Nucleotide Mono Research Articles

Class III Polyphosphate Kinase 2 Enzymes Catalyze the Pyrophosphorylation of Adenosine-5'-Monophosphate.

Polyphosphate kinase 2 (PPK2) transfer phosphate from inorganic polyphosphate to nucleotides. According to their activity, PPK2 enzymes are classified into three groups. Among them, class III enzymes catalyze both the phosphorylation of nucleotide mono- to diphosphates and di- to triphosphates by using polyphosphate, which is a very inexpensive substrate. Therefore, class III enzymes are very attractive for use in biotechnological applications. Despite several studies on class III enzymes, a detailed mechanism of how phosphate is transferred from the polyphosphate to the nucleotide remains to be elucidated. Herein, it is reported that PPK2 class III enzymes from two different bacterial species catalyze the phosphorylation of adenosine mono- (AMP) into triphosphate (ATP) not only through step-by-step phosphorylation, but also by pyrophosphorylation. These are the first PPK2 enzymes that have been shown to possess polyphosphate-dependent pyrophosphorylation activity.

Capillary electrophoresis coupled to mass spectrometry employing hexafluoro-2-propanol for the determination of nucleosides and nucleotide mono-, di- and tri-phosphates in baby foods

The present work describes a method for the simultaneous determination of unmodified nucleosides and nucleotide mono-, di- and tri-phosphates by capillary electrophoresis coupled to mass spectrometry (CE-MS). The use of hexafluoro-2-propanol (HFIP) in the separation medium, and as an additive to the sheath liquid of the electrospray interface (ESI), generated a highly efficient and sensitive method. Instrumental limits of detection in the range of 14–53ngmL−1 for nucleosides and 7–23, 20–49 and 64–124ngmL−1 for nucleotide mono-, di-, and tri-phosphates, respectively, were found. Sample treatment involved diluting an aliquot of baby food with ultra-high quality water and applying centrifugation-assisted ultrafiltration (CUF). The proposed method was validated and used to analyse a variety of baby food samples (16 in total) such as fish, meat, fruits, and baby dairy desserts that may endogenously contain these analytes.

Hydrophilic interaction chromatography coupled to tandem mass spectrometry in the presence of hydrophilic ion-pairing reagents for the separation of nucleosides and nucleotide mono-, di- and triphosphates

A fast and efficient method for the simultaneous separation of highly polar compounds, in this case nucleosides and nucleotide mono-, di- and triphosphates, using hydrophilic interaction chromatography coupled with tandem mass spectrometry (HILIC–MS/MS) is proposed. This new separation method revealed the possibilities of the formation of hydrophilic ion-pairing compounds.Three stationary phases (HILIC XBridge-Amide, HILIC-CoreShell and ZIC-HILIC) were assayed for the separation of 20 target analytes, and a detailed study of the composition of the mobile phase was made using different salts at different concentrations in a organic-rich mobile phase. We report that in order to prevent the adsorption of nucleotides on the LC–MS setup and to enhance their retention on the HILIC stationary phase, a mobile phase containing hexafluoro-2-propanol and different cations should be used. Four cations were evaluated: ammonium, diethylammonium, triethylammonium and tetrabutylammonium. The results revealed the formation of an ionic-association compound between the phosphorylated analytes and the cationic ion-pairing reagents, whose retention increased with the polarity of the cationic ion-pairing reagent.HILIC XBridge-Amide was found to be the most suitable column for the separation of these analytes, and the optimized mobile phase consisted of an ACN/UHQ water mixture (3min of isocratic elution using 82:18%, v/v and then a fast gradient from 18% to 22% of water) with 100mM hexafluoro-2-propanol and 50mM diethylamine (wwpH 9–wspH 10). In a total analysis time of 8min, good results were achieved in terms of resolution. Under these optimum conditions, a further comprehensive study of the retention mechanism was carried out.

