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)

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

The reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole [NBD-Cl] with purified eel electrophax Na+ and K+ stimulated adenosine triphosphatase [(Na-K)ATPase] has been monitored by changes in the (Na-K)ATPase activity, the K+ stimulated p-nitrophenyl phosphatase [PNPase] activity, and the protein ultraviolet absorption spectrum. The NBD-Cl reacts with two tyrosine residues per mol of enzyme (approximately 6-7 nmol/mg of protein), as judged by changes in protein absorption spectra and incorporation of [14C]NBD-Cl. The modified tyrosine groups are located on the Mr = 95 000 polypeptide chain and react at different rates. Only one tyrosine modification is necessary for complete inhibition of (Na-K)ATPase activity, although both must be modified for complete inhibition of PNPase activity. Reversal of these modifications by 2-mercaptoethanol restores 65% of both activities. Na+ increases the rate of tyrosine modification, K+ decreases the rate, and ATP affords the more reactive tyrosine group complete protection. NBD-Cl modification of approximately 6-7 nmol of tyrosine groups/mg of protein results in a large decrease in ATP affinity as judged by equilibrium binding. These results are compared with similar results obtained from NBD-Cl modification of the coupling factors of oxidative phosphorylation and photophosphorylation. A model is presented suggesting an asymmetric arrangement of two 95 000 polypeptide chains with a single tyrosine residue at the ATP site.
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PMID:Reaction of (Na-K)ATPase with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole: evidence for an essential tyrosine at the active site. 14 73

Effect of cAMP on the activity of polynucleotide phosphorylase (PNPase) was studied in polyribosome fraction of rat liver tissue. Intraperitonel administration of cAMP or of theophilline into rats distinctly decreased the PNPase activity in the polyribosome fraction. The cAMP (1-10(-4) M) inhibited the enzymatic activity only by 8% in polyribosome fraction in vitro, as it was estimated by the reaction of phosphorolysis of endogenous RNA and polyA added. Any attempts were proved to be uncucessful to reveal cAMP, ATP-dependent proteinkinase in rat liver, responsible for the decrease in the PNPase activity in the polyribosome fraction. The cAMP inhibited the increase in the PNPase activity, coupled with protein biosynthesis in polyribosomes. Moreover, cAMP caused a decrease in the PNPase activity in reaction of polyA phosphorolysis and did not affect the rate of endogenous RNA phosphorolysis in polyribosome fraction, isolated from postmitochondrial fraction after incubation for 15 min at 30 degrees. The 3',5'-cyclo AMP (2-10(-6)-2-10(-4) M) stimulated incorporation of 14C-leucine into acid-insoluble material, when postmitochondrial fraction was incubated under the same conditions. The data obtained suggest that cAMP either inhibits specifically the PNPase synthesis or represses the coupled with protein biosynthesis formation of active "heavy" type of PNPase from less active "light" type.
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PMID:[Effect of cyclic-3',5'-AMP on rat liver polynucleotide phosphorylase activity]. 18 2

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

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

E. coli tryptophanyl-tRNA synthetase can form a complex with Blue-dextran Sepharose, in the presence or in the absence of Mg++. In its absence, the complex is dissociated by either ATP or cognate tRNATrp. However, in the presence of Mg++, only tRNATrp can dissociate the complex whereas ATP has no effect. E. coli total tRNA or tRNAMet, at the same concentration, cannot displace the synthetase from the complex. It is suggested that the Blue-dextran binds to the synthetase through its tRNA binding domain. This hypothesis is supported by previous findings with polynucleotide phosphorylase showing that Blue-dextran Sepharose can be used in affinity chromatography to recognize a polynucleotide binding site of the protein. The selective elution by its cognate tRNA of Trp-tRNA synthetase bound to Blue-dextran Sepharose provides a rapid and efficient purification of the enzyme. Examples of other synthetases and nucleotidyl transferases are also discussed.
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PMID:Blue dextran Sepharose chromatography of the tryptophanyl-tRNA synthetase of E. coli: a potential application for the purification of the enzyme. 37 31

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

In the phosphorolytic degradation catalyzed by chicken liver PNPase (E.C. 2.4.2.1) inosine appears to behave as a better substrate than xanthosine. Hypoxanthine, xanthine, guanine and purine (1 X 10(-1)M) appear to be inhibitors of the pigeon liver PNPase, whereas allopurinol, ATP, ITP, CTP and UTP (1. X 10(-3) M) do not inhibit the enzyme. Both PNPase activities exhibit the same optimum temperature (37-40 degrees C). Chicken liver PNPase optimum pH is in the range 6.5-7, whereas that of pigeon liver is in the range 7-7.5. Lineweaver-Burk plots for the inosine phosphorolysis catalyzed by chicken liver PNPase yielded straight lines if substrate concentrations were lower than 1 X 10(-4) M but concave downward curves at higher concentrations. This activation increases when the homogenates are stored at 4 degrees C and pH = 7 during 24 h or more; pigeon liver PNPase does not show this activation phenomenon.
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PMID:[Purine metabolites in the activity of purine nucleoside phosphorylase (author's transl)]. 82 98

A simple method for the preparation of adenosine 5'-[beta-32 P]triphosphate is described. When [32P]orthophosphate was incubated with polyadenylate and phosphoenolpyruvate in the presence of polynucletide phosphorylase (EC 2.7.7.8) and pyruvate kinase (EC 2.7.1.40), up to 75% of 32P radioactivity was recovered in ATP. [32P]ATP was purified to 99.5% radiochemical purity by chromatography on polyethyleneimine-cellulose thin-layer plates. Analysis of hydrolysis products of [32P]ATP with apyrase (EC 3.6.1.5) indicates that 32P in the beta-phosphate position accounts for all 32P label in ATP.
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PMID:Enzymic preparation of adenosine 5'-[beta-2P]triphosphate. 88 81

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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