]: the ion particle energy and the ion energy spectrum are controlled to meet requirements for a future compact laser ion accelerator for ion cancer therapy or for other purposes. The energy efficiency from the laser to ions is improved by using a solid target with a fine sub-wavelength structure or a near-critical density gas plasma. The ion beam collimation is performed by holes behind the solid target or a multi-layered solid target. The control of the ion energy spectrum and the ion particle energy, and the ion beam bunching would be successfully realized by a multistage laser-target interaction.

  14. Cascaded radiation pressure acceleration

    SciTech Connect

    Pei, Zhikun; Shen, Baifei E-mail: zhxm@siom.ac.cn; Zhang, Xiaomei E-mail: zhxm@siom.ac.cn; Wang, Wenpeng; Zhang, Lingang; Yi, Longqing; Shi, Yin; Xu, Zhizhan

    2015-07-15

    A cascaded radiation-pressure acceleration scheme is proposed. When an energetic proton beam is injected into an electrostatic field moving at light speed in a foil accelerated by light pressure, protons can be re-accelerated to much higher energy. An initial 3-GeV proton beam can be re-accelerated to 7 GeV while its energy spread is narrowed significantly, indicating a 4-GeV energy gain for one acceleration stage, as shown in one-dimensional simulations and analytical results. The validity of the method is further confirmed by two-dimensional simulations. This scheme provides a way to scale proton energy at the GeV level linearly with laser energy and is promising to obtain proton bunches at tens of gigaelectron-volts.

  15. Accelerators, Beams And Physical Review Special Topics - Accelerators And Beams

    SciTech Connect

    Siemann, R.H.; /SLAC

    2011-10-24

    Accelerator science and technology have evolved as accelerators became larger and important to a broad range of science. Physical Review Special Topics - Accelerators and Beams was established to serve the accelerator community as a timely, widely circulated, international journal covering the full breadth of accelerators and beams. The history of the journal and the innovations associated with it are reviewed.

  16. Influence of protein hydrolysis on the growth kinetics of β-lg fibrils.

    PubMed

    Kroes-Nijboer, Ardy; Venema, Paul; Bouman, Jacob; van der Linden, Erik

    2011-05-17

    Recently it was found that protein hydrolysis is an important step in the formation of β-lactoglobulin fibrils at pH 2 and elevated temperatures. The objective of the present study was to further investigate the influence of hydrolysis on the kinetics of fibril formation. Both the hydrolysis of β-lactoglobulin and the growth of the fibrils were followed as a function of time and temperature, using SDS polyacrylamide gel electrophoresis and a Thioflavin T fluorescence assay. As an essential extension to existing models, the quantification of the effect of the hydrolysis on the fibrillar growth was established by a simple polymerization model including a hydrolysis step.

  17. Investigation of the Polymorphs and Hydrolysis of Uranium Trioxide

    SciTech Connect

    Sweet, Lucas E.; Blake, Thomas A.; Henager, Charles H.; Hu, Shenyang Y.; Johnson, Timothy J.; Meier, David E.; Peper, Shane M.; Schwantes, Jon M.

    2013-04-01

    This work focuses on progress in gaining a better understanding of the polymorphic nature of the UO3-water system, one of several important materials associated with the nuclear fuel cycle. The UO3-water system is complex and has not been fully characterized, even though these species are common throughout the fuel cycle. Powder x-ray diffraction, Raman and fluorescence characterization was performed on polymorphic forms of UO3 and UO3 hydrolysis products for the purpose of developing some predictive capability of estimating process history and utility, e.g. for polymorphic phases of unknown origin. Specifically, we have investigated three industrially relevant production pathways of UO3 and discovered a previously unknown low temperature route to β-UO3. Pure phases of UO3, hydrolysis products and starting materials were used to establish optical spectroscopic signatures for these compounds.

  18. Catalysis of a Flavoenzyme-Mediated Amide Hydrolysis

    SciTech Connect

    Mukherjee, Tathagata; Zhang, Yang; Abdelwahed, Sameh; Ealick, Steven E.; Begley, Tadhg P.

    2010-09-13

    A new pyrimidine catabolic pathway (the Rut pathway) was recently discovered in Escherichia coli K12. In this pathway, uracil is converted to 3-hydroxypropionate, ammonia, and carbon dioxide. The seven-gene Rut operon is required for this conversion. Here we demonstrate that the flavoenzyme RutA catalyzes the initial uracil ring-opening reaction to give 3-ureidoacrylate. This reaction, while formally a hydrolysis reaction, proceeds by an oxidative mechanism initiated by the addition of a flavin hydroperoxide to the C4 carbonyl. While peroxide-catalyzed amide hydrolysis has chemical precedent, we are not aware of a prior example of analogous chemistry catalyzed by flavin hydroperoxides. This study further illustrates the extraordinary catalytic versatility of the flavin cofactor.

