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Query: EC:2.7.7.8 (polynucleotide phosphorylase)
 723 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Several oligonucleotides of defined sequence were synthesized using 2'(3')-O-dihydrocinnamoyl-nucleoside 5'-diphosphates (DHC-NDP) as substrates for polynucleotide phosphorylase [EC 2.7.7.8] from Thermus thermophilus. The enzyme catalyzed the transfer of one nucleotidyl residue from each of the 2'(3')-O-dihydrocinnamoyl esters of CDP, UDP, and GDP to the 3'-terminus of the primer triadenosine diphosphate, (Ap)2A. The products were shown to be (Ap)3C, (Ap)3U, and (Ap)3G by enzymatic analysis.
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PMID:Enzymatic synthesis of oligonucleotides of defined sequence. The "single addition" of 2(3)-O-dihydrocinnamoyl-nucleoside 5'-diphosphate to a primer oligonucleotide catalyzed by a thermophilic polynucleotide phosphorylase. 0 79

O2-Ethyl-UDP and O4-methyl-UDP have been prepared and copolymerized in various proportions with UDP or CDP, using polynucleotide phosphorylase. The copolymers were used as templates for DNA-dependent RNA polymerases in the presence of Mn2+. Both of the O-alkylated uridines caused a similar misincorporations. When copolymerized with U they led to incorporation of CMP and GMP into the poly(A). No AMP or UMP incorporation seemed to be caused by the introduction of O-alkyluridines into either poly(U) or poly(C). The mispairing of O2- and O4-alkyluridine to behave like C or G represents mutagenic events. O2 alkylation of U or T is, in contrast to O4 alkylation, a relatively frequent result of treatment of double-stranded nucleic acids with N-nitroso alkylating agents. In single-stranded nucleic acids both O2 and O4 alkylations of U and T occur to similar extents. Thus, the observed mutagenic effects of O2 and O4 alkylation of U may be involved in the high carcinogenicity of these alkylating agents.
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PMID:Preparation and template activities of polynucleotides containing O2- and O4-alkyluridine. 27 2

Native Escherichia coli polynucleotide phosphorylase can be retained on blue-dextran--Sepharose. The bound enzyme cannot be displaced by its mononucleotide substrates such as ADP, UDP, CDP, GDP and IDP, but it is easily eluted by its polymeric substrates. Under identical conditions, lactate dehydrogenase, bound on blue-dextran--Sepharose, is not eluted by poly(I) but can be specifically displaced by NADH. On the other hand, the trypsinized polynucleotide phosphorylase, known to be an active enzyme which has lost its polynucleotide site, does not bind to the affinity column. The native polynucleotide phosphorylase can also be tightly bound to poly(U)--agarose and displaced from it only by high salt concentration. The trypsinized enzyme is not bound at all on poly(I)--AGAROSe. Moreover, the native enzyme linked on blue-dextran--Sepharose, remains active indicating a free access of nucleoside diphosphates to the active center. These results taken together show that the dye ligand is not inserted onto the mononucleotide binding site and suggest rather that it binds to the polynucleotide binding region. The implications of this study and the application of blue-dextran--Sepharose affinity chromatography to other proteins having affinity for nucleic acids are discussed.
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PMID:Blue-dextran--Sepharose affinity chromatography: recognition of a polynucleotide binding site of a protein. 34 36

On incubation of cells of E. coli B and MRE 600 (logariphmic phase of growth), treated with toluene in presence of a mixture 14C-nucleoside-5'-diphosphates, Mg2+ or Mn2+ and tris HCl buffer pH 8.0, intracellular synthesis of heteropolyribonucleotide was observed. The synthesis was catalyzed by polynucleotide phosphorylase (PNPase, E. C. 2.7.7.8). An increase in GDP concentration in the medium distinctly decreased the incorporation of other NDP into the polymer (poly-AGUC). If the ratio of ADP, UDP, CDP, GDP in the medium was 1:1:1:0.2, the composition of nitrogenous bases in the heteropolymer produced reflected completely the NDP concentrations in the incubation mixture. Addition of different amino acids (1-lysine, 1-histidine, glycine, 1-phenylalanine) and their mixtures stimulated poly-AGUC synthesis markedly and caused an appreciable alteration in the nucleotide composition of the poly-AGUC synthesized. This phenomenon resembled the effect of amino acids on the activity of partially purified PNPase and on RNA synthesis, catalized by the enzyme in vitro. These data suggest that in bacterial cell, i. e. in vivo, PNPase synthesizes specific RNA polyribonucleotide sequences, participating in protein synthesis or in its regulation.
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PMID:[Nucleotide composition of RNA, synthesized by polynucleotide phosphorylase, in toluene-treated cells of Escherichia coli]. 76 93

