Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

The E. coli polynucleotide phosphorylase-catalysed reaction of the deoxynucleoside 5'-diphosphates of 5-methyldeoxycytidine, N4-hydroxydeoxycytidine, deoxyuridine and 5-mercurideoxyuridine with the primers d(pT-T-A-G) and d(pT-T-T-T-T-T) have been studied under conditions where the primer is extended, predominantly, by one or two nucleotide residues. In experiments with 5-mercurideoxyuridine 5'-diphosphate, no 5-mercurideoxy-uridine-containing oligonucleotides were produced. The other three nucleotide analogs were found to be good substrates for E. coli PNPase and the conditions established for synthesis with these analogs will allow the construction of a number of biologically useful types of oligodeoxyribonucleotide.
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PMID:Enzymatic synthesis of oligodeoxyribonucleotides of defined sequence. Polynucleotide phosphorylase catalysed synthesis using pyrimidine analog-containing deoxyribonucleoside 5'-diphosphates. 35 64

The kinetic model of carbohydrate metabolism has been expanded to include: (a) the accumulation of alpha and beta-cellulose, insoluble cell-wall glycogen and mucopolysaccharide; (b) the role of RNA turnover as a source of carbon for end-product synthesis and as a buffer regulating the level of uridine nucleotides in this metabolic network; and (c) the role of purine-nucleoside phosphorylase, 5'-AMP nucleotidase, nucleosidediphosphate kinase and polynucleotide phosphorylase. One of many predictions based on this model is that cells differentiating in the presence of glucose will produce sorocarps with an abnormally high trehalose to cellulose ratio. External perturbation of either the model or of developing cells by glucose increases the levels of sorocarp trehalose and glycogen, 5-fold and 6-fold respectively. Evaluation of the experimental data and the simulation analyses have allowed several predictions to be made concerning the compartmentation of metabolites and the permeability of cells to glucose during differentiation.
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PMID:Fourth expansion and glucose perturbation of the Dictyostelium kinetic model. 55 94

An affinity analog with a 5-bromoacetamido uridine 5'-phosphate moiety bonded to the 3' end of A-U-G has been prepared with the aid of polynucleotide phosphorylase. This 3'-modified, chemically reactive A-U-G analog was used to probe the ribosomal codon binding site. The yield of the reaction depended strongly on the ribosomal source and was sensitive to salt-washing ribosomes. The major crosslinking product was identified to be protein S1. Since the reaction of this 3'-modified A-U-G programmed ribosomes for Met-tRNA-Met-M binding, it is concluded that protein S1 is located at or near the 3'-side of the ribosomal codon binding site.
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PMID:Location of protein S1 of Escherichia coli ribosomes at the 'A'-site of the codon binding site. Affinity labeling studies with a 3'-modified A-U-G analog. 82 27

The kinetics of 3H-uridine incorporation into measles-infected Vero cells demonstrated that maximum virus-specific RNA synthesis occurred between 16 and 20 h after infection. Sedimentation analysis on sucrose gradients revealed the presence of four species of RNA having sedimentation coefficients 4S, 12 to 26S, 28 to 36S and 50S. Annealing studies showed that RNA sedimenting in the 12 to 36S regions was 100% complementary in base sequence to nucleocapsid 50S RNA, and at least 96% of the 50S genomic RNA was transcribed during virus replication. Polynucleotide binding experiments ane ribonuclease treatment indicated that poly(A) sequences were associated with the intracellular 12 to 26S, 28 to 36S and 50S RNAs. Denaturation of intracellular 50S RNA followed by sucrose gadient centrifugation demonstrated that this was a mixture of genomic 50S and heterogeneous RNAs which sedimented at 4 to 40S. The genomic RNA did not contain poly(A) sequences, and these are presumably associated with the heterogeneously sedimenting RNAs. The size of poly(A) sequences present on the 12 to 36S RNAs was estimated to be in the range of 70 to 140 nucleotides. Treatment of the 12 to 36S RNAs and their poly(A) sequences with polynucleotide phosphorylase indicated that the poly(A) was located on the 3' end of the RNAs, but that under the experimental conditions used this was protected by the secondary structure of the molecules.
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PMID:Rolyadenylic acid [poly(A)] sequences associated with measles virus intracellular ribonucleic acid (RNA) species. 88 16