  19. Hydrolysis kinetics of tulip tree xylan in hot compressed water.

    PubMed

    Yoon, Junho; Lee, Hun Wook; Sim, Seungjae; Myint, Aye Aye; Park, Hee Jeong; Lee, Youn-Woo

    2016-08-01

    Lignocellulosic biomass, a promising renewable resource, can be converted into numerous valuable chemicals post enzymatic saccharification. However, the efficacy of enzymatic saccharification of lignocellulosic biomass is low; therefore, pretreatment is necessary to improve the efficiency. Here, a kinetic analysis was carried out on xylan hydrolysis, after hot compressed water pretreatment of the lignocellulosic biomass conducted at 180-220°C for 5-30min, and on subsequent xylooligosaccharide hydrolysis. The weight ratio of fast-reacting xylan to slow-reacting xylan was 5.25 in tulip tree. Our kinetic results were applied to three different reaction systems to improve the pretreatment efficiency. We found that semi-continuous reactor is promising. Lower reaction temperatures and shorter space times in semi-continuous reactor are recommended for improving xylan conversion and xylooligosaccharide yield. In the theoretical calculation, 95% of xylooligosaccharide yield and xylan conversion were achieved simultaneously with high selectivity (desired product/undesired product) of 100 or more.

  20. Enzymatic hydrolysis of cellulosic materials: a kinetic study

    SciTech Connect

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

    1984-01-01

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

  1. Role of bifidobacteria in the hydrolysis of chlorogenic acid

    PubMed Central

    Raimondi, Stefano; Anighoro, Andrew; Quartieri, Andrea; Amaretti, Alberto; Tomás-Barberán, Francisco A; Rastelli, Giulio; Rossi, Maddalena

    2015-01-01

    This study aimed to explore the capability of potentially probiotic bifidobacteria to hydrolyze chlorogenic acid into caffeic acid (CA), and to recognize the enzymes involved in this reaction. Bifidobacterium strains belonging to eight species occurring in the human gut were screened. The hydrolysis seemed peculiar of Bifidobacterium animalis, whereas the other species failed to release CA. Intracellular feruloyl esterase activity capable of hydrolyzing chlorogenic acid was detected only in B. animalis. In silico research among bifidobacteria esterases identified Balat_0669 as the cytosolic enzyme likely responsible of CA release in B. animalis. Comparative modeling of Balat_0669 and molecular docking studies support its role in chlorogenic acid hydrolysis. Expression, purification, and functional characterization of Balat_0669 in Escherichia coli were obtained as further validation. A possible role of B. animalis in the activation of hydroxycinnamic acids was demonstrated and new perspectives were opened in the development of new probiotics, specifically selected for the enhanced bioconversion of phytochemicals into bioactive compounds. PMID:25515139

  2. Novel agents for enzymatic and fungal hydrolysis of stevioside

    PubMed Central

    Milagre, H.M.S.; Martins, L.R.; Takahashi, J.A.

    2009-01-01

    A comparative study on the potential of some biological agents to perform the hydrolysis of stevioside was carried out, aiming at establishing an alternative methodology to achieve the aglycon steviol or its rearranged derivative isosteviol, in high yields to be used in the preparation of novel bioactive compounds. Hydrolysis reactions were performed by using filamentous fungi (Aspergillus niger, Rhizopus stolonifer and Rhizopus arrhizus), a yeast (Saccharomyces cerevisiae) and enzymes (pancreatin and lipases PL250 and VFL 8000). Pancreatin showed the best hydrolytic activity, furnishing isosteviol at 93.9% of yield, at pH 4.0, using toluene as a co-solvent. Steviol was produced using both pancreatin at pH 7.0 (20.2% yield) and A. niger at pH 7 (20.8% yield). PMID:24031374

  3. Simultaneous pretreatment and enzymatic hydrolysis of forage biomass

    SciTech Connect

    Henk, L.; Linden, J.C.

    1993-12-31

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

  4. Enzymatic hydrolysis and fermentation of agricultural residues to ethanol

    SciTech Connect

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

    1984-01-01

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

  5. Enzymatic hydrolysis of corn stover process development and evaluation

    SciTech Connect

    Perez, J.; Wilke, C.R.; Blanch, H.W.

