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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)

We report here the presence of two enzymatic activities associated with highly purified preparations of polynucleotide phosphorylase from Micrococcus luteus. The first, a nuclease activity, which is not separated from the phosphorylase on hydroxylapatite, may be due to substitution of H2O for phosphate in the phosphorolysis reaction. The second activity, a deoxyadenylate kinase, the bulk of which is not resolved from the phosphorylase using gel filtration, sucrose density gradient centrifugation, DEAE-Sephadex, or hydroxylapatite chromatography, may represent a new activity of polynucleotide phosphorylase or be due to an enzyme which is tightly bound to the phosphorylase. Several properties of the kinase are described and its possible significance with respect to the overall enzyme mechanism is discussed.
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PMID:A deoxyadenylate kinase activity associated with polynucleotide phosphorylase from Micrococcus luteus. 18 14

A simple method for the preparation of adenosine 5'-[beta-32 P]triphosphate is described. When [32P]orthophosphate was incubated with polyadenylate and phosphoenolpyruvate in the presence of polynucletide phosphorylase (EC 2.7.7.8) and pyruvate kinase (EC 2.7.1.40), up to 75% of 32P radioactivity was recovered in ATP. [32P]ATP was purified to 99.5% radiochemical purity by chromatography on polyethyleneimine-cellulose thin-layer plates. Analysis of hydrolysis products of [32P]ATP with apyrase (EC 3.6.1.5) indicates that 32P in the beta-phosphate position accounts for all 32P label in ATP.
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PMID:Enzymic preparation of adenosine 5'-[beta-2P]triphosphate. 88 81

Mutants having low levels of polynucleotide phosphorylase activity grow poorly at 45 C. All revertants isolated for their ability to grow better at that temperature also regained higher levels of polynucleotide phosphorylase and the ability to be induced for tryptophanase. Thus, a physiological role is implied for the enzyme polynculeotide phosphorylase.
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PMID:Polynucleotide phosphorylase has a role in growth of Escherichia coli. 457 Jul 76

Polyadenylylated mRNA was purified from poly(I).poly(C)- and cycloheximide-superinduced human fibroblast (FS-4) cultures. The mRNA was subjected to electrophoresis through an agarose/CH3HgOH gel, and human fibroblast beta 1 and beta 2 interferon mRNAs were isolated. Each mRNA preparation was phosphorolyzed at 0 degrees C for 20 min by using a molar excess of polynucleotide phosphorylase to produce RNAs lacking poly(A) and then incubated at 37 degrees C for varying lengths of time to allow the phosphorylase to further digest the deadenylylated RNA from the 3' end in a processive and synchronous manner. Removal of the poly(A) (less than or equal to 100 residues) and approximately 100 adjacent residues from human fibroblast beta 1 interferon mRNA (native length, 900 residues, including a 3'-noncoding region of 203 residues) did not alter the translational activity or the functional stability of this mRNA in Xenopus oocytes, whereas deletion of the poly(A) and approximately 200 adjacent residues decreased its translational efficiency. On the other hand, removal of the poly(A) (approximately 200 residues) and approximately 200 adjacent residues from human fibroblast beta 2 interferon mRNA (native length, 1300 residues) did not alter the translational activity or the functional stability of this molecule in oocytes. Thus, neither the poly(A) nor large segments of the 3'-noncoding region (which includes the hexanucleotide A-A-U-A-A-A sequence, at least in the case of beta 1 mRNA) are required for the maintenance of the functional stability of human beta 1 and beta 2 interferon mRNAs in Xenopus oocytes.
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PMID:Translational activity and functional stability of human fibroblast beta 1 and beta 2 interferon mRNAs lacking 3'-terminal RNA sequences. 616 16

Restoration of the ability to catabolise the purine nucleosides in phenotypic revertants of Escherichia coli K-12 mutants defective in deoD encoded purine nucleoside phosphorylase (PNPase 1) is the result of regulatory pndR mutations for synthesis of a second purine nucleoside phosphorylase (PNPase 2). In pndR+ strains synthesis of PNPase 2 is induced by xanthosine; in pndR mutants catabolising all purine nucleosides synthesis of this enzyme is constitutive; in other pndR mutants only catabolising some of purine nucleosides, this catabolisible nucleosides, namely, deoxyinosine, deoxyadenosine as well as, in some cases, inosine and adenosine, act as inducers of PNPase 2 synthesis. In some pndR mutants with inducible PNPase 2, xanthosine is a stronger inducer, in others it is weaker, in comparison with pndR+ strains. In bacterial cells PNPase 2 catalyses the phosphorolytic cleavage of adenosine, inosine, deoxyinosine, guanosine, deoxyguanosine and xanthosine, though in crude extracts adenosine and deoxyadenosine phosphorylase activities of the enzyme are not expressed.
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PMID:[Regulatory mutants for the synthesis of a 2d purine nucleoside phosphorylase in Escherichia coli K-12. I. Synthesis inducers and the substrate specificity of purine nucleoside phosphorylase in pndR mutants]. 643 6

