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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)

A new ribonuclease has been isolated from Escherichia coli. The enzyme is present in the 100,000 times g supernatant fraction and has been purified over 200-fold. Studies of the enzyme reveal that: 1. The enzyme shows a marked preference for oligoribonucleotides; indeed, the reaction rate is inversely proportional to the chain length of the substrate. The enzyme does not attack polynucleotides even at high concentrations of enzyme and has no detectable DNase activity. 2. The enzyme is stimulated strongly by Mn2+, less strongly by Mg2+, and not at all by Ca2+ and monovalent cations. 3. The enzyme is purified free of RNase I, RNase II, RNase III, polynucleotide phosphorylase, and other known ribonucleases of E. coli. The enzyme displays identical properties when isolated from mutants of E. coli that are deficient in the above ribonucleases. 4. The enzyme has a marked thermostability, a point of further distinction from RNase II.
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PMID:A novel oligoribonuclease of Escherichia coli. I. Isolation and properties. 24 Aug 24

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

Procedures for the controlled addition of one or more deoxyribonucleotide residues to the 3' end of an oligodeoxyribonucleotide primer are described. Polynucleotide phosphorylase (EC 2.7.7.8), purified from Escherichia coli B, catalyzes the reaction using a deoxyribonucleoside 5'-diphosphate as substrate, with Mn2+ as cofactor. Reaction occurs rapidly in aqueous solution, and no protecting groups are required, simplifying recovery and purification of the products. The concentrations of sodium chloride and manganous chloride in the incubation mixture are critical to obtaining good yield of the required product. Primers of chain length from 3 to 12 have been extended by up to 9 deoxyribonucleotide residues to obtain oligodeoxyribonucleotides of chain length up to 13. Yields of single addition products varied from 8 to 59%. Factors which influence these yields are discussed. The effects of added polyamines and some organic solvents on the reaction are described. Spermidine or dimethylsulfoxide in the incubation medium tend to favor the addition of several residues of deoxyribonucleotide to the primer.
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PMID:Enzymatic synthesis of oligodeoxyribonucleotides of defined sequence. 63 85

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

The 6-aza analogues of toyocamycin and sangivamycin were prepared as potential cytotoxic agents. The toyocamycin analogue (4-amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine-3-carbonitrile) could not be obtained directly from its O-acetylated precursor but was accessible via 4-amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine-3-thiocarboxamide. The identity of the nitrile was verified by its ultraviolet, infrared, and mass spectra, and by its conversion to the corresponding 3-carboxamide and thiocarboxamide when treated with water or hydrogen sulfide, respectively. Bioassay of the synthetic compounds in comparison with 4-amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (6-azatubercidin) and 4-amino-2-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine revealed that the 3-thiocarboxamido derivative was more cytotoxic to the growth of mouse fibroblasts than 6-azatubercidin, effecting killing of 3T6 cells at less than or equal to 1 mug/ml. 4-Amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (but not its 2-ribofuranosyl isomer) was shown to act as a substrate for adenosine deaminase from calf intestinal mucosa with an apparent Km of 125 (vs. 20 for adenosine) and the corresponding 5'-diphosphate of 6-azatubercidin was polymerized by polynucleotide phosphorylase (Micrococcus luteus) in the presence of Mn2+ to afford a homopolymer and copolymers with adenosine. The copolymers directed the binding of [3H]lysyl-tRNA to the A-site of ribosomes from Escherichia coli, but could not be used for the synthesis of polylsine in a cellfree system. The copolymer consiting of adenosine and 6-azatubercidin in a 2:1 ratio was found to form a 1:1 complex with poly(uridylic acid) at 4degreesC.
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PMID:Synthesis and biological activity of pyrazolo[3,4,-d]pyrimidine nucleosides and nucleotides related to tubercidin, toyocamycin, and sangivamycin. 76 33

2'-O-o-Nitrobenzyluridine, -cytidine and -adenosine were phosphorylated with phosphoryl chloride to the corresponding 5'-phosphates and led to 5'-diphosphates by the method of Moffatt and Khorana. These 2'-O-oNB-nucleoside 5'-diphosphates were incubated with a primer CpApA and polynucleotide phosphorylase in the presence of Mn2+. Tetranucleotides CpApApU, CpApApC and CpApApA were obtained after photosensitive removal of oNB groups in yields of 54-70%.
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PMID:Polynucleotides. XLII1. Limited addition of 2'O-onitrobenzyl nucleotides to the 3'-end of ribooligonucleotide with polynucleotide phosphorylase. 100 16

Polymerization of 2'-O-methylcytidine-5'-diphosphate (CmDP) with polynucleotide phosphorylase in the presence of Mn2+ proceeds with 65% yield after 72 h, and in the presence of Mg2+ the yield does not exceed 10%. Phosphorolysis of poly 2'-O-methylcytidylic acid and poly 2'-O-methyluridylic acid, as well as exchange of the beta-phosphate group of CmDP in the presence of Mn2+ and Mg2+, proceed with a yield of only a few percent. A possible mechanism of Mn2+ action on CmDP polymerization is discussed.
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PMID:Role of Mn2+ in the reaction of polynucleotide phosphorylase with 2'-O-methylated substrates. 115 44