A number of synthetic methods for the preparation of the 2-O-(alpha-methoxyethyl) derivatives of the 5-diphosphates of adenosine, cytidine, guanosine, and uridine have been studied in order to provide nucleotide substrates that can be applied to the synthesis of specific oligoribonucleotides using polynucleotide phosphorylase. The reaction of nucleoside 5-diphosphates with methyl vinyl ether for a limited time produces low yields of the corresponding 2-O-(alpha-methoxyethyl) derivatives because the rate of methoxyethylation of the 3-hydroxyl groups. A study of the rates of acidic hydrolysis of alpha-methoxyethyl groups in the 2 and 3 positions of nucleosides and nucleotides has been made, and the results obtained form the basis of a more efficient method for the synthesis of the blocked nucleoside diphosphates. The method involves the reaction of nucleoside 5-diphosphates with methyl vinyl ether to give the corresponding 2,3-di-O-(alpha-methoxyethyl)nucleoside 5-diphosphates, and exploits the fact that, in the acidic hydrolysis of these derivatives, the rate of removal of the 3-methoxyethyl group is about twice that of the group in the 2 position. Alternative syntheses were based on the phosphorylation of methoxyethylated nucleosides and nucleotides. The derivatives, 2-O- and 2,3-di-O-(alpha-methoxyethyl)uridine, were prepared by the methoxyethylation of 3,5-di-O-acetyluridine and 5-O-acetyluridine followed by removal of the acetyl groups. The corresponding guanosine derivatives were made by the synthetic routes: (i) guanosine leads to O-2,O-3,O-5,N-2-tetrabenzoylguanosine leads to 2-N-benzoylguanosine leads to O3-acetyl-N-2,O5-dibenzoylguanosine leads to 2-O-(alpha-methoxyethyl)guanosine, and (ii) 2,3-O-isopropylideneguanosine leads to N-2,O5-diacetyl-2,3-O-isopropylideneguanosine leads to N-2,O-5-diacetylguanosine leads to 2,3-di-O-(alpha-methoxyethyl)guanosine. These methoxyethylated nucleosides were converted to the corresponding 5-phosphates by reaction with cyanoethyl phosphate and dicyclohexylcarbodiimide, and then to the corresponding 5-diphosphates by subsequent reaction with 1,1-carbonyldiimidazole and inorganic phosphate.
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PMID:"Single Addition" substrates for the synthesis of specific oligoribonucleotides with polynucleotide phosphorylase. Synthesis of 2'-(alpha-methoxyethy) nucleoside 5'-diphosphates. 114 95

In this paper we examine the binding of Escherichia coli transcription termination factor rho to single-stranded RNA. Random polyribonucleotide copolymers containing low ratios of the fluorescent base 1,N6-ethenoadenosine have been synthesized using polynucleotide phosphorylase. Binding of rho to these polynucleotides elicits a significant increase in fluorescence, thus allowing either the direct monitoring of the titration of these polynucleotides with rho or measurement of the competitive displacement of the protein from these probes with other nucleic acids, even in the presence of biologically significant concentrations of ATP. By these techniques, it is shown that the binding site size (n) of rho protein to polynucleotides is 13(+/- 1) nucleotide residues per rho monomer (or 78(+/- 6) nucleotide residues per rho hexamer). Binding constants (K) and co-operativity parameters (omega) for the binding of rho to these polynucleotides have been measured as a function of nucleotide composition and of salt concentration. The results show that the affinity of rho for cytosine residues is quite strong and salt concentration independent, whilst binding to uridine residues is somewhat weaker and very salt concentration dependent. Poly(rC) and poly(dC) bind to rho competitively and with equal affinity and site size, although poly(rC) is the strongest cofactor for activating rho-dependent ATPase and poly(dC) has no ATPase cofactor activity at all. It is also shown that ATP (or ADP or ATP-gamma-S) binding does not change the binding site size of rho on RNA nor decrease its affinity for RNA binding. Circular dichroism measurements of rho binding to phage R17 RNA suggest that the affinity (K omega) of rho for RNA may be increased by ATP. The possible significance of these results for models of rho-dependent transcription termination is discussed in the companion paper.
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PMID:Interactions of Escherichia coli transcription termination factor rho with RNA. I. Binding stoichiometries and free energies. 245 Oct 28

