Acid-soluble degradation products of ribonucleic acid in Escherichia coli and the role of nucleotidases in their catabolism.

The fate of the internally formed nucleotides resulting from the degradation of ribonucleic acid was studied. Prelabeled Escherichia coli cells were submitted to carbon starvation, and the acid-soluble products were separated by thin-layer chromatography. It was determined that free bases constitute some 75% of the end product, the balance consisting of nucleoside diphosphates, 5'-nucleoside monophosphates, 3'-nucleoside monophosphates, and nucleosides. The majority of degradation products, including phosphorylated derivatives, were excreted into the medium. The amount of products in the pool remained constant. The soluble products formed by E. coli mutants lacking either 5'-nucleotidase (Ush-) or 3'-nucleotidase (Cpd-) were compared with those produced by the parental strain with both enzymes. The results obtained indicated that 5'-nucleotidase is involved in the degradation of internally foromed nucleotides but that 3'-nucleotidase takes no part in the process.     

Partial purification and properties of rat liver mitochondrial polynucleotide phosphorylase

1. Polynucleotide phosphorylase was partially purified from the inner membrane of rat liver mitochondria. 2. The partially purified particulate enzyme catalyses phosphorolysis of poly(A), poly(C), poly(U) and RNA to nucleoside diphosphates. 3. It is devoid of nucleoside diphosphate-polymerization activity. 4. Variable amounts of ADP/P(i)-exchange activity are associated with the polynucleotide phosphorylase and are probably due to a different enzyme. 5. ADP is the preferred substrate for exchange, and little or no reaction occurs with other nucleoside diphosphates, but ATP/P(i)-exchange takes place at one-third the rate observed with ADP. 6. The partially purified enzyme is free from the phosphatases found in the crude mitochondrial inner membrane, but is associated with an endonuclease activity and some adenylate kinase activity; no cytidylate kinase activity analogous to the latter was detectable.     

Fingerprinting nonradioactive ribonucleic acid with the aid of polynucleotide phosphorylase

We describe a method for obtaining radioactive fingerprints from nonradioactive ribonucleic acid. Fragments derived by T1 ribonuclease digestion of RNA are dephosphorylated with bacterial alkaline phosphatase. When these fragments are used as primers for the reaction of primer dependent polynucleotide phosphorylase with [α-³²P]GDP in the presence of T1 ribonuclease the 3′-hydroxyl group of each fragment becomes phosphorylated. The degree of phosphorylation is reasonably uniform. The method has been applied to T1 ribonuclease digests of Escherichia coli tRNA^Met_f; the oligonucleotides were further analyzed by spleen phosphodiesterase digestion. In a similar manner fingerprints of pancreatic ribonuclease digests of RNA can be obtained, when [α-³²P]UDP, polynucleotide phosphorylase and pancreatic ribonuclease are used.     

Catabolic pathways of purine ribonucleotides and deoxyribonucleotides in lymphocytes

Deficiency of either one of the subsequent purine catabolic enzymes adenosine deaminase or purine nucleoside phosphorylase results in immunodeficiency disease in humans. However, the mechanism by which impairment of purine metabolism may cause immunodeficiency is unclear. In the present work we have studied the catabolism of purine ribonucleotides and deoxyribonucleotides in T lymphocytes to better understand the role of purine nucleoside phosphorylase and adenosine deaminase in the immune function. It was found that purine deoxyribonucleotides are degraded via catabolic pathways distinctly different from those used for purine ribonucleotide degradation. Thus both adenine and guanine ribonucleotides are deaminated to IMP whereas purine deoxyribonucleotides are exclusively dephosphorylated to the corresponding deoxyribonucleosides. These findings may explain the relatively higher degradation rates of purine deoxyribonucleotides in mammalian cells as compared to purine ribonucleotides. The catabolism of purine nucleotides is tightly linked to the active purine nucleoside cycles which consist of the phosphorolysis of purine nucleosides and deoxyribonucleosides to their corresponding bases, their salvage to monophosphates and back to the corresponding ribonucleosides. The above observations also imply that a possible role of the purine nucleoside cycles is to convert purine deoxyribonucleotides into their corresponding ribonucleotide derivatives. Deficiencies of purine nucleoside phosphorylase or of adenosine deaminase activities, enzymes which participate or lead to the purine nucleoside cycles, thus result in a selective impaired deoxyribonucleotide catabolism and immunodeficiency.     

Escherichia coli K-12 mutants capable of catabolizing purine nucleosides in the absence of purine nucleoside phosphorylase]

Strains of Escherichia coli K-12 defective in purine nucleoside phosphorylase (pup gene) formed on the medium with inosine as the source of carbon and energy phenotypical reversions for the ability of utilizing inosine as source of carbon or purines. The phenotypical suppression of the purine nucleoside phosphorylase deficiency is the result of the mutations (called pnd), which are mapped on the chromosome of E. coli beyond the region of the structural pup-gene location and have phenotypic manifestation distinct from that of pup+ allele: a) pnd mutants divide into some groups for the ability of utilizing several purine nucleosides, including xantosine that cannot be metabolized by pnd+ strains of E. coli; b) pnd mutations do not restore the ability of purine auxotrophs (pur) defective in purine nucleoside phosphorylase (pup) and adenine phosphoribosyltransferase (apt) to grow on the medium with adenine as the sole source of purines. Cell-free extracts of pnd mutants fail to degrade the guanine nucleosides in the absence of phosphate or arsenate ions. These data (and also the ability of pnd mutants to utilize both purine ribonucleosides and deoxyribonucleosides) seem to indicate that the activities induced by pnd mutations are phosphorylase activities.     

