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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 reduction of nucleic acid by an endogenous polynucleotide phosphorylase and ribonuclease in cells of Brevibacterium JM98A (ATCC 29895) was studied. A simple process was developed for the activation of the endogenous RNA-degrading enzyme(s). RNA degradation was activated by the presence of Pi with 14.2 mumol of ribonucleoside 5'-monophosphate per g of cell mass accumulating extracellularly. The optimum pH for degradation of RNA was 10.5 and the optimum temperature was 55 to 60 degrees C. Enzymatic activity was inhibited by the presence of Ca2+, Zn2+, or Mg2+. Although some of the RNA-degrading enzymatic activity was associated with the ribosomal fraction, most was soluble. Both polynucleotide phosphorylase and ribonuclease activities were identified.
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PMID:Reduction of endogenous nucleic acid in a single-cell protein. 3 4

In crude extracts of T2L phage-infected Escherichia coli cells an enzyme activity was found that produced poly(A) from ATP as substrate. Purification of the extract led to the isolation of two enzymes, a polynucleotide phosphorylase and an ATPase. The polynucleotide phosphorylase possessed the same properties as the well-known enzyme from uninfected cells and its molecular weight was about 265 000. The ATPase was purified to over 90% purity; its molecular weight was estimated to be about 165 000 with three subunits of 55 000. The characterization of this enzyme showed that it was different from any ATPase known so far. Mg2+ cannot be replaced by Ca2+, as it can from the membrane-bound ATPases. The only product yielded by the enzyme was ADP; it was very specific for ATP, other ribonucleotide triphosphates being practically unaffected. The rate of ATP splitting was found to be very high, the turnover number being 2.51 X 10(4) min-1 at 37 degrees C. Even at 0 degree C the enzyme was still active. The optimal assay conditions for ATPase turned out to be very similar to those of polynucleotide phosphorylase. Thus the combination of the two enzymes very efficiently produced poly(A) from ATP. In this combination the polynucleotide phosphorylase was the rate-limiting enzyme, since its turnover number was about 40 times lower than that of the ATPase. The evaluation of a variety of properties of the poly(A)-synthesizing constituent found in the crude extracts led us to conclude that this activity arises from the combined action of ATPase and polynucleotide phosphorylase, and is not due to a poly(A) polymerase.
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PMID:Poly(A) synthesis in T2L phage-infected Escherichia coli. A combination of polynucleotide phosphorylase and ATPase. 12 62

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

Poly(4-thiouridylic acid) [poly(s4U)] synthesized by polymerization of 4-thiouridine 5'-diphosphate with Escherichia coli polynucleotide phosphorylase (EC 2.7.7.8) acts as messenger RNA in vitro in a protein-synthesizing system from E. coli. It stimulates binding of Phe-tRNA to ribosomes both in the presence of EF-Tu-Ts at 5 mM Mg2+ concentration and nonenzymatically at 20 mM Mg2+ concentration. It codes for the synthesis of polyphenylalanine. Poly(s4U) competes with poly(U) for binding to E. coli ribosomes. Light of 330 nm photoactivates poly(s4U) thus making it a useful photoaffinity label for the ribosomal mRNA binding site. Upon irradiation of 70-S ribosomal complexes, photoreaction occurs with ribosomal proteins as well as 16-S RNA. Ribosomes pre-incubated with R17 RNA are protected against the photoaffinity reaction. The labelling of 16-S RNA can be reduced by treatment of ribosomes with colicin E3.
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PMID:Poly(4-thiouridylic acid) as messenger RNA and its application for photoaffinity labelling of the ribosomal mRNA binding site. 32 11

Poly(A) synthesis and degradation have been examined in Escherichia coli cells made permeable to nucleotides by treatment with toluene. Although newly synthesized poly(A) is normally rapidly degraded in this system, extraction of the soluble portion of the cell effectively eliminates this process without affecting poly(A) synthesis. Poly(A) synthesis in this system displays many properties associated with poly(A) synthesis by purified poly(A) polymerase in vitro including a lag in polymerization, stimulation by increased ionic strength, and a low Mg2+ optimum. As with the purified enzyme, this system uses both ADP and ATP as substrates, requires conversion of ATP to ADP, and is strongly inhibited by dADP, orthophosphate, and pyrophosphate. In contrast to the purified poly(A) polymerase, the permeable cell system displays some properties suggestive of in vivo poly(A) metabolism. Thus, the permeable cells require an endogenous RNA primer for activity, the poly(A) product remains with the cells, and the reaction is greatly stimulated by polyamines. This system should prove extremely useful for studies of poly(A) metabolism in E. coli. A surprising feature of these studies was the finding that mutant strains deficient in polynucleotide phosphorylase were unable to synthesize poly(A). The possible roles of polynucleotide phosphorylase and poly(A) in E. coli are discussed.
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PMID:Synthesis and degradation of poly(A) in permeable cells of Escherichia coli. 35 56

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

Polyguanylic acid (poly(G)) was synthesized from GDP in a yield of 60-75% by Thermus thermophilus polynucleotide phosphorylase (polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8) at 70 degrees C, pH 8.5 in the presence of Mg2+. The yield was dependent on the ratio of GDP to Mg2+, but was independent of the concentrations of enzyme or substrate. The maximal rate of GDP polymerization was obtained when the ratio of GDP to Mg2+ was 3:1. However, by prolonged incubation, the higher initial ratio of over 4:1 was preferred because of the rapid consumption of GDP in the reaction mixture. Poly(G) prepared by 1 h incubation was heterogeneous in size from 5 S to over 23 S, but by prolonged incubation of 19 h the size of product converged to 9 S as judged by sucrose density gradient centrifugation. Its chain length was determined by terminal nucleoside analysis to be 200 nucleotides long.
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PMID:An efficient synthesis of polyguanylic acid by a thermophilic polynucleotide phosphorylase. 88 4

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

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


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