Conversion of uridine and cytidine to their 5'-O-tosyl derivatives, followed by cyanation with tetraethylammonium cyanide, reduction and deamination, led to isolation of the hitherto unknown homouridine (1-(5'-deoxy-beta-D-allofuranosyl)uracil) and homocytidine (1-(5'-deoxy-beta-D-allofuranosyl)cytosine), analogues of uridine and cytidine in which the exocyclic 5'-CH2OH chain is extended by one carbon to CH2CH2OH. Homocytidine was also phosphorylated to its 6'-phosphate and 6'-pyrophosphate analogues. In addition, it was converted, via its 2,2'-anhydro derivative, to arahomocytidine, an analogue of the chemotherapeutically active araC. The structures of all the foregoing were established by various criteria, including 1H and 13C NMR spectroscopy, both of which were also applied to analyses of the solution conformations of the various compounds, particularly as regards the conformations of the exocyclic chains. The behaviour of the homo analogues was examined in several enzymatic systems. Homocytidine was a feeble substrate, without inhibitory properties, of E. coli cytidine deaminase. Homocytidine was an excellent substrate for wheat shoot nucleoside phosphotransferase; while homouridine was a good substrate for E. coli uridine phosphorylase. Although homoCMP was neither a substrate, nor an inhibitor, of snake venom 5'-nucleotidase, homoCDP was a potent inhibitor of this enzyme (Ki approximately 6 microM). HomoCDP was not a substrate for M. luteus polynucleotide phosphorylase. None of the compounds exhibited significant activity vs herpes simplex virus type 1, or cytotoxic activity in several mammalian cell lines.
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PMID:Pyrimidine homoribonucleosides: synthesis, solution conformation, and some biological properties. 303 11

The reaction of the tetranucleotide, pA-A(2)-A, with 2'(3')-0-(alpha-methoxyethyl)uridine 5'-diphosphate, Mg(2+) ions, and M. luteus polynucleotide phosphorylase followed by mild acid treatment to remove the blocking groups results in a 49% yield of the desired single addition product, pA-A(3)-U, together with smaller amounts of pA-A-U, pA-A-A, pA-A(2)-U, pA-A(2)-A, pA-A(3)-A, pA-A(4)-U, and pA-A(4)-A. The side products are thought to arise from the phosphorolysis of the acceptor molecule by the inorganic phosphate formed in the reaction mixture and from subsequent additions to the various oligonucleotide species by the resulting adenosine 5'-diphosphate. A system developed for the removal of inorganic phosphate as it is formed in the synthesis involves the addition to the reaction mixture of calf spleen nucleoside phosphorylase and nicotinamide riboside and, under these conditions, pA-A(3)-U can be prepared in 90% yield with essentially no side products. Under similar conditions, pA-A(3)-A, pA-A(3)-G, and pA-A(3)-C may be prepared from pA-A(2)-A and the appropriate blocked nucleoside diphosphate in yields of 85-94%. The incubation of pA-A(2)-A alone with polynucleotide phosphorylase exhibits the phenomenon of "transnucleotidation" in that the molecule is partially converted to oligonucleotides of smaller and larger chain lengths. In the presence of the phosphate removal system, however, the tetranucleotide is not attacked by the enzyme, and thus, "transnucleotidation" appears to be simply a combination of phosphorolytic and addition reactions catalyzed by trace amounts of inorganic phosphate contaminating the enzyme and/or the substrate.
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PMID:'Single addition' and 'transnucleotidation' reactions catalyzed by polynucleotide phosphorylase. Effect of enzymatic removal of inorganic phosphate during reaction. 428 Oct 80

1. Treatment of Micrococcus lysodeikticus polynucleotide phosphorylase (nucleoside diphosphate-polynucleotide nucleotidyltransferase) with trypsin causes a preferential loss of its cytidine diphosphate and uridine diphosphate polymerization activities. 2. The phosphorolytic activity of the enzyme towards polycytidylic acid is unaffected in conditions in which the cytidine diphosphate-polymerization activity without added primer is virtually abolished. 3. The treated enzyme retains its altered pattern of activities when purified fivefold by gel filtration. 4. The effect on the cytidine diphosphate-polymerization activity is due, in part, to a large increase in primer requirement as a result of proteolysis, and is qualitatively independent of the state of purity of the polynucleotide phosphorylase. 5. The enzyme is protected from trypsin degradation by nucleic acids, polynucleotides and nucleoside disphosphates. 6. A similar, but less marked differential effect, is caused by alpha-chymotrypsin.
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PMID:The effect of trypsin digestion on the activities of polynucleotide phosphorylase. 605 26

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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