We have characterized a chloroplast processing activity that catalyzes the conversion of the plastid cytochrome b6/f subunit IV (pet D) mRNA 3' end precursor to the mature RNA possessing a 3' inverted repeat (IR). In a chloroplast soluble protein extract, the activity requires Mg2+ or Mn2+, but not K+. In the absence of Mg2+, the pet D 3' IR-RNA product does not accumulate, and UV-cross-linking indicates that the 3' IR-RNA precursor binds several new proteins in addition to those previously characterized as part of the 3' IR-RNA: protein complex in vitro. In contrast, high concentrations of Zn2+ or Cu2+ suppress protein binding and inhibit the processing reaction. The purified exoribonuclease polynucleotide phosphorylase (E.C.2.7.7.8) is not efficient in processing the pet D 3' IR-RNA precursor, whereas Escherichia coli ribonuclease II rapidly processes the pet D IR-RNA precursor to a product of a size similar to that of the mature 3' IR-RNA, but also rapidly degrades the mature RNA in the absence of chloroplast extract. We therefore conclude that the maturation of the pet D mRNA in vitro requires specific chloroplast enzymes which process the mRNA 3' end precursor in the absence of efficient transcription termination. The chloroplast enzyme activities are biochemically distinct from their bacterial counterparts. We also note that specific chloroplast components may be required to stabilize the mature pet D mRNA 3' end against further exonucleolytic degradation.
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PMID:Chloroplast mRNA 3' end maturation is biochemically distinct from prokaryotic mRNA processing. 248 89

Novel RNA polymerase activities (termed type II reaction) can be found in toluene-treated Escherichia coli with Ca2+, Fe2+, or endogenously bound cations, probably Mg2+. These activities are distinguishable from the well characterized DNA-dependent RNA polymerase (type I reaction) by: (i) their divalent cation requirements, i.e., the classical enzyme is activated by exogenously added Mn2+, Mg2+, or CO2+ ions; (ii) their relative resistance to inhibition by actinomycin D, rifampicin, and streptolydigin; (iii) their selective synthesis of low molecular weight RNA; (iv) their sensitivity to inhibition by arabinonucleoside 5'-triphosphates or deoxyribonucleoside 5'-triphosphates; and (v) the strict requirement for ATP in Ca2+ and bound cation-activated reactions. The Ca2+-activated and endogenous RNA polymerase activities are inhibited by orthophosphate. The properties of the type II RNA polymerase(s) are compared with those of polynucleotide phosphorylase, and dnaG gene product, and the RNA polymerase described by Ohasa and Tsugita.
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PMID:Divalent cation-activated RNA synthesis in toluene-treated Escherichia coli. 617 Apr 2

1. Polynucleotide phosphorylase [polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8] was purified to near homogeneity from the photosynthetic bacterium, Rhodospirillum rubrum. The purified enzyme had a molecular weight of approximately 160,000, and consisted of two equivalent subunits of approximately 76,000 daltons. It catalyzed the three reactions described below. 2. In the exchange reaction of the beta-phosphate of nucleoside diphosphates with Pi by the purified enzyme in the presence of 3.3 mM Pi, 6.7 mMCl2, and 0.33 mM or 1.0 mM nucleotide at pH 8.0 and 20 degrees C, ADP, GDP, and CDP, and CDP were better substrates than UDP, while IDP and deoxyribonucleoside diphosphates hardly served as substrates. The ADP-Pi exchange activity was significantly inhibited by high concentrations of either ADP or Pi. 3. In the polymerization reaction of ribonucleoside diphosphates by the purified enzyme in the presence of 6.7 mM nucleotide and 6.7 mM MgCl2 at pH 8.0 and 20 degrees C, ADP was the best substrate; the activities relative to that with ADP were 55% with UD, 51% with CDP, and 48% with IDP, while GDP hardly served as a substrate, 4. In the phosphoryolysis reaction of polynucleoside diphosphates by the purified enzyme in the presence of 1.0 mM polynucleotide, 6.7 mM Pi, and 6.7 mM MgCl2 at pH 8.0 and 20 degrees C, poly[U] was the best substrate; the activities relative to that with poly[U] were 32% with poly[A], 28% with poly[I], 21% with poly[C], and 2% with yeast RNA, while poly[G] and yeast DNA hardly served as substrates. 5. The three kinds of activities of the purified enzyme described above were stimulated by divalent cations such as Mg2+, Mn2+, Cd2+, and Co2+.
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PMID:Purification and properties of polynucleotide phosphorylase from photosynthetic bacterium Rhodospirillum rubrum. 676 23


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