Phosphorylase polynucleotides

Synthetic oligonucleotides may be used as "primers and be elongated stepwise with the aid of polynucleotide phosphorylase (PNPase) and nucleoside diphosphates. 

Similarly, Ikehara, Tazawa, and Fukui (51) have found that the nucleotides 8-bromo and 8-oxoadenosine 5 -diphosphate, 8-bromo-, 8-oxo, and 8-dimethylaminoguanosine 5 -diphosphate are all inactive as substrates for homopolymer synthesis catalyzed by polynucleotide phosphorylase from Escherichia coli. Some of the results were later confirmed by Kapuler, Monny, and Michelson (52), who found that neither 8-bromo- nor 8-oxoguanosine 5 -diphosphate was active as a substrate for homopolymerization with polynucleotide phosphorylases isolated both irom Azotobacter vinelandii and . coli. 

They did find that these compounds behaved kinetically as competitive inhibitors of polymerization of the normal substrates e.g., guanosine 5 -diphosphate. These authors suggested that the successful completion of the polynucleotide phosphorylase reaction requires that the nucleotide be capable of assuming the anti conformation. Also, Kapuler and Reich (53) have found that both 8-bromo- and 8-oxoguanosine 5 -triphosphates are very poor substrates in the E. coli RNA polymerase reaction and are competitive inhibitors with respect to guanosine 5 -triphosphate as a substrate. 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

After more than 20 years, Walde et al. (1994) returned in a way to coacervate experiments, although using other methods. Walde (from the Luisi group) repeated nucleotide polymerisation of ADP to give polyadenylic acid, catalysed by polynucleotide phosphorylase (PNPase). But instead of Oparin s coacervates, the Zurich group used micelles and self-forming vesicles. They were able to demonstrate that enzyme-catalysed reactions can take place in these molecular structures, which can thus serve as protocell models. Two different supramolecular systems were used ... 

The experiments were carried out using Ci4-phosphatidylcholine (PC) vesicles. The biochemical reaction which was planned to occur in the vesicles was the aforementioned RNA polymerisation reaction involving the enzyme polynucleotide phosphorylase (PNPase), which Oparin and co-workers had used many years ago in their work on coacervates. PNPase and added ADP then form oligonucleotides in the vesicles. 

PHOSPHOFRUCTOKINASE 3-PHOSPHOGLYCERATE KINASE 5-PHOSPHOMEVALONATE KINASE PHOSPHORYLASE KINASE POLYNUCLEOTIDE 5 -HYDROXYL KINASE... 

ACTIN ASSEMBLY KINETICS POLYNUCLEOTIDE S -HYDROXYL-KINASE POLYNUCLEOTIDE PHOSPHORYLASE Polyol dehydrogenase,... 

Figure 10.3 Enzymatic synthesis of poly(adenylic acid) in self-reproducing oleate liposomes (redrawn from Walde et al., 1994a). (a) The ADP penetrates (sluggishly) the liposome bilayer, (b) in the presence of polynucleotide phosphorylase, ADP is converted in poly(A), which remains entrapped in the liposome, (c) Polycondensation of ADP goes on simultaneously with the self-reproduction of liposomes (A is the membrane precursor, oleic anhydride, which, once added, induces the self-reproduction of liposomes S, surfactant, in this case oleate, which is the hydrolysis product of A on the bilayer E is polynucleotide phosphorylase).

Glucose oxidase, in combination with peroxidase or lactoperoxidase Polynucleotide phosphorylase... 

Polynucleotide Phosphorylase Makes Random RNA-like Polymers... 

In 1955, Marianne Grunberg-Manago and Severo Ochoa discovered the bacterial enzyme polynucleotide phosphorylase, which in vitro catalyzes the reaction... 

Polynucleotide phosphorylase was the first nucleic acid-synthesizing enzyme discovered (Arthur Kornberg s discovery of DNA polymerase followed soon thereafter). 

The reaction catalyzed by polynucleotide phosphorylase differs fundamentally from the polymerase activities discussed so far in that it is not template-dependent. The enzyme uses the 5 -diphosphates of ribonucleosides as substrates and cannot act on the homologous 5 -triphos-phates or on deoxyribonucleoside 5 -diphosphates. The RNA polymer formed by polynucleotide phosphorylase contains the usual 3, 5 -phosphodiester linkages, which can be hydrolyzed by ribonuclease. The reaction is readily reversible and can be pushed in the direction of breakdown of the polyribonucleotide by increasing the phosphate concentration. The probable function of this enzyme in the cell is the degradation of mRNAs to nucleoside diphosphates. 

Because the polynucleotide phosphorylase reaction does not use a template, the polymer it forms does not have a specific base sequence. The reaction proceeds equally well with any or all of the four nucleoside diphosphates, and the base composition of the resulting polymer reflects nothing more than the relative concentrations of the 5 -diphosphate substrates in the medium. 