Rapid and Efficient Synthesis of Nucleoside Polyphosphates and Their Conjugates Using Sulfonyl Imidazolium Salts

This unit describes a method for preparing nucleoside polyphosphates and their conjugates such as nucleoside triphosphates, symmetrical and unsymmetrical dinucleoside polyphosphates and sugar nucleotides. The protocols employ sulfonyl imidazolium salts (SnISs) as coupling reagents. These reagents are prepared in two steps in very high yield from commercially available materials and can be stored for at least 1 year at -20°C. The tetra-n-butylammonium salts of nucleoside mono-, di-, or triphosphates are reacted with the SnISs to form reactive phosphoryl imidazolium donors. These donors are reacted with the tetra-n-butylammonium salts of pyrophosphate, nucleotide mono- or diphosphates or sugar-1-phosphates to give the nucleoside polyphosphates and their conjugates in excellent yields.

Interaction of ATP with acid-denatured cytochrome c via coupled folding-binding mechanism

The non-native conformations of the cytochrome c (cyt c) are believed to play key roles in a number of physiological processes. Nucleotides are supposed to act as allosteric effectors in these processes by regulating structural transitions among different conformations of cyt c. To understand the interaction between acid denatured cytochrome c and nucleotides, spectroscopic and calorimetric techniques were utilized to observe the structural features of the induced conformation and the energetics of interaction of acid denatured cyt c with different nucleotides. Structure induction in the acid denatured cyt c was observed on the addition of the ∼1mM nucleotide tri-phosphates (ATP/GTP/CTP) at 25°C, however, not in the presence of 1mM nucleotide mono and diphosphates. ATP-bound cyt c at pH 2.0 is likely to have a conformation that has intact α-helical domain. However, Met80-Fe(III) axial bond is still ruptured. Observed thermodynamics reflect interaction between nucleotide and cyt c via coupled binding–folding mechanism. DSC data suggest the preferential binding of the ATP to the folded conformation with respect to the acid denatured cyt c. ITC data indicate that the exothermic folding of cyt c was accompanied by endothermic binding of ATP to cyt c.

Characterization of AtNST-KT1, a novel UDP-galactose transporter from Arabidopsis thaliana

Nucleotide sugar transporters (NST) mediate the transfer of nucleotide sugars from the cytosol into the lumen of the endoplasmatic reticulum and the Golgi apparatus. Because the NSTs show similarities with the plastidic phosphate translocators (pPTs), these proteins were grouped into the TPT/NST superfamily. In this study, a member of the NST-KT family, AtNST-KT1, was functionally characterized by expression of the corresponding cDNA in yeast cells and subsequent transport experiments. The histidine-tagged protein was purified by affinity chromatography and reconstituted into proteoliposomes. The substrate specificity of AtNST-KT1 was determined by measuring the import of radiolabelled nucleotide mono phosphates into liposomes preloaded with various unlabelled nucleotide sugars. This approach has the advantage that only one substrate has to be used in a radioactively labelled form while all the nucleotide sugars can be provided unlabelled. It turned out that AtNST-KT1 represents a monospecific NST transporting UMP in counterexchange with UDP-Gal but did not transport other nucleotide sugars. The AtNST-KT1 gene is ubiquitously expressed in all tissues. AtNST-KT1 is localized to Golgi membranes. Thus, AtNST-KT1 is most probably involved in the synthesis of galactose-containing glyco-conjugates in plants.

Dissolved organic phosphorus in the coastal ocean: Reassessment of available methods and seasonal phosphorus profiles from the Eel River Shelf

