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At least 19 recordsLinked to original sources
PubMed1975

Kinetic studies on the phosphorolysis of polynucleotides by polynucleotide phosphorylase.

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.

Binding Sites↗
PubMed1976

Study on the structure-function relationship of polynucleotide phosphorylase: model of a proteolytic degraded polynucleotide phosphorylase.

It is already known that modification of E. coli polynucleotide phosphorylase by endogenous proteolysis induces drastic changes in both phosphorolysis and polymerisation reactions. The structural parameters of the proteolysed polynucleotide phosphorylase are described. The phosphorolysis of polynucleotide, which is quite progressive for the native enzyme, is shown to be only partially progressive for the degraded enzyme, owing to the loss of polymer attachment sites.

Escherichia coli↗
PubMed1975

Structure and synthesis of a lipid-containing bacteriophage. A polynucleotide-dependent polynucleotide-pyrophosphorylase activity in bacteriophage PM2.

A polymerase activity is associated with protein IV, a protein which is associated with the DNA in bacteriophage PM2. The native enzyme unit is probably a dimer. Manganese ions are required for the polymerisation reaction and there is a well-defined Mn2+ optimum at 2.5 mM. The pH optimum is at 8.1, the temperature optimum at 28 degrees C. The activity is a polynucleotide-pyrophosphorylating reaction in the presence of ribo- or deoxyribonucleoside triphosphates. The polymerisation reaction is stimulated in the presence of nuclei- acids or polynucleotides as effectors. The product is not covalently linked to the effector.

Bacteriophages↗
PubMed1975

Polynucleotides. XXVIII. Stimulation of the binding of aminoacyl-tRNA to ribosomes by tri- and polynucleotide analogs.

Messenger activity of synthetic tri- and polynucleotide analogs was studied by binding of 14C-labeled aminoacyl-tRNAs to ribosomes in the presence of the analogs. Synthetic messengers used were: poly(A) analogs in which adenosine was replaced by tubercidine (I), 3-deazaadenosine (II), 1-deazaadenosine (III) and 2-methyladenosine (IV); copolymers of adenosine and aristeromycin (V); cyclic triadenylate (VI); the heptanucleotide of 6,2'-O-cyclouridine (VII); the pentanucleotide of 8,2'-S-cycloadenosine (VIIIa); A-U-G analogs in which adenosine was replaced by 8,2'-O- and S-cycloadenosine (VIII), 8,5'-O- and S-cycloadenosine (IX); 8-oxyadenosine (x); 8-bromoadenosine (XI) and formycine (XII). Among these oligo- and polynucleotides, analogs which contained nucleotides of anti conformation having appropriate bases for Watson-Crick type hydrogen bonding stimulated the binding of corresponding tRNAs to ribosomes.

Adenosine↗
PubMed1977

Osmium-labeled polynucleotides. The reaction of osmium tetroxide with deoxyribonucleic acid and synthetic polynucleotides in the presence of tertiary nitrogen donor ligands.

Osmium tetroxide in the presence of pyridine or 2,2'-bipyridine has been found to react completely with the pyrimidine moieties (thymine, uracil, and cytosine) in polynucleotides. Pyrimidine osmate ester moieties, L2OSO4-pyrimidine, were formed. The OSO4 has added across the 5,6 double bond and L=pyridine or 1/2-bipyridine. The pyridine derivatives were not stable and decomposed slowly after the OSO4-pyridine reagent was removed by gel chromatography. Labeled poly(uridylic acid) lost osmium completely during gel chromatography unless the eluent contained a high concentration of pyridine. The products formed between OSO4-bipyridine and polynucleotides were much more stable and the OS label was retained during and after gel chromatography. Both the OSO4-pyridine and OSO4-bipyridine reagents reacted more rapidly than the OSO4-CN-reagent.

Binding Sites↗
PubMed1976

Polynucleotides. XLII1. Limited addition of 2'O-onitrobenzyl nucleotides to the 3'-end of ribooligonucleotide with polynucleotide phosphorylase.

2'-O-o-Nitrobenzyluridine, -cytidine and -adenosine were phosphorylated with phosphoryl chloride to the corresponding 5'-phosphates and led to 5'-diphosphates by the method of Moffatt and Khorana. These 2'-O-oNB-nucleoside 5'-diphosphates were incubated with a primer CpApA and polynucleotide phosphorylase in the presence of Mn2+. Tetranucleotides CpApApU, CpApApC and CpApApA were obtained after photosensitive removal of oNB groups in yields of 54-70%.

Benzyl Compounds↗
PubMed1976

Interaction of nucleic acids with electrically charged surfaces. II. Conformational changes in double-helical polynucleotides.