    1981-12-01

    The hydrolysis of acid treated corn stover with cellulase from T. reesei Rut-C-30 was evaluated. Experiments were conducted with substrate concentrations of 5 to 25% by weight, enzyme activities of 0.5 to 7 IU/ml and residence times of 24 to 48 hours. Maximum conversion was 55% for specific cellulase activity of 25 to 30 IU/g. Optimum cellobiase activity for minimum cellobiose production was found to be approximately 1.8 cellobiase units to 1 FPA unit. Hydrolysis experiments with steam exploded corn stover led to a maximum conversion of 80%, significantly higher than the results obtained for acid treated substrate. Steam exploded corn stover was demonstrated as a suitable carbon source for growth of T. reesei in submerged cultures.

  6. Alcohol fermentation of sweet potato. Membrane reactor in enzymatic hydrolysis

    SciTech Connect

    Azhar, A.; Hamdy, M.K.

    1981-06-01

    Use of ultrafiltration membrane systems in stirred cell and in thin-channel systems for immobilizing enzyme (sweet potato intrinsic and crystalline /beta/-amylase) in hydrolysis of sweet potato through a continuous operation mode were studied. Both the filtration rate and reducing sugars, produced as the result of enzymatic hydrolysis, decreased with the filtration time. THe immobilized enzymes in the thin-channel system showed a much better performance compared to that in the stirred cell system. Addition of crystalline sweet potato /beta/-amylase to the sweet potato increased both the filtration rate and reducing-sugars content. Alcoholic fermentation of the filtrate resulted in an alcohol content of 4.2%. This represented fermentation of 95% of the sugars with an efficiency of 88%. 17 refs.

  7. Alcohol fermentation of sweet potato. Membrane reactor in enzymic hydrolysis

    SciTech Connect

    Azhar, A.; Hamdy, M.K.

    1981-01-01

    Use of ultrafiltration membrane systems in stirred cell and in thin-channel systems for immobilizing enzyme (sweet potato intrinsic and crystalline beta-amylase) in hydrolysis of sweet potato through a continuous operation mode were studied. Both the filtration rate and reducing sugars, produced as the result of enzymic hydrolysis, decreased with the filtration time. The immobilized enzymes in the thin-channel system showed a much better performance compared to that in the stirred cell system. Addition of crystalline sweet potato beta-amylase to the sweet potato increased both the filtration rate and reducing-sugars content. Alcohol fermentation of the filtrate resulted in an alcohol content of 4.2%. This represented fermentation of 95% of the sugars with an efficiency of 88%.

  8. Snapshots of the maltose transporter during ATP hydrolysis

    SciTech Connect

    Oldham, Michael L.; Chen, Jue

    2011-12-05

    ATP-binding cassette transporters are powered by ATP, but the mechanism by which these transporters hydrolyze ATP is unclear. In this study, four crystal structures of the full-length wild-type maltose transporter, stabilized by adenosine 5{prime}-({beta},{gamma}-imido)triphosphate or ADP in conjunction with phosphate analogs BeF{sub 3}{sup -}, VO{sub 4}{sup 3-}, or AlF{sub 4}{sup -}, were determined to 2.2- to 2.4-{angstrom} resolution. These structures led to the assignment of two enzymatic states during ATP hydrolysis and demonstrate specific functional roles of highly conserved residues in the nucleotide-binding domain, suggesting that ATP-binding cassette transporters catalyze ATP hydrolysis via a general base mechanism.

  9. Large electrostatic accelerators

    SciTech Connect

    Jones, C.M.

    1984-01-01

    The increasing importance of energetic heavy ion beams in the study of atomic physics, nuclear physics, and materials science has partially or wholly motivated the construction of a new generation of large electrostatic accelerators designed to operate at terminal potentials of 20 MV or above. In this paper, the author briefly discusses the status of these new accelerators and also discusses several recent technological advances which may be expected to further improve their performance. The paper is divided into four parts: (1) a discussion of the motivation for the construction of large electrostatic accelerators, (2) a description and discussion of several large electrostatic accelerators which have been recently completed or are under construction, (3) a description of several recent innovations which may be expected to improve the performance of large electrostatic accelerators in the future, and (4) a description of an innovative new large electrostatic accelerator whose construction is scheduled to begin next year. Due to time and space constraints, discussion is restricted to consideration of only tandem accelerators.