Methods for quantifying total dissolved phosphorus (TDP) were reevaluated. High‐temperature ashing/hydrolysis (Ash/Hydrol.) and Acid Persulfate methods were examined for their ability to quantitatively convert a range of organic‐P compounds to orthophosphate. The Ash/Hydrol. method recovered on average 99% of organic‐P compounds tested, including phosphonates (compounds with C‐P bonds). Contrary to concerns voiced in prior studies, we found no P loss due to volatilization in seawater samples. Poor recovery by the Acid Persulfate method was observed for two compound classes: phosphonates and phospholipids. The Ash/Hydrol. method is recommended for routine TDP analyses.The hydrolysis of dissolved organic phosphorus (DOP) during the phosphomolybdate‐blue reaction and the subsequent overestimation of dissolved inorganic phosphorus (DIP) have long been a concern. Extent of hydrolysis of standard DOP compounds was evaluated both during this reaction and over long‐term acidified (pH 1.0)/refrigerated and unacidified/frozen storage. Eleven of 12 compounds tested hydrolyzed <2% during the phosphomolybdate‐blue reaction. Phospholipid, phosphosugar, and nucleotide mono‐ and diphosphate standards stored acidified/refrigerated over 2.5 months were <5% hydrolyzed; compounds with higher energy phosphate bonds (e.g., phosphocreatine) hydrolyzed to a greater extent. A natural seawater sample showed no hydrolysis over 80 d, however, suggesting that refrigerated storage of acidified samples is a viable option.Seasonal DOP profiles from the Eel River Shelf quantified by the Ash/Hydrol. and Acid Persulfate methods yielded comparable results, indicating that the DOP pool at this site did not contain significant phospholipids or phosphonates, which are underrecovered by the Persulfate method. Recoveries using a third method, Nitrate Oxidation, were on average 7% lower. Surface‐water DOP exceeded DIP in summer and was apparently related to biological productivity, underscoring the need to include organic species when evaluating nutrient limitation and creating nutrient budgets.

Structure and NTPase activity of the RNA-translocating protein (P4) of bacteriophage φ6

The RNA polymerase complex of bacteriophage φ6 comprises four proteins, P1, P2, P4 and P7, and forms the core of the virion. Protein P4 is a non-specific NTPase that provides the energy required for RNA translocation (packaging). Characterization of purified recombinant P4 shows that the protein assembles into stable hexamers in the presence of ADP and divalent cations. Image averaging of electron micrographs reveals this hexamer as a slightly skewed ring with outer and inner diameters of 12 and 2 nm, respectively. NTPase activity of P4 is associated only with the hexameric form. Ca 2+and Zn 2+and non-specific single-stranded RNA stimulate the NTPase activity, while Mg 2+acts as a non-competitive inhibitor, presumably via a separate Mg 2+binding site. Binding affinities of different nucleotide mono-, di- and triphosphates and non-hydrolyzable analogs indicate that the β-phosphate moiety is required for substrate binding. A slight preference for binding of purine nucleotides is also observed. Analysis of P4 by CD and Raman spectroscopy indicates an α/β subunit fold that is altered only slightly by hexamer assembly. Raman markers of P4 secondary and tertiary structures are also largely invariant to nucleotide exchange and hydrolysis, suggesting that the mechanism of RNA translocation involves movement of subunits relative to one another rather than large scale changes in the α/β subunit fold. The stoichiometry of P4 in the mature φ6 virion is estimated as 120 copies. Because the recombinant P4 hexamers exhibit hydrodynamic and enzymatic properties that are identical to those of P4 oligomers released from native φ6, we propose that P4 occurs as hexamers in the native viral core particle.

Oligosapphyrins: receptors for the recognition and transport of nucleotide di- and tri-phosphates

Covalently connected oligosapphyrins, in particular two trimers (linear and branched) and a tetramer, the syntheses of which are detailed, have been found to be effective receptors for the binding and through-model-membrane transport of nucleotide mono-, di- and tri-phosphates.

Capillary electrophoresis-electrospray ionization-mass spectrometry

New developments are described in the area of capillary electrophoresis combined with electrospray ionization-mass spectrometry (CE—ESI-MSD), a promising new method for the separation and mass spectrometric characterization of labile, polar or ionic constituents of biological mixtures. the varous experimental modes of capillary electrophoresis are reviewed. The ESI-MS technique is described and the significance of its combination with capillary electrophoresis is illustrated by MS analysis of large biomelecules. The design and performance optimization of a new ESI interface are discussed, including the influence of electrophoretic and MS operating parameters. Various examples from our laboratory illustrating the range of present application of this interface and direction of future development are presented. These include negative iion electrospray mass spectra of nucleotide co-enzymes, nucleotide mono-, di- and triphosphates and positive ion spectra of biologically important oligopeptides and proteins of M r > 75 kilodaltons.