The influence of adsorption of double-stranded (ds) DNA, ds RNA and homopolymeric pairs at a mercury electrode on conformation of these polynucleotides was studied. Changes in the polarographic reducibility of polynucleotides, which were followed by means of normal pulse polarography and linear sweep peak voltammetry at the dropping mercury electrode were exploited to indicate conformational changes. It was found that, as a consequence of adsorption of ds polynuclotides on the negatively charged electrode conformational changes similar to denaturation take place in a narrow potential region around -1.2 V (the region U). After sufficiently long time of the contact with the electrode (under our conditions about 10 s) these changes reach limiting values, which can approach total denaturation. Upon adsorption of ds polynucleotides on the electrode charged to more positive potentials than the region U either (1) no conformational changes occur or (2) only a small part of the polynucleotide (probably labile regions of the ds molecule) is very quickly denatured - the remainder of the molecule preserves its ds structure. Conformational changes of adsorbed ds polynucleotides are influenced by factors which change the stability of ds polynucleotides in solution. It is supposed that denaturation of ds polynucleotides in the region U might result from the strains connected with the repulsion of certain segments of the molecule anchored on the electrode from the negatively charged surface.

Animals↗
PubMed1977

Effects of polyamines on the degradation of ribonucleic acids by polynucleotide phosphorylase of Micrococcus luteus.

The effects of polyamines on the breakdown of synthetic polynucleotides [poly(A), poly(C), and poly(U)] by polynucleotide phosphorylase [polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8] from Micrococcus luteus have been studied. Although the breakdown of all the synthetic polynucleotides tested was stimulated by polyamines, the degree of stimulation by polyamines was in the order poly(C) greater than poly(A) greater than poly(U) at pH 7.5. However, the difference in degree of stimulation among polynucleotides decreased as the pH or monovalent cation concentration was increased. In the presence of heparin, an inhibitor of polynucleotide phosphorylase hydrolysis of polynucleotides, spermidine clearly stimulated the breakdown of poly(C) and poly(A), while the breakdown of poly(U) was stimulated only slightly by the addition of spermidine. Although binding of [14C]spermine to polynucleotide phosphorylase was observed by gel filtration, the amount of spermine bound to the enzyme was much less than that to RNA.

Binding Sites↗
PubMedDate not supplied

[Effect of the protein product of phage f1, gene 5, on heat denaturation of synthetic polynucleotides].

The influence of phage f1 gene 5 protein on melting of the synthetic polynucleotides has been investigated, using UV-spectroscopy. In our experiments we have varied the proteins concentration. It has been shown, that the protein lowers the melting temperature of the studied polynucleotides (d/A--Tn dAndTn, rAndTn, rAn.r n, dAn.rn). The melting temperatures and the shapes of melting curves of various polynucleotides differ when the same protein concentrations are used. We have shown that the protein binds to the double-stranded polynucleotides, containing ribo-ribo-, deoxyribo-ribo-chains. The difference in melting temperatures and shapes of melting curves was explained using the data about the differences in the secondary structure of these polynucleotides. Only for d/A-Tn renaturation was observed after sample cooling. It may reflect the single-stranded hairpin structure of this polynucleotide.

Bacteriophages↗
PubMedDate not supplied

[Compact form of synthetic polynucleotides. Relationship between secondary structure and circular dichroism spectra].

The formation of compact particles from synthetic double- and triplestranded polynucleotides in water-salt solutions, containing poly(ethylene glycol) (PEG) has been investigated. CD spectra of compact particles are characterized by intense bands (positive or negative) in the region of 270 nm, compact particles being divided into two families--psi- and psi+--according to the CD band sign. The amplitude of the CD band at 270 nm increases with the increase of CPEG. Heating of a solution, containing compact particles, results in a disappearance of the CD band, the "melting" of compact particles as revealed by the CD method occuring prior to the melting of the secondary structure of the corresponding polynucleotide. It is concluded that intense CD bands, which are characteristic of the compact form of synthetic polynucleotides, arise (similar to the case of DNA or dsRNA) from regular arrangement of polynucleotide chains in compact particles. The question, concerning the relation between parameters of the secondary structure of polynucleotides and their belonging either to psi- or to psi+ family is discussed. The factors, which could account for the appearance of intense bands in CD spectra of compact particles are also considered.

Circular Dichroism↗
PubMedDate not supplied

[Unwinding effect of F1 gene 5 protein on double-stranded polynucleotides and DNA].

The effect of gene 5 protein from bacteriophage f1 on melting of double-stranded polynucleotides and DNAs has been investigated using the UV-spectroscopy method. A dependence of the melting temperature of polynucleotide (DNA)-gene 5 protein complexes upon the polynucleotide (DNA) GC-pair content has been detected. Using experimental data and examining some model systems we came to the supposition that the lowering of melting temperature of polynucleotide (DNA) induced by this protein is probably stipulated by intercalation of the protein tyrosyl residues into one of the chains of polynucleotide (DNA) double helix.

Coliphages↗
PubMedDate not supplied

[Comparative study of slow 1H to 3H exchange in synthetic polynucleotides of A- and B-type conformations].

