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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 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

1. Trypsin digestion of Micrococcus lysodeikticus polynucleotide phosphorylase (nucleoside diphosphate-polynucleotide nucleotidyltransferase) causes a progressive increase in electrophoretic mobility in polyacrylamide gels of the single active degradation product. 2. A marked increase in primer requirement for CDP polymerization occurs before a more mobile product is formed. 3. alpha-Chymotrypsin digestion yields a product that separates into several active species on polyacrylamide-gel electrophoretograms. 4. No separation of ADP-and CDP-polymerization activities occurs during electrophoresis after either trypsin or alpha-chymotrypsin treatment.
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PMID:A study by polyacrylamide-gel electrophoresis of the effect of proteolysis on Micrococcus lysodeikticus polynucleotide phosphorylase. 570 78

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

Genome comparison permits identification of chromosome regions conserved during evolution. Bacillus subtilis and Escherichia coli are so distant that there exists very few conserved landmarks in their genome organisation. Analysis of the conserved cmk rpsA cluster pinpointed the importance of cytosine nucleotide metabolism. In these bacteria, mRNA turnover provides an efficient means to fulfil the need for CDP as a precursor of DNA synthesis. The cmk rpsA operon is responsible for CDP synthesis. This function is self-explained in the case of the cmk gene (which codes for cytidylate kinase). The case of rpsA, that codes for ribosomal protein S1, is more subtle. It is suggested here that S1 is a RNA-binding protein helping polynucleotide phosphorylase (PNPase, known to be phylogenetically related to S1) to degrade mRNA, or helper molecule involved in other RNase activities. This provides an explanation for the elusive function of PNPase, which generates nucleoside diphosphates (not monophosphates) when degrading RNA. This also accounts for the discovery that the B. subtilis comR gene product is PNPase. This article briefly discusses the availability of cytosine nucleotides in eukaryotes, and suggests that they are derived from phospholipids turnover. Finally, the GC content of genomes is discussed in this new light.
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PMID:Comparison between the Escherichia coli and Bacillus subtilis genomes suggests that a major function of polynucleotide phosphorylase is to synthesize CDP. 917 91

We examined the activity of polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli in phosphorolysis using substrates derived from the rpsO-pnp operon of S. coelicolor. The Streptomyces and E. coli enzymes were both able to digest a substrate with a 3' single-stranded tail although E. coli PNPase was more effective in digesting this substrate than were the Streptomyces enzymes. The kcat for the E. coli enzyme was ca. twofold higher than that observed with the S. coelicolor enzyme. S. coelicolor PNPase was more effective than its E. coli counterpart in digesting a substrate possessing a 3' stem-loop structure, and the Km for the E. coli enzyme was ca. twice that of the S. coelicolor enzyme. Electrophoretic mobility shift assays revealed an increased affinity of S. coelicolor PNPase for the substrate possessing a 3' stem-loop structure compared with the E. coli enzyme. We observed an effect of nucleoside diphosphates on the activity of the S. coelicolor PNPase but not the E. coli enzyme. In the presence of a mixture of 20 microM ADP, CDP, GDP, and UDP, the Km for the phosphorolysis of the substrate with the 3' stem-loop was some fivefold lower than the value observed in the absence of nucleoside diphosphates. No effect of nucleoside diphosphates on the phosphorolytic activity of E. coli PNPase was observed. To our knowledge, this is the first demonstration of an effect of nucleoside diphosphates, the normal substrates for polymerization by PNPase, on the phosphorolytic activity of that enzyme.
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PMID:Kinetics of polynucleotide phosphorylase: comparison of enzymes from Streptomyces and Escherichia coli and effects of nucleoside diphosphates. 1796 56

A new, homogeneous, high-throughput-compatible assay method is described for the fluorescence-based quantitation of nanomolar concentrations of ribonucleoside diphosphates (rNDPs). The principle of the method is the conversion of the rNDPs to RNA by the enzyme polynucleotide phosphorylase (EC 2.7.7.8) and detection of the RNA by the increased fluorescence of a commercial nucleic acid detection dye. A commercial RNA homopolymer complementary to the RNA product is included to increase the sensitivity for ADP and UDP. Standard curves for nanomolar concentrations of ADP, UDP, GDP, and CDP are shown. The assay detected 75 nM ADP produced by the pyruvate kinase-catalyzed phosphorylation of pyruvate with a signal-to-baseline ratio of 2.8. The assay may be used in either a continuous or a discontinuous mode.
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PMID:High-throughput, homogeneous, fluorescence intensity-based measurement of adenosine diphosphate and other ribonucleoside diphosphates with nanomolar sensitivity. 2157 Sep 43


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