Effect of DNA polymerase I and DNA helicase II on the turnover rate of UvrABC excision nuclease.

UvrABC excision nuclease (UvrA, UvrB, and UvrC proteins) of Escherichia coli removes nucleotide mono- and diadducts from DNA in the form of oligonucleotides 12 or 13 bases long. We find that the purified enzyme dissociates from DNA very slowly, if at all, in the absence of other proteins implicated in excision repair. Addition of DNA polymerase I and helicase II (UvrD protein) to the reaction mixture stimulates the turnover rate of the excision nuclease to a level comparable to that observed in vivo.

MULTIENZYME RNA SYNTHETASE COMPLEXES: METABOLIC CHANNELLING OF NUCLEOTIDE MONO- AND DIPHOSPHATE PRECURSORS INTO RNA: 110

The uptake of exogenous nucleoside tracers into RNA proceeds without equilibration of the tracer with the cellular ribonucleotide triphosphate pools. Kinetic analysis of the tracer progression into RNA suggests that the functional nucleotide RNA-precursor pool is very small, so that simple compartmentation into nuclear and cytoplasmic pools fails to account for these results. We have tested the hypothesis that RNA synthesis is dependent on a multienzyme complex (similar to the proposed replitase) that functions as a metabolic channel. Nuclear lysates of mouse lymphoma S49 cells and human HL-60 promyelocytic leukemia cells were separated on sucrose gradients (20-60%) and fractions analyzed for RNA polymerase and nucleotide kinase activities. At least three fractions with high molecular weights (> 106 Daltons) were identified that were capable of incorporating mono-, di- and triphosphates (U+C+A+G) into acid-insoluble macromolecules. The mono- and diphosphates did not seem to equilibrate with the triphosphate pools in the medium, which suggests metabolic channeling. Tracer incorporation was dependent on the presence of all four bases as their nucleotides in the medium, while DNA template was not required in these fractions. These results provide first evidence for the existence of multienzyme RNA synthetase complexes that preferentially accept distant precursors, i.e. nucleotide mono- and diphosphates. Supported by CA 34304.

High-performance liquid chromatography analysis of purine nucleosides in human gingival crevicular fluid

Samples of human crevicular fluid were collected and analysed by high-performance liquid chromatography (HPLC) for purine nucleotides, nucleosides and bases. The complex chromatograms, had concentrations of compounds in two regions. Region (I) corresponded to purine bases and nucleosides, adenine and hypoxanthine were specifically identified. In region (II), where purine nucleotide mono- and diphosphates are generally observed, IMP and AMP were identified. In one subject with advanced periodontitis, additional compounds in region II were observed; crevicular fluid in gingivitis had the majority of their purine-containing compounds in region I.

Phenylalanyl-tRNA synthetase from escherichia coli MRE-600 : Activation by nucleotides and affinity modification of the effector binding sites