The rate of 1H leads to 3H exchange between water and C(8)H-groups of purinic residues in synthetic polynucleotides in wide temperature range measured. At temperatures below their Tm the rate of the exchange is shown to be lower as compared with that in corresponding mononucleotides. In the case of polynucleotides of A-conformation (poly(A).poly(U), poly(A).2poly(U) and poly(dA).2poly(dT), and poly(G).poly(C) the exchange is retarded by a factor of 5.7--7.5, whereas in the case of those of B-conformation (poly(dA).poly(dT), poly(dA--dT).poly(dA--dT) and poly(dG).poly(dC)) the exchange is retarded only by a factor of 2.3--2.5. Assuming the ylide mechanism of exchange the retardation is interpreted as a consequence of sterical hidrance in polynucleotides helical structure, which hampers contacts between purinic C(8)H-groups and OH-ions of solvent. Analysis of atomic arrangement around C(8)H-group and interatomic distances calculated on the basis of published atomic coordinates support our general conclusion that the sterical hindrance is more significant in the A-form as compared with that in the B-form. Elucidated correlation between the degree of the retardation in purine-containing polynucleotides and their conformation in solution allows to estimatf the type of conformation of polynucleotide with unknown structure on account of the slow 1H leads to 3H exchange data.

Hydrogen↗
PubMed1976

Incorporation of 5'-amino-5'-deoxythymidine5'-phosphate in polynucleotides by use of DNA polymerase I and a phiX174 DNA template.

An aqueous solution of 5'-amino-5'-deoxythymidine 5'-triphosphate, prepared by incubation of equimolar solutions of 5'-amino-5'-deoxythymidine and sodium trimetaphosphate, stimulates synthesis of acid-precipitable polynucleotides in a system containing single-strand phiX174 DNA template, random oligonucleotide primers, dATP, dCTP,dGTP, Escherichia coli DNA polymerase I, and either magnesium or manganese ion. Approximately onefold synthesis on the template can be achieved and each of the indicated reagents is essential for extensive synthesis. The reaction is slower than the corresponding reaction of dTTP as a consequence of a lower V max and a higher Km for the amino analogue. That aminodeoxythymidine phosphate is incorporated into the synthetic polynucleotides was shown by a double-labeling experiment with [14C]dATP and [32P]-5'-amino-5'-deoxythymidine 5'-triphosphate and by the unusually high lability of the phosphoramidate polynucleotides toward acid. The phosphoramidate polynucleotides range in size from about 100 nucleotide units to well over a thousand nucleotide units, and the size is increased by addition of DNA ligase to the system. These experiments indicate that synthetic polynucleotides in which oligonucleotide blocks have been joined by means of phosphoramidate bonds should prove useful as primers for enzymatic syntheses with DNA polymerase I.

Coliphages↗
PubMed1975

Poly(A) synthesis in T2L phage-infected Escherichia coli. A combination of polynucleotide phosphorylase and ATPase.

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.

Adenosine Triphosphatases↗
PubMed1978

Blue-dextran--Sepharose affinity chromatography: recognition of a polynucleotide binding site of a protein.

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.

Chromatography, Affinity↗
PubMed1990

Related domains in yeast tRNA ligase, bacteriophage T4 polynucleotide kinase and RNA ligase, and mammalian myelin 2',3'-cyclic nucleotide phosphohydrolase revealed by amino acid sequence comparison.

Related domains containing the purine NTP-binding sequence pattern have been revealed in two enzymes involved in tRNA processing, yeast tRNA ligase and phage T4 polynucleotide kinase, and in one of the major proteins of mammalian nerve myelin sheath, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). It is suggested that, similarly to the tRNA processing enzymes, CNPase possesses polynucleotide kinase activity, in addition to the phosphohydrolase one. It is speculated that CNPase may be an authentic mammalian polynucleotide kinase recruited as a structural component of the myelin sheath, analogously to the eye lens crystallins. Significant sequence similarity was revealed also between the N-terminal regions of yeast tRNA ligase and phage T4 RNA ligase. A tentative scheme of the domainal organizations for the three complex enzymes is proposed. According to this model, tRNA ligase contains at least three functional domains, in the order: N-ligase-kinase-phosphohydrolase-C, whereas polynucleotide kinase and CNPase encompass only the two C-terminal domains in the same order.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMedDate not supplied

Block-units method for conformational calculations of large nucleic acid chains. I. Block-units approximation of atomic structure and conformational energy of polynucleotides.

A new block-units method for rapid conformational calculation of large nucleic acid fragments has been developed. Atomic structure of polynucleotides has been approximated by the block-units structure. Each monomer of the polynucleotide consists of three one-center block units: phosphate, ribose, and nucleic base. Full conformational energy of the polynucleotide is separated into two parts. The first part is the energy of the short-range interactions between adjacent block units and is calculated on the basis of the potential energy surface model of the dinucleotide fragment pXp (where X = A, G, T, U, C). The second part is the energy of the middle- and long-range interactions between separated block units, and is calculated as a sum of the effective interaction energies between centers of the block units. The present block-units method is in agreement within the range of +/- 0.5 kcal/mol with the method of the atom-atom potentials, but the former is 30-100-fold faster. The block-units method is recommended for screening of the probable conformations of the large polynucleotide systems.

Chemical Phenomena↗