  10. Analyzing radial acceleration with a smartphone acceleration sensor

    NASA Astrophysics Data System (ADS)

    Vogt, Patrik; Kuhn, Jochen

    2013-03-01

    This paper continues the sequence of experiments using the acceleration sensor of smartphones (for description of the function and the use of the acceleration sensor, see Ref. 1) within this column, in this case for analyzing the radial acceleration.

  11. Fructan Hydrolysis Drives Petal Expansion in the Ephemeral Daylily Flower.

    PubMed

    Bieleski, R. L.

    1993-09-01

    Dry weight, water content, soluble carbohydrate content, and carbohydrate composition of daylily (Hemerocallis hybrid cv Cradle Song) flower petals were monitored in the 3 d leading up to full opening and in the first day of senescence. Timing of events was related to the time (hour 0) when flower expansion was 60% complete. Petal dry weight increased linearly from hour -62 (tight bud) to hour 10 (fully developed flower), then fell rapidly to hour 34 as senescence advanced. Increase in water content was proportional to dry weight increase from hour -62 to hour -14, but was more rapid as the bud cracked and the flower opened, giving an increase in fresh weight/dry weight ratio. Soluble carbohydrate was 50% of petal dry weight up to hour 10, then decreased during senescence to reach 4% by hour 34. Up until hour -14, fructan accounted for 80% of the soluble carbohydrate in the petals, whereas hexose accounted for only 2%. Fructan hydrolysis started just prior to bud crack at hour -14, reaching completion by hour 10 when no detectable fructan remained, and fructose plus glucose accounted for more than 80% of the total soluble carbohydrate. The proportion of sucrose remained constant throughout development. Osmolality of petal cell sap increased significantly during fructan hydrolysis, from 0.300 to 0.340 osmolal. Cycloheximide applied to excised buds between hour -38 and hour -14 halted both fructan hydrolysis and flower expansion. The findings suggest that onset of fructan hydrolysis, with the concomitant large increase in osmoticum, is an important event driving flower expansion in daylily. PMID:12231928

  12. Ultrasound-enhanced enzymatic hydrolysis of poly(ethylene terephthalate).

    PubMed

    Pellis, Alessandro; Gamerith, Caroline; Ghazaryan, Gagik; Ortner, Andreas; Herrero Acero, Enrique; Guebitz, Georg M

    2016-10-01

    The application of ultrasound was found to enhance enzymatic hydrolysis of poly(ethylene terephthalate) (PET). After a short activation phase up to 6.6times increase in the amount of released products was found. PET powder with lower crystallinity of 8% was hydrolyzed faster when compared to PET with 28% crystallinity. Ultrasound activation was found to be around three times more effective on powders vs. films most likely due to a larger surface area accessible to the enzyme. PMID:27481467

  13. Effect of bovine serum albumin (BSA) on enzymatic cellulose hydrolysis.

    PubMed

    Wang, Hui; Mochidzuki, Kazuhiro; Kobayashi, Shinichi; Hiraide, Hatsue; Wang, Xiaofen; Cui, Zongjun

    2013-06-01

    Bovine serum albumin (BSA) was added to filter paper during the hydrolysis of cellulase. Adding BSA before the addition of the cellulase enhances enzyme activity in the solution, thereby increasing the conversion rate of cellulose. After 48 h of BSA treatment, the BSA adsorption quantities are 3.3, 4.6, 7.8, 17.2, and 28.3 mg/g substrate, each with different initial BSA concentration treatments at 50 °C; in addition, more cellulase was adsorbed onto the filter paper at 50 °C compared with 35 °C. After 48 h of hydrolysis, the free-enzyme activity could not be measured without the BSA treatment, whereas the remaining activity of the filter paper activity was approximately 41 % when treated with 1.0 mg/mL BSA. Even after 96 h of hydrolysis, 25 % still remained. Meanwhile, after 48 h of incubation without substrate, the remaining enzyme activities were increased 20.7 % (from 43.7 to 52.7 %) and 94.8 % (from 23.3 to 45.5 %) at 35 and 50 °C, respectively. Moreover, the effect of the BSA was more obvious at 35 °C compared with 50 °C. When using 15 filter paper cellulase units per gram substrate cellulase loading at 50 °C, the cellulose conversion was increased from 75 % (without BSA treatment) to ≥90 % when using BSA dosages between 0.1 and 1.5 mg/mL. Overall, these results suggest that there are promising strategies for BSA treatment in the reduction of enzyme requirements during the hydrolysis of cellulose.