FEBS LettersVolume 109, Issue 1 p. 13-17 Full-length articleFree Access Phenylalanyl-tRNA synthetase from escherichia coli MRE-600 Activation by nucleotides and affinity modification of the effector binding sites O.I. Lavrik, O.I. Lavrik Institute of Organic Chemistry, Novosibirsk 630090, USSRSearch for more papers by this authorG.A. Nevinsky, G.A. Nevinsky Institute of Organic Chemistry, Novosibirsk 630090, USSRSearch for more papers by this author O.I. Lavrik, O.I. Lavrik Institute of Organic Chemistry, Novosibirsk 630090, USSRSearch for more papers by this authorG.A. Nevinsky, G.A. Nevinsky Institute of Organic Chemistry, Novosibirsk 630090, USSRSearch for more papers by this author First published: January 01, 1980 https://doi.org/10.1016/0014-5793(80)81300-7Citations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 V.S. Prasolov, A.M. Kritsin, S.N. Mikhailov, V.L. Florentyev, Dokl. AN SSSR, 221, (1975), 1226– 1228. 2 I.I. Gorshkova, O.I. Lavrik, Molekul. Biol., 9, (1975), 887– 892. 3 G.K. Kovaleva, L.L. Ivanov, I.A Madoyan, O.O. Favorova, E.S. Severin, N.N. Gulyaev, L.A. Baranova, Z.A. Shabarova, N.I. Sokolova, L.L. Kisselev, Biokhimiya, 43, (1978), 525– 534. 4 D.V. Santi, P.V. Dannenberg, K.A. Montgomery, Biochemistry, 18, (1971), 4821– 4824. 5 D.V. Santi, V.A. Pena, J. Med. Chem., 16, (1973), 273– 280. 6 G.A. Nevinsky, O.I. Lavrik, O.O. Favorova, L.L. Kisselev, Bioorg. Khim., 5, (1979), 352– 364. 7 M.P. Stulberg, J. Biol. Chem., 242, (1967), 1060– 1064. 8 L.S. Sandakchiev, V.K. Starostina, L.E. Stephanovich, V.M. Chuchaev, Molekul. Biol., 1, (1967), 463– 466. 9 O. Silberrad, B.J. Smart, J. Chem. Soc., 89, (1906), 170– 171. 10 G. Fayat, S. Blanquet, F. Dessen, G. Batelier, J.P. Waller, Biochimie, 56, (1974), 35– 41. 11 V.G. Budker, D.G. Knorre, V.V. Kravchenko, O.I. Lavrik, G.A. Nevinsky, N.M. Teplova, FEBS Lett., 49, (1974), 159– 162. 12 V.Z. Akhverdyan, L.L. Kisselev, D.G. Knorre, O.I. Lavrik, G.A. Nevinsky, Dokl. AN SSSR, 226, (1976), 698– 701. 13 V.N. Ankilova, D.G. Knorre, V.V. Kravchenko, O.I. Lavrik, G.A. Nevinsky, FEBS Lett., 60, (1975), 172– 175. 14 O.I. Lavrik, G.A. Nevinsky, I.A. Ryazankin, Molekul. Biol., 13, (1979), 5– in press. Citing Literature Volume109, Issue1January 01, 1980Pages 13-17 ReferencesRelatedInformation

Heart phosphofructokinase. Allosteric kinetics following affinity labeling modification of the enzyme by 8-[m-(m-fluorosulfonylbenzamido) benzylthio] adenine.

An adenine analog 8-[m-(m-fluorosulfonylbenzamido)benzylthio]adenine (FSB-adenine) reacts covalently with sheep heart phosphofructokinase. Under conditions optimal for allosteric kinetics the modified enzyme is less sensitive to inhibition by ATP and insensitive to activation by AMP, cyclic AMP, and ADP. The concentration of fructose-6-P necessary for half-maximal activity is markedly decreased, while the cooperativity to the same substrate is not changed under the same conditions. The modified enzyme is more stable at pH 6.5 when compared with the native enzyme. Changes in the allosteric kinetics of the enzyme are proportional to the extent of modification reaching maximal effect when 3.2 mol of the reagent were bound/mol of tetrameric enzyme. Affinity labeling of the enzyme by the adenine derivative does not affect significantly the catalytic site. This is evidenced by the demonstration that under assay conditions optimal for Michaelian kinetics neither the Km for ATP nor for fructose-6-P is significantly changed following chemical modification. Maximal activity of the modified enzyme was 60% of the native enzyme. ADP gives the best protection, while AMP gives less protection against modification by the reagent. ATP slows the rate of the reaction and causes a slight decrease in maximum binding of the reagent to the enzyme. Modification of the enzyme caused a marked reduction of AMP and ADP binding. The evidence indicates that the modified site is a nucleotide mono- and diphosphate activation site.

Further studies on the properties of violet-colored acid phosphatase from soybean.