Polyriboadenylate polymerase was isolated from Escherichia coli PR7 (RNase I-, pnp) in good yield and high purity. The enzyme catalyzes the polymerization of ATP and ADP. These polymerizations show an initial lag which can be removed by the addition of poly(A). However, poly(A) does not function as a primer. UDP and CDP can also serve as substrates but with decreased efficiency. The polymerization of CDP is enhanced by the presence of an oligonucleotide which again does not function as a primer. Polymerization of [gamma-32P]ATP or [beta-32P]ADP result in products with no radioactivity. The product formed from [alpha-32P]ATP on hydrolysis with alkali yields labeled pAp and 2',3'-AMP; thus the enzyme synthesizes poly(A) chains de novo. During the polymerization of ATP, no burst of free ADP can be detected and the time course of phosphate release from ATP ro ADP follows very closely the kinetics of polymerization. dATP and dADP are effective inhibitors of poly(A) synthesis from either ATP or ADP. Sulfhydryl reagents inhibit only the polymerization of ATP and the inhibition is fully reversed by dithiothreitol. However, the enzyme can be protected from sulfhydryl reagents by preincubation with either ATP or ADP in the absence of Mg2+ which is required for polymerization. Studies using acrylamide gel electrophoresis indicate that the polymerization activity with either ATP or nucleoside diphosphates resides in the same protein. The enzyme catalyzes the following exchanges: 32Pi into ADP, 32Pi into ATP, and [14C] ADP into ATP in the presence of phosphate. While the enzyme catalyzes the phosphorolysis of its own product, (pAp-(Ap)nA), it fails to cleave the dephosphorylated product, (Ap(Ap)nA), or ribosomal RNA or tRNA in the presence of inorganic phosphate. The differences and similarities between poly(A) polymerase and polynucleotide phosphorylase are discussed. Based on the 32P exchange studies and other properties of poly(A) polymerase, a plausible mechanism for its action is proposed.
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PMID:Further studies on the isolation and properties of polyriboadenylate polymerase from Escherichia coli PR7 (RNase I-, pnp). 78 66

A thermophilic polynucleotide phosphorylase lacking polynucleotide phosphoryltic activity was purified from Thermus thermophilus HB-8 strain. The enzyme is an altered form of the native polynucleotide phosphorylase, probably attacked by the proteinase(s) of this extreme thermophile during the purification process. This modified enzyme lacks phosphorolytic activity to poly(A) while retaining weak activity to phosphorolyse tetranucleotides or hexanucleotides. The purified enzyme was shown to be homogenous by electrophoretic analysis in polyacrylamide gel. This enzyme had a molecular weight of 190 000 as calculated both from electrophoresis on polyacrylamide gel and from the Stoke's radius derived from the gel filtration pattern and the sedimentation coefficient. The enzyme was separated into three polypeptide chains by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate; their molecular weights were calculated to be 92000, 73000 and 35000. The enzyme was thermophilic and thermotolerant, exhibiting its maximal activity at 70 degrees C. The four ribonucleoside diphosphates (ADP, GDP, UDP and CDP) were polymerized to the extent of 7-S size.
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PMID:Thermophilic polynucleotide phosphorylase from Thermus thermophilus. Purification and properties of an altered form of enzyme which lacks phosphorolytic activity to polynycleotide. 89 51

A method has been developed for the routine synthesis of 2'(3')-o-monoacyl ribonucleoside 5'-diphosphates for stepwise synthesis of oligoribonucleotides with Escherichia coli polynucleotide phosphorylase. The use of triethyl orthoisovalerate allows the facile preparation of 2'(3')-o-isovaleryl-UDP, -CDP, -ADP, -GDP, -IDP, -EPLISON-APD, eplison-CDP, and N6-isopentenyl-ADP. The synthesis of N6-isopentenyl-ADP from ADP by N1-alkylation and the Dimroth rearrangement to N6 is reported. The effects of several factors including the nature of the divalent cation, pH, SALT CONCENTRATION, AND TIME ON THE EFFICIENCY OF THE POLYNUCLEOTIDE PHPSPHORYLASE CATALYZED SINGLE ADDITIONS OF THE 2'(3')-O-ISOVALERYL RIBONUCLEOSIDE 5'-DIPHOSPHATES TO AN OLIGORIBONUCLEOTIDE PRIMER ARE REPORTED. The syntheses of many tetranucleoside triphosphates and two pentanucleoside tetraphosphates in yields of 20-75 per cent are reported. The 2'(3')-o-isovaleryl derivatives of IDP, eplison-ADP, eplison-CDP, and N6-isopentenyl-ADP were all accepted by polynucleotide phosphorylase as substrates for the monoaddition reaction. The extension of the method to include the syntheses of oligoribonucleotides containing modified nucleosides offers a means of studying the role s of these modification by the use of relatively simple model compounds.
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PMID:Stepwise enzymatic oligoribonucleotide synthesis including modified nucleotides. 109 Mar