  14. Novel Penicillium cellulases for total hydrolysis of lignocellulosics.

    PubMed

    Marjamaa, Kaisa; Toth, Karolina; Bromann, Paul Andrew; Szakacs, George; Kruus, Kristiina

    2013-05-10

    The (hemi)cellulolytic systems of two novel lignocellulolytic Penicillium strains (Penicillium pulvillorum TUB F-2220 and P. cf. simplicissimum TUB F-2378) have been studied. The cultures of the Penicillium strains were characterized by high cellulase and β-glucosidase as well moderate xylanase activities compared to the Trichoderma reesei reference strains QM 6a and RUTC30 (volumetric or per secreted protein, respectively). Comparison of the novel Penicillium and T. reesei secreted enzyme mixtures in the hydrolysis of (ligno)cellulose substrates showed that the F-2220 enzyme mixture gave higher yields in the hydrolysis of crystalline cellulose (Avicel) and similar yields in hydrolysis of pre-treated spruce and wheat straw than enzyme mixture secreted by the T. reesei reference strain. The sensitivity of the Penicillium cellulase complexes to softwood (spruce) and grass (wheat straw) lignins was lignin and temperature dependent: inhibition of cellulose hydrolysis in the presence of wheat straw lignin was minor at 35°C while at 45°C by spruce lignin a clear inhibition was observed. The two main proteins in the F-2220 (hemi)cellulase complex were partially purified and identified by peptide sequence similarity as glycosyl hydrolases (cellobiohydrolases) of families 7 and 6. Adsorption of the GH7 enzyme PpCBH1 on cellulose and lignins was studied showing that the lignin adsorption of the enzyme is temperature and pH dependent. The ppcbh1 coding sequence was obtained using PCR cloning and the translated amino acid sequence of PpCBH1 showed up to 82% amino acid sequence identity to known Penicillium cellobiohydrolases.

  15. DWPF integrated cold runs revised technical bases for precipitate hydrolysis

    SciTech Connect

    Landon, L.F.

    1992-06-01

    The report defines new precipitate hydrolysis process operating parameters for DWPF Chemical runs assuming the precipitate feed simulants to be processed reflect the decision to implement a final wash of the tetraphenylborate slurry before transfer to DWPF (i.e. the Late Wash Facility). Control of the nitrite content of the tetraphenylborate slurry to 0.01M or less has eliminated the need for hydroxylamine nitrate (HAN) during hydrolysis. Consequently, the oxidant nitrous oxide will not be generated. However, nitric oxide (NO) is expected to be generated (reaction of formic acid with nitrite) and some fraction of the NO can be expected to be oxidized to nitrogen dioxide. The rate of NO generation with low nitrite feed has not been quantified at this time nor is the extent to which the NO is oxidized to NO{sub 2} known. A mass spectrometer is being installed in the Precipitate Hydrolysis Experimental Facility (PHEF) which will enable the NO generation rate to be defined as well as the extent to which the NO is oxidized to NO{sub 2}. There is some undocumented data available for C{sub 6}H{sub 6}/NO and C{sub 6}H{sub 6}/NO{sub 2} with N{sub 2} as the diluent but no similar data for CO{sub 2}. Development of test data in the required time frame is not possible. However, MOC`s will be estimated for benzene/NO/NO{sub 2}/CO{sub 2} gas mixtures (the MOC is expected to be approximately 60% less than for the HAN process). Once these data are obtained, and NO/NO{sub 2} concentration profiles are obtained from PHEF hydrolysis process demonstrations, a flammability control strategy for the DWPF Salt Processing Cell will be developed. Implementation of the HAN process purge strategy upon startup of the SPC with the late wash process would be conservative.

  16. Allergenicity of Peanut Proteins is Retained Following Enzymatic Hydrolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rationale: Hydrolysis of peanut proteins by food-grade enzymes may reduce allergenicity and could lead to safer forms of immunotherapy. Methods: Light roasted peanut flour extracts were digested with pepsin (37°C, pH 2), Alcalase (60°C pH 8), or Flavourzyme (50°C, pH 7) up to 1 hr, or sequentially w...

  17. Regiospecific Ester Hydrolysis by Orange Peel Esterase - An Undergraduate Experiment.

    NASA Astrophysics Data System (ADS)

    Bugg, Timothy D. H.; Lewin, Andrew M.; Catlin, Eric R.

