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 release of lipoteichoic acid and mesosomal vesicles to the supernatant buffer during the formation of spherical, osmotically fragile bodies was studied using Streptococcus faecalis ATCC 9790. Autolytic N-acetylmuramidase action was permitted to take place in exponential-phase cells incubated in a buffer which provides an exceptional degree of osmotic stabilization. Both lipoteichoic acid and mesosomal vesicles were relatively rapidly released to the supernatant buffer. Most of the cellular content of lipoteichoic acid (and mesosomal vesicles) was found in the supernatant buffer at incubation times when the cells still retained over 75% of their cell wall. [14-C]- or [3-H]glycerol was used as a label for both cellular lipoteichoic acids and lipid-glycerol. Glycerol in lipoteichoic acid was quantitated after phenol-water and chloroform-methanol treatments and identified by products of acid hydrolysis and its ability to be precipitated by (i) antibodies specific for the polyglycerol-phosphate backbone, (ii) antibodies to the streptococcal group D antigen, and (iii) concanavalin A. Evidence was obtained that lipoteichoic acid was not associated with isolated mesosomal vesicles. Centrifugation of supernates at 200,000 X g sedimented membranous (mesosomal) vesicles and nearly all of the lipid-glycerol present, whereas essentially all of the lipoteichoic acid remained in the supernatant. The sedimented mesosomal vesicles differed from protoplast membrane in their higher lipid-phosphorus to protein ratio and in the absence of detectable levels of two enzymatic activities found in protoplast membranes, adenosine triphosphatase and polynucleotide phosphorylase. Both types of membranes were found to contain DD-carboxypeptidase and LD-transpeptidase activities at nearly the same specific activities. No evidence was obtained for the association of autolytic N-acetylmuramidase activity with either type of membrane preparation.
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PMID:Cellular localization of lipoteichoic acid in Streptococcus faecalis. 80 56

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

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

A thermophilic polynucleotide phosphorylase lacking polynucleotide phosphoryltic activity was purified from Thermus thermophilus HB-8 strain. The enzyme is an altered form of the native polynucleotide phosphorylase, probably attacked by the proteinase(s) of this extreme thermophile during the purification process. This modified enzyme lacks phosphorolytic activity to poly(A) while retaining weak activity to phosphorolyse tetranucleotides or hexanucleotides. The purified enzyme was shown to be homogenous by electrophoretic analysis in polyacrylamide gel. This enzyme had a molecular weight of 190 000 as calculated both from electrophoresis on polyacrylamide gel and from the Stoke's radius derived from the gel filtration pattern and the sedimentation coefficient. The enzyme was separated into three polypeptide chains by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate; their molecular weights were calculated to be 92000, 73000 and 35000. The enzyme was thermophilic and thermotolerant, exhibiting its maximal activity at 70 degrees C. The four ribonucleoside diphosphates (ADP, GDP, UDP and CDP) were polymerized to the extent of 7-S size.
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PMID:Thermophilic polynucleotide phosphorylase from Thermus thermophilus. Purification and properties of an altered form of enzyme which lacks phosphorolytic activity to polynycleotide. 89 51

The polymerization of 2'deoxy-2'-fluoro-cytidine-diphosphate (dCflDP) by polynucleotide phosphorylase is barely detectable in the presence of Mg++ under usual experimental conditions for polymerization of nucleoside diphosphates. High concentrations of enzyme have to be used to accomplish the synthesis. Mn++ is a better activator than Mg++ for the reaction. cCflDP inhibits the polymerization of CDP and has a Km=8.8X10-3M, six times higher than CDP.- The polymer, poly (dCfl), ressembles in many respects poly(C), but not poly(dC): the acid selfstructure forms at similar pK's; interaction with poly(I) yields a 1:1 complex the CD spectrum of which is similar to that of poly(I).poly(C). Finally, the Tm's of poly(I).poly(dCfl) are comparable to those of poly(I).poly(C).
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PMID:Poly-2'-deoxy-2'-fluoro-cytidylic acid: enzymatic synthesis, spectroscopic characterization and interaction with poly-inosinic acid. 89 78