Nucleic acid enzymology of extremely halophilic bacteria. Halobacterium cutirubrum polynucleotide phosphorylase

1. Polynucleotide phosphorylase was purified 200-fold from Halobacterium cutirubrum. 2. It is membrane-associated and can be solubilized by sonication. 3. The purified enzyme requires a high ionic strength for both stability and activity. 4. It is Mn(2+)-dependent, has all three typical polynucleotide phosphorylase activities and is specific for nucleoside diphosphates. 5. The enzyme is of low molecular weight.     

32P-Postlabeling Assay for Free Radical-Induced Dna Damage: The Formamido Remnant of Thymine

A prominent lesion in DNA exposed to oxidative free radicals results from the degradation of thymine leaving a formamido remnant. A ³²P-postlabeling assay has been developed for the detection of the formamido lesion. The assay is based on the circumstance that the lesion prevents hydrolysis by nuclease PI of the phosphoester bond 3' to the damaged nucleoside. Thus, a nuclease PI plus acid phosphatase digest of DNA generates mostly nucleosides whereas the formamido lesion is rendered as a modified dinucleoside monophosphate. Dinucleoside monophosphates, but not nucleosides, are apt substrates for ³²P-postlabeling by polynucleotide kinase. The assay was applied to calf thymus DNA X-irradiated in oxygenated solution. The formamido lesion could be detected down to a dose of a few Gy.     

Enzymology and genetic regulation of a cyclic nucleotide-binding phosphodiesterase-phosphomonoesterase from Aspergillus nidulans.

A cyclic nucleotide-binding phosphohydrolase that possesses both a phosphomonoesterase and a phosphodiesterase catalytic function has been partially purified from Aspergillus nidulans. The enzyme hydrolyzes both p-nitrophenylphosphate and bis-(p-nitrophenyl)-phosphate. o'-Nucleoside monophosphates are the best physiological phosphomonesterase substrates but 5'- and 2'-nucleoside monophosphates are also hydrolyzed. The enzyme catalyzes the hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, and 2',3'- and 3'5'-cyclic nucleotides, but not of ribonucleic acid, deoxyribonucleic acid, or nicotinamide adenine dinucleotide. The enzyme has acid pH optima and is not activated by divalent cations. Nucleosides and nucleotides inhibit the enzyme. Cyclic nucleotides are competitive inhibitors of the phosphodiesterase-phosphomonoesterase. The enzyme can occur extracellularly. The phosphodiesterase-phosphomonoesterase is present at high levels in nitrogen-starved mycelium, and it is strongly repressed during growth in media containing ammonium or glutamine and weakly repressed during growth in glutamate-containing medium. Experiments with various area mutants show that this regulatory gene is involved in the control of the enzyme. No evidence for regulation of the enzyme by carbon or phosphorus starvation has been found.     

Guanosine triphosphate catabolism in purine nucleoside phosphorylase deficient human B lymphoblastoid cells

GTP catabolism induced by sodium azide or deoxyglucose was studied in purine nucleoside phosphorylase (PNP) deficient human B lymphoblastoid cells. In PNP deficient cells, as in control cells, guanylate was both dephosphorylated and deaminated but dephosphorylation was the major pathway. Only nucleosides were excreted during GTP catabolism by PNP deficient cells and the main product was guanosine. The level of nucleoside excretion was largely affected by intracellular orthophosphate (P_i) level. In contrast, normal cells excreted nucleosides only at low P_i level while at high P_i levels, purine bases (guanine and hypoxanthine) were exclusively excreted. PNP deficiency had no effect on the extent of GMP deamination.     

Nucleoside phosphotransferase activity of human colon carcinoma cytosolic 5'-nucleotidase.

A cytosolic 5'-nucleotidase, acting preferentially on IMP and GMP, has been isolated from human colon carcinoma extracts. This enzyme activity catalyzes also the transfer of the phosphate group of 5'-nucleoside monophosphates (mainly, 5'-IMP, 5'-GMP, and their deoxycounterparts) to nucleosides (preferentially inosine and deoxyinosine, but also nucleoside analogs, such as 8-azaguanosine and 2',3'-dideoxyinosine). It has been proposed that the enzyme mechanism involves the formation of a phosphorylated enzyme as an intermediate which can transfer the phosphate group either to water or to the nucleoside. The enzyme is activated by some effectors, such as ATP and 2,3-diphosphoglycerate. Results indicate that the effect of these activators is mainly to favor the transfer of the phosphate of the phosphorylated intermediate to the nucleoside (i.e., the nucleoside phosphotransferase activity). This finding is in accordance with previous suggestions that cytosolic 5'-nucleotidase cannot be considered a pure catabolic enzyme.     

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.     

Ribonucleic acid destruction and synthesis during intraperiplasmic growth of Bdellovibrio bacteriovorus.

During growth