    1997-01-01

    A simple but effective experiment has been developed to demonstrate the regiospecificity of enzyme catalysis using an esterase activity easily isolated from orange peel. The experiment involves the preparation of diester derivatives of para-, meta- and ortho-hydroxybenzoic acid (e.g. methyl 4-acetoxy-benzoic acid). The derivatives are incubated with orange peel esterase, as a crude extract, and with commercially available pig liver esterase and porcine pancreatic lipase. The enzymatic hydrolysis reactions are monitored by thin layer chromatography, revealing which of the two ester groups is hydrolysed, and the rate of the enzyme-catalysed reaction. The results of a group experiment revealed that in all cases hydrolysis was observed with at least one enzyme, and in most cases the enzymatic hydrolysis was specific for production of either the hydroxy-ester or acyl-acid product. Specificity towards the ortho-substituted series was markedly different to that of the para-substituted series, which could be rationalised in the case of pig liver esterase by a published active site model.

  18. Hydrolysis of organonitrate functional groups in aerosol particles

    SciTech Connect

    Liu, Shang; Shilling, John E.; Song, Chen; Hiranuma, Naruki; Zaveri, Rahul A.; Russell, Lynn M.

    2012-10-19

    Organonitrate (ON) groups are important substituents in secondary organic aerosols. Model simulations and laboratory studies indicate a large fraction of ON groups in aerosol particles, but much lower quantities are observed in the atmosphere. Hydrolysis of ON groups in aerosol particles has been proposed recently. To test this hypothesis, we simulated formation of ON molecules in a reaction chamber under a wide range of relative humidity (0% to 90%). The mass fraction of ON groups (5% to 20% for high-NOx experiments) consistently decreased with increasing relative humidity, which was best explained by hydrolysis of ON groups at a rate of 4 day-1 (lifetime of 6 hours) for reactions under relative humidity greater than 20%. In addition, we found that secondary nitrogen-containing molecules absorb light, with greater absorption under dry and high-NOx conditions. This work provides the first evidence for particle-phase hydrolysis of ON groups, a process that could substantially reduce ON group concentration in the atmosphere.

  19. The Competing Mechanisms of Phosphate Monoester Dianion Hydrolysis

    PubMed Central

    2016-01-01

    Despite the numerous experimental and theoretical studies on phosphate monoester hydrolysis, significant questions remain concerning the mechanistic details of these biologically critical reactions. In the present work we construct a linear free energy relationship for phosphate monoester hydrolysis to explore the effect of modulating leaving group pKa on the competition between solvent- and substrate-assisted pathways for the hydrolysis of these compounds. Through detailed comparative electronic-structure studies of methyl phosphate and a series of substituted aryl phosphate monoesters, we demonstrate that the preferred mechanism is dependent on the nature of the leaving group. For good leaving groups, a strong preference is observed for a more dissociative solvent-assisted pathway. However, the energy difference between the two pathways gradually reduces as the leaving group pKa increases and creates mechanistic ambiguity for reactions involving relatively poor alkoxy leaving groups. Our calculations show that the transition-state structures vary smoothly across the range of pKas studied and that the pathways remain discrete mechanistic alternatives. Therefore, while not impossible, a biological catalyst would have to surmount a significantly higher activation barrier to facilitate a substrate-assisted pathway than for the solvent-assisted pathway when phosphate is bonded to good leaving groups. For poor leaving groups, this intrinsic preference disappears. PMID:27471914

  20. Hydrolysis of cellulose catalyzed by novel acidic ionic liquids.

    PubMed

    Zhuo, Kelei; Du, Quanzhou; Bai, Guangyue; Wang, Congyue; Chen, Yujuan; Wang, Jianji

    2015-01-22

    The conversion of cellulosic biomass directly into valuable chemicals becomes a hot subject. Six novel acidic ionic liquids (ILs) based on 2-phenyl-2-imidazoline were synthesized and characterized by UV-VIS, TGA, and NMR. The novel acidic ionic liquids were investigated as catalysts for the hydrolysis of cellulose in 1-butyl-3-methylimidazolium chloride ([Bmim]Cl). The acidic ionic liquids with anions HSO4(-) and Cl(-) showed better catalytic performance for the hydrolysis of cellulose than those with H2PO4(-). The temperature and dosage of water affect significantly the yield of total reducing sugar (TRS). When the hydrolysis of cellulose was catalyzed by 1-propyl sulfonic acid-2-phenyl imidazoline hydrogensulfate (IL-1) and the dosage of water was 0.2g, the TRS yield was up to 85.1% within 60 min at 100°C. These new acidic ionic liquids catalysts are expected to have a wide application in the conversion of cellulose into valuable chemicals. PMID:25439867