Poly (2'-azido-2'-deoxyinosinic acid), [poly (Iz)], was synthesized from 2'-azido-2'-deoxyinosine diphosphate by the action of polynucleotide phosphorylase. Poly (Iz) has UV absorption properties similar to poly (I) and hypochromicity of 11% at 0.15M Na+ and neutrality. In solutions of high Na+ ion concentration, poly (Iz) forms a multi-stranded complex and its Tm at 1.0M Na+ ion concentration was 43 degrees. Upon mixing with poly (C), poly (Iz) forms a 1:1 complex having a Tm lower than that of poly (I)-poly (C) complex in the same conditions. The effect of substitution at the 2'-position of the poly (I) strand was discussed in relation to the interferon-inducing activity.
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PMID:Polynucleotides. XLV Synthesis and properties of poly(2'-azido-2'-deoxyinosinic acid). 90 87

The nucleotide analogues, O6-methyl- and O6-ethylguanosine diphosphate, have been synthesized and polymerized to high-molecular-weight homopolymers with polynucleotide phosphorylase. The ultraviolet spectra of these polymers show marked hypochromicity, which suggests that they possess considerable secondary structures. Graphs of optical denisty vs. temperature in 0.15 M NaC1 indicate that cooperative melting occurs for both polymers, and that the secondary structure of poly(O6-methylguanosine monophosphate) is somewhat more stable than that of poly (O6-ethyl-guanosine monophosphate). Mixing experiments show that these analogue polymers no longer form helical structures with poly(C), nor do thye form helices with poly(U). We would conclude from these data that environmental mutagens and carcinogens which react at the O6 position of guanine not only disrupt normal base-pairing relationships, but may also affect the secondary structure of nucleic acids.
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PMID:Synthesis and properties of poly(O6-methylguanylic acid) and poly(O6-ethylguanylic acid). 96 38

The synthesis of poly(2-amino-6-chloropurinylic acid) [poly(n2cl6Pu)] by the polynucleotide phosphorylase catalyzed polymerization of 2-amino-6-chloro-9-(beta-D-ribofuranosyl)purine 5'-diphosphate and its chemical conversion to poly(6-thioguanylic acid) [poly(s6G)] is described. Poly(s6G) was found to form a relatively unstable complex with poly(C), the properties of which were incompatible with those previously reported for the same complex prepared by another method [Darlix, J.L., Fromageot, P., and Reich, E. (1973), Biochemistry 12, 914]. It was found that poly(s6G) could be thermally converted to a copolymer of which with poly(C) was strikingly similar to that reported earlier for poly(s6G)-poly(C).
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PMID:Polyribonucleotides containing thiopurines: synthesis and properties of poly (6-thioguanylic acid). 97 65

Under the conditions that RNA ligase converts the tetranucleotide, pA-A2-A, to larger polynucleotides, no such polymerization can be detected with the derivative, pA-A2-A(MeOEt), that possesses a terminal 2'-0-(alpha-methoxyethyl) group. The protection against self condensation offered by the methoxyethyl group in this system allows the specific joining of donor and acceptor oligonucleotides in reaction mixtures containing equimolar concentrations of the two species. Thus, the enzyme, together with ATP, converts equimolar quantities of A-A2-A and pA-A2-A(MeOEt) to A-A6-A(MeOEt) in 55% yield, while a similar reaction with A-A2-A and pU-U2-U(MeOEt) results in a 40% yield of A-A3-U3-U(MeOEt). The intermediate in these ligations is a disubstituted pyrophosphate composed of the donor molecule and the adenylate moiety deriving from ATP. In the case of the intermediate arising from the blocked adenosine tetranucleotide, the assigned structure, A5'pp5'A-A2-A(MeOEt), has been confirmed by chemical synthesis. The pyrophosphate derivative is able to participate in joining reactions in the absence of ATP. These observations constitute an efficient approach to the synthesis of larger polynucleotides from a specific series of oligonucleotide blocks since (i), the methoxyethyl group can be easily introduced into each oligonucleotide using the single addition reaction catalyzed by polynucleotide phosphorylase in the presence of a 2'-0-(alpha-methoxyethyl)nucleoside 5'-diphosphate, and (ii), the blocking group may be readily removed under mild conditions after each successive ligation reaction. Two other octanucleotides, I-I2-A-U3-U and U-U2-C-I3-A, have also been synthesized by this method, and these molecules correspond (with I substituting for G) to sequences appearing near the 3' terminus of the 6S RNA transcribed from phage lambda DNA. The terminal 3'-phosphate group serves equally well as a blocking group for specific ligation reactions in that the ligase converts equimolar amounts of A-A2-A and pA-A2-Ap to A-A6-Ap in 50% yield.
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PMID:The use of terminal blocking groups for the specific joining of oligonucleotides in RNA ligase reactions containing equimolar concentrations of acceptor and donor molecules. 100 14


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