The diastereomers of adenosine 5'-O-(1-thiodiphosphate) (ADP alpha S) have been tested as substrates for the polymerization reaction of primer-independent polynucleotide phosphorylase from Micrococcus luteus. The preferred substrate is ADP alpha S(Sp), which has a similar Km and a greatly reduced Vmax when compared to the natural substrate ADP. The other diastereomer, ADP alpha S(Rp), is preferentially cleaved by a polyphosphate kinase activity (present with the phosphorylase) that may be responsible for the removal of the 5'-beta-phosphate during de novo polymerization, leading to the observed 5'-phospho-poly(A). Inhibitor studies suggest that the kinase and de novo polymerization sites are not coincident. During de novo polymerization of the diastereomeric mixture, ADP alpha S(Rp) is selectively used to form 5' termini, whereas ADP alpha S(Sp) serves to support the chain elongation. Thus there are two stereochemically distinct subsites for initiating polymerization. ADP beta S functions as a substrate for polynucleotide phosphorylase with kinetic properties similar to those of ADP, indicating that removal of the beta-phosphate (a thiophosphate) is not a kinetically important step and probably occurs after polymerization is complete. The average chain length of the polymeric product is considerably smaller for ADP alpha S vs. ADP beta S or ADP, suggesting that the degree of processivity of the polymerization is determined by competition between the rate of polymerization and the rate of dissociation of the growing chain.
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PMID:On the mechanism of de novo polymerization by form I polynucleotide phosphorylase of Micrococcus luteus. 709 93

The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation by polynucleotide phosphorylase (PNPase). In Escherichia coli, polyadenylation is performed mainly by poly(A)-polymerase (PAP) I or by PNPase in its absence. While trying to purify the chloroplast PAP by following in vitro polyadenylation activity, it was found to copurify with PNPase and indeed could not be separated from it. Purified PNPase was able to polyadenylate RNA molecules with an activity similar to that of lysed chloroplasts. Both activities use ADP much more effectively than ATP and are inhibited by stem-loop structures. The activity of PNPase was directed to RNA degradation or polymerization by manipulating physiologically relevant concentrations of P(i) and ADP. As expected of a phosphorylase, P(i) enhanced degradation, whereas ADP inhibited degradation and enhanced polymerization. In addition, searching the complete Arabidopsis genome revealed several putative PAPs, none of which were preceded by a typical chloroplast transit peptide. These results suggest that there is no enzyme similar to E. coli PAP I in spinach chloroplasts and that polyadenylation and exonucleolytic degradation of RNA in spinach chloroplasts are performed by one enzyme, PNPase.
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PMID:Polynucleotide phosphorylase functions as both an exonuclease and a poly(A) polymerase in spinach chloroplasts. 1146 23

Polymer formation is arguably one of the essential factors that allowed the emergence, stabilisation and spread of life on Earth. Consequently, studies concerning biopolymers could shed light on the origins of life itself. Of particular interest are RNA and polysaccharide polymers, the archetypes of the contrasting proposed evolutionary scenarios and their respective polymerases. Nucleic acid polymerases were hypothesised, before their discovery, to have a functional similarity with glycogen phosphorylase. Further identification and characterisation of nucleic acid polymerases; particularly of polynucleotide phosphorylase (PNPase), provided experimental evidence for the initial premise. Once discovered, frequent similarities were found between PNPase and glycogen phosphorylase, in terms of catalytic features and biochemical properties. As a result, PNPase was seen as a model of primitive polymerase and used in laboratory precellular systems. Paradoxically, however, these similarities were not sufficient as an argument in favour of an ancestral common polymerisation mechanism prior to polysaccharides and polyribonucleotides. Here we present an overview of the common features shared by polymer phosphorylases, with new proposals for the emergence of polysaccharide and RNA polymers.
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PMID:Polymer phosphorylases: clues to the emergence of non-replicative and replicative polymers. 2178 67