Polynucleotide phosphorylase reversibly forms RNA-like polymers from ribonucleoside 5 -diphosphates, adding or removing ribonucleotides at the 3 -hydroxyl end of the polymer. The enzyme degrades RNA in vivo. 

The mechanism of action of spleen exonuclease is similar to that seen for venom exonuclease (19-21) but different from the processive type of attack exhibited by E. coli RNase II, sheep kidney exonuclease, and polynucleotide phosphorylase (22, 23), in which cases each polynucleotide molecule is completely degraded before the enzymes attack a new molecule. The results of Bernardi and Cantoni (12) contradict the previous beliefs that the enzyme has an intrinsic, though weak, endonucleolytic activity (5) and that a phosphate group in a terminal 5 position makes a polynucleotide chain completely resistant to the enzyme (15, 24, 25). 

The first enzyme discovered that could catalyze polynucleotide synthesis was a bacterial enzyme called polynucleotide phosphorylase. This enzyme, isolated by Severo Ochoa and Marianne Grunberg-Manago in 1955, could make long chains of 5 -3 -linked polyribonucleotides starting from nucleoside diphosphates. However, there was no template requirement for this synthesis, and the sequence was uncontrollable except in a crude way by adjusting the relative concentrations of different nucleotides in the starting materials. 

In addition to the cellular enzyme(s) that catalyzes DNA-directed RNA synthesis, cellular enzymes are involved in polyribonucleotide synthesis that do not use a template. Some of the properties of these enzymes are summarized in table 28.5. We have already mentioned polynucleotide phosphorylase in this chapter, and in chapter 26 we discussed the importance of DNA primase to DNA synthesis. 

Polynucleotide phosphorylase was used to produce RNA of random sequence, the composition of which reflected the mixture of nucleoside diphosphates in the reaction mixture. Mixed polynucleotides containing two bases were used in the incorporating system and shown to incorporate a pattern of amino acids consistent with a triplet code, but the observed incorporation could not define the code sequence. 

Ochoa and Grunberg-Manago discovered polynucleotide phosphorylase. 

Phosphorolysis of ribonucleic acid with polynucleotide phosphorylase gives a mixture of the diphosphates of the four common nucleosides, which are transformed into triphosphates with enolpyruvate phosphate and pyruvate kinase. This mixture may be used as such as a source of uridine triphosphate in the preparation of the nucleotide-sugar uridine 5 -(a-D-glucopy-ranosyl diphosphate) ( uridine-diphosphate-glucose, UDP-Glc), or as a... 

Polymerization activity of proteinoid or polynucleotide-phosphorylase has been compared in ADP solution at pH 8.5 48). The results show that the activity of the neutral proteinoid is approximately 20 times lower than that of the enzyme, and the lower molecular-weight fraction of the proteinoid has negligible activity. The polymerization by polynucleotide-phosphorylase is increased approximately three times when Leuchs polylysine is supplied, polylysine and enzyme yield a heterogeneous solution 48). 

Liebl, V., Novak, V., Bejsovcova, L., Masinovskij, Z., Oparin, A. I. Polymerization of radioactive adenosine diphosphate by polynucleotide-phosphorylase or by proteinoids in microsystems, in Origin of Life Proceedings of the Second ISSOL Meeting, the Fifth ICOL Meeting (ed.) Noda, H., p. 363, Japan, Bus. Cent. Acad. Soc. Japan 1978... 

To demonstrate polymerase activity in a model cell, Chakrabarti et al. [79] encapsulated polynucleotide phosphorylase in vesicles composed of dimyris-toylphosphatidylcholine (DMPC). This enzyme can produce RNA from nucleoside diphosphates such as adenosine diphosphate (ADP) and does not require a template, so it has proven useful for initial studies of encapsulated polymerase activity (Fig. 10a). Furthermore, DMPC liposomes are sufficiently permeable so that 5-10 ADP molecules per second enter each vesicle. Under these conditions, measurable amounts of RNA in the form of polyadenylic acid were synthesized and accumulated in the vesicles after several days incubation. The enzyme-catalyzed reaction could be carried out in the presence of a protease external to the membrane, demonstrating that the vesicle membrane protected the encapsulated enzyme from hydrolytic degradation. Similar behavior has been observed with monocarboxylic acid vesicles [80], and it follows that complex phospholipids are not required for an encapsulated polymerase system to function. 

Growing membrane systems have been used to obtain artificial infrabiological systems. Walde et al. [47] have carried out the synthesis of polyadenylic acid in self-reproducing vesicles [48], in which the enzyme polynucleotide phosphorylase carried out the synthesis of poly-A, and membrane vesicle multiplication was due to the hydrolysis of externally provided oleic anhydride to oleic acid. The snag is that the enzyme component is not auto-catalytic. Enzymatic RNA replication in vesicles [49] suffers from the same problem. It is also not known whether redistribution of the entrapped enzymes into newly formed vesicles occurs or not. An affirmative answer would be evidence for vesicle reproduction by fission. 

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