Optimal conditions of homopolyribonucleotide (poly-A-14C) synthesis in toluene-treated E. coli cells under incubation with ADP-14C, Mg2+ and tris. HCl buffer (pH 8.0) are studied. Optimal Mg2+ concentration was 0.75.-10(-3) M. Heterogeneity of the isolated poly-A-14C from E. coli cell was demonstrated by means of sucrose density gradient (5-20%) centrifugation and polyacrylamide gel electrophoresis. Actinomycin D was found not to affect the reaction rate of polymerization of ADP-14C, UDP-14C and GDP-14C, catalyzed by polynucleotide phosphorylase in toluene-treated E. coli cells.
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PMID:[Isolation and characteristics of homopolyribonucleotide synthesized in toluene-treated E. coli cells]. 110 77

The type of RNA is studied, which is degraded by polynucleotide phosphorylase (PNPase) in the fraction of free ribosomes and ribosomes released from endoplasmic reticulum membranes with Triton X-100. Beta-32P labelled ADP, UDP, GDP and CDP are found among the degradation products of endogenous RNA of free and bound ribosomes in vitro in the presence of 32P-ortophosphate. An analysis of molar ratio of beta-32P-NDP isolated revealed that PNPase degrades RNA of GC type in both ribosome fractions. The amount of PNPase-degraded RNA in bound ribosimes is 4-fold as high as that in free ribosomes under the same conditions. Analysis of stable 32P-RNA and rapidly labelled 32-P-dRNA, isolated from bound ribosomes after the incubation with and without inorganic phosphate, revealed that PNPase attacks the 28S fragment of RNA, which consists of about 370 nucleotides, and dRNA having a sedimentation coefficient less than 12S. The rate of dRNA degradation is considerably higher than that of rRNA. 5'-RNAase, hydrolysing synthetic homopolyribonucleotides to oligonucleotides with free 3'-OH terminal group, apparently participates, together with PNPase, in dRNA and rRNA degradation.
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PMID:Study of the type of RNA, degraded by polynucleotide phosphorylase in polyribosomal fraction of rat liver. 121 63

Spin-labeled copolymers of 4-thiouridine and uridine (ls4U,U)n] that contain various amounts of spin label (l) were synthesized by either (i) chemical alkylation of the 4-thiouridine-uridine copolymers (s4U,U)n prepared by copolymerizing 4-thiouridine 5'-diphosphate (s4UDP) and UDP or (ii) copolymerization of spin-labeled s4UDP with UDP using polynucleotide phosphorylase. The effect of (s4U,U)n and (ls4U,U)n on avian myeloblastosis virus (AMV) RNA-dependent DNA polymerase (RNA-dependent DNA nucleotidyltransferase, EC 2.7.7.7; reverse transcriptase) was studied to determine whether the presence of potentially reactive thiol groups or spin labels enhances the inhibitory properties of the copolymers as compared to (U)n. Inhibition by (s4U,U)n gradually increases as the percentage of thiolation increases. Enhanced inhibition by (s4U,U)n appears to be due to the interaction of the thiol groups of (s4U,U)n with the thiol group(s) of the polymerase, because inhibition by (s4U,U)n (8% thiolated) in the presence of dithiotreitol resembles that by (U)n. In contrast, inhibition by (ls4U,U)n containing 3% spin label resembles that by (U)n; however, increasing the spin label to 6% or 12% results in enhanced inhibition by (ls4U,U)n as compared to that by (U)n, and dithiothreitol has no effect on enhanced inhibition by (ls4U,U)n. These results suggest that the mechanism of inhibition observed with (ls4U,U)n with a ls4U:U ratio > 1:33 differs from the mechanism for (s4U,U)n and involves complex formation between the spin label and the essential Zn2+ of RNA-dependent DNA polymerase.
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PMID:Reactivity of reverse transcriptase toward (s4U,U)n copolymers and spin-labeled nucleic acid lattices. 615 32


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