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)

Poly (2'-amino-2'-deoxyadenylic acid) [poly (Aa)] was prepared from chemically synthesized 2'-amino-2'-deoxy-ADP by the catalysis of polynucleotide phosphorylase. Poly (Aa) showed a similar UV absorption spectra to poly (A), but quite different CD spectra at pH 7.0 and 5.7. At the former pH it showed a single negative Cotton band and at the latter a curve with a large splitting of bands. Acid titration of poly (Aa) suggested protonated form below pH 7.0. Temperature absorption profiles and their dependency on sodium ion concentration suggested an ordered structure for poly (Aa) which is stabilized by stacking of bases and intrastrand interaction between 2'-amino and internucleotidic phosphate groups. Poly (Aa) forms a 1:2 complex with poly (U) at neutrality and its Tm was 45 degrees in the presence of 0.15M sodium ion.
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PMID:Polynucleotides. XLVI. 1 Synthesis and properties of poly (2'-amino-2'-deoxyadenylic acid). 1 2

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

An isotopic shift of the (31)P nuclear magnetic resonance due to (18)O bonded to phosphorus of 0.0206 ppm has been observed in inorganic orthophosphate and adenine nucleotides. Thus, the separation between the resonances of (31)P(18)O(4) and (31)P(16)O(4) at 145.7 MHz is 12 Hz and, in a randomized sample containing approximately 50% (18)O, all five (16)O-(18)O species are resolved and separated from each other by 3 Hz. Not only does this yield the (18)O/(16)O ratio of the phosphate but, more important, the (18)O-labeled phosphate in effect can serve as a double label in following phosphate reactions, for oxygen in all cases and for phosphorus, provided the oxygen does not exchange with solvent water. Thus, it becomes possible to follow labeled phosphorus or labeled oxygen continuously as reactions proceed. Rate studies involving (i) phosphorus and (ii) oxygen are illustrated by continuous monitoring of the exchange reactions between (i) the beta phosphate of ADP and inorganic phosphate catalyzed by polynucleotide phosphorylase and (ii) inorganic orthophosphate and water catalyzed by yeast inorganic pyrophosphatase. In the ADP-P(i) exchange, the P(i) ((18)O(4)) yielded an alpha P((16)O(3) (18)O) and a beta P((18)O(4)), proving that bond cleavage occurs between the alpha P and the alpha-beta bridge oxygen. Among the many additional potential uses of this labeling technique and its spectroscopic observation are: (i) different labeling of each phosphate group of ATP, (ii) to follow rate of transfer of (18)O from a nonphosphate compound such as a carboxylic acid to a phosphate compound, and (iii) to follow the rate of scrambling (for example, of the beta-gamma bridge oxygen of ATP to nonbridge beta P positions) and simultaneously the rate of exchange of the gamma P nonbridge oxygens with solvent water in various ATPase reactions.
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PMID:Isotopic (18O) shift in 31P nuclear magnetic resonance applied to a study of enzyme-catalyzed phosphate--phosphate exchange and phosphate (oxygen)--water exchange reactions. 20 29

Native Escherichia coli polynucleotide phosphorylase can be retained on blue-dextran--Sepharose. The bound enzyme cannot be displaced by its mononucleotide substrates such as ADP, UDP, CDP, GDP and IDP, but it is easily eluted by its polymeric substrates. Under identical conditions, lactate dehydrogenase, bound on blue-dextran--Sepharose, is not eluted by poly(I) but can be specifically displaced by NADH. On the other hand, the trypsinized polynucleotide phosphorylase, known to be an active enzyme which has lost its polynucleotide site, does not bind to the affinity column. The native polynucleotide phosphorylase can also be tightly bound to poly(U)--agarose and displaced from it only by high salt concentration. The trypsinized enzyme is not bound at all on poly(I)--AGAROSe. Moreover, the native enzyme linked on blue-dextran--Sepharose, remains active indicating a free access of nucleoside diphosphates to the active center. These results taken together show that the dye ligand is not inserted onto the mononucleotide binding site and suggest rather that it binds to the polynucleotide binding region. The implications of this study and the application of blue-dextran--Sepharose affinity chromatography to other proteins having affinity for nucleic acids are discussed.
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PMID:Blue-dextran--Sepharose affinity chromatography: recognition of a polynucleotide binding site of a protein. 34 36

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

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 kinetics of the phosphorolysis of polynucleotide (as differentiated from oligonucleotide) by polynucleotide phosphorylase of Micrococcus luteus has been investigated. Double reciprocal plots of initial velocity against either inorganic phosphate or polynucleotide concentration are linear, and furthermore, the affinity of the enzyme for either substrate is unaffected by the presence of the other. dADP, an analogue of ADP product, is a competitive inhibitor with respect to Pi and polynucleotidy. (Ap)tA-cyclic-p is a competitive inhibitor with respect to Pi. The results are almost identical with both primer-independent (Form-I) and primer-dependent (Form-T) enzymes, although the various kinetic constants differ. On the vasis of these data a rapid equilibrium random Bi Bi mechanism is proposed. The demonstration of two different inhibitor constants for dADP and the difference between the Michaelis and the inhibitor constant for polyadenylic acid in polynucleotide phosphorolysis indicate at least two binding sites for polyadenylic acid and dADP on M. luteus polynucleotide phosphorylase. Its is suggested that in the phosphorolysis of long chain polymers the second binding site permits the polynucleotide to snap right back into position after removal of I mononucleotide unit and thus leads to the observed processive degradation. A general discussion of oligonucleotide and polynucleotide phosphorolysis and the differences between Form-I and Form-T enzymes in de novo synthesis and degradation of polynucleotides is presented.
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PMID:Kinetic studies on the phosphorolysis of polynucleotides by polynucleotide phosphorylase. 107 70

A method has been developed for the routine synthesis of 2'(3')-o-monoacyl ribonucleoside 5'-diphosphates for stepwise synthesis of oligoribonucleotides with Escherichia coli polynucleotide phosphorylase. The use of triethyl orthoisovalerate allows the facile preparation of 2'(3')-o-isovaleryl-UDP, -CDP, -ADP, -GDP, -IDP, -EPLISON-APD, eplison-CDP, and N6-isopentenyl-ADP. The synthesis of N6-isopentenyl-ADP from ADP by N1-alkylation and the Dimroth rearrangement to N6 is reported. The effects of several factors including the nature of the divalent cation, pH, SALT CONCENTRATION, AND TIME ON THE EFFICIENCY OF THE POLYNUCLEOTIDE PHPSPHORYLASE CATALYZED SINGLE ADDITIONS OF THE 2'(3')-O-ISOVALERYL RIBONUCLEOSIDE 5'-DIPHOSPHATES TO AN OLIGORIBONUCLEOTIDE PRIMER ARE REPORTED. The syntheses of many tetranucleoside triphosphates and two pentanucleoside tetraphosphates in yields of 20-75 per cent are reported. The 2'(3')-o-isovaleryl derivatives of IDP, eplison-ADP, eplison-CDP, and N6-isopentenyl-ADP were all accepted by polynucleotide phosphorylase as substrates for the monoaddition reaction. The extension of the method to include the syntheses of oligoribonucleotides containing modified nucleosides offers a means of studying the role s of these modification by the use of relatively simple model compounds.
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PMID:Stepwise enzymatic oligoribonucleotide synthesis including modified nucleotides. 109 Mar


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