The properties of the acid phosphatase isolated in a homogeneous state from soybean (Glycine max) have been investigated in detail. The enzyme catalyzed the hydrolysis of a wide variety of phospholylated compounds including phosphomonoesters, nucleotide mono-, di-and triphosphate, and inorganic pyrophosphate. No activity was detected for nicotinamide adenine dinucleotide and diphenyl phosphate. The enzyme activity was inhibited by orthophosphate, arsenate, fluoride, molybdate, and heavy metal ions including Cu2+, Zn2+, Hg2+ and Ag+. Treatment of the enzyme with chelating agents, reducing agents and oxidizing agents resulted in inactivation of the enzyme. The reduction of the absorbance at 540 nm of the enzyme was observed in parallel with the loss of the enzyme activity by the treatment with ethylenediaminetetraacetic acid and rongalit, but not with H2O2. The enzyme had a molecular weight of approximately 240000. Polyacrylamide disc gel electrophoresis in the presence of sodium dodecyl sulfate suggested that the enzyme dissociated into subunits with molecular weight of approximately 60000. The amino acid and carbohydrate composition of the enzyme were also determined.

Formation in Vitro of Uridine-5′-(oligosaccharide)-1-pyrophosphates

Bovine testicular β-galactosidase catalyzed the enzymatic transfer of galactose residues from appropriate β-galactosides to UDP-N-acetylglucosamine to yield nucleotide disaccharides. The disaccharide portion of the nucleotides was a mixture of isomers of O-β-galactopyranosyl-(1→6, 1→3, and 1→4)-N-acetylglucosamine in ratios approximating 1:8:9, respectively. Nucleotide disaccharides were also obtained when UDP-N-acetylgalactosamine and UDP-glucose served as acceptors for β-galactosyl residues. Synthesis of a family of nucleotide trisaccharides, UDP-N-acetylglucosamine-(β,1→6; β,1→3; and β,1→4)-galactose-N-acetylneuraminic acid, was achieved when goat colostrum sialyltransferase was incubated with UDP-N-acetylglucosamine-[3H]galactose and CMP-N-acetylneuraminic acid. The results of the present studies suggest a pathway for the enzymatic synthesis of the UDP-disaccharides and UDP-trisaccharides found in milk and colostrum. The preparation of tritium-labeled UDP-disaccharides and 14C-labeled UDP-trisaccharides as well as conditions for the resolution of nucleotide mono-, di-, and trisaccharides on gel filtration columns are described.

Studies on the Extracellular Alkaline Phosphatase of Micrococcus sodonensis: I. ISOLATION AND CHARACTERIZATION

Abstract An orthophosphate-repressible extracellular alkaline phosphatase has been isolated from cultures of Micrococcus sodonensis. The enzyme is pure as judged by molecular sieve chromatography, disc gel electrophoresis, and ultracentrifugation, and possesses the following properties: (a) molecular weight, 80,000; (b) pH optimum, pH 9 to 9.5; (c) competitive inhibitors, orthophosphate, arsenite, and arsenate; (d) substrate specificity, hydrolyzes a wide variety of phosphorylated compounds including phosphomonoesters, nucleotide mono-, di-, and triphosphates, and inorganic pyrophosphate. Enzymatic activity is dependent upon the presence of free sulfhydryl group(s); derivatization of the enzyme with 1 eq of p-chloromercuribenzoate results in the loss of approximately 75% of its catalytic activity. Activity of the enzyme exhibits an absolute requirement for divalent cation with the following relative reactivations of ethylenediaminetetraacetate-inactivated enzyme: Ca++, 90; Mn++, 22; Co++, 15; Sr++, 13; and Cu++, Mg++, Zn++, l1. The Km for Ca++ was estimated to be 1.5 x 10-5 m and the Kdissociation = 1.5 x 10-5 m as determined by equilibrium dialysis. Assuming a molecular weight of 80,000, the Ca++ content of the enzyme was estimated to be 8 g atoms per mole. Removal of Ca++ from the enzyme resulted in a marked increase in susceptibility of the enzyme to inactivation by heat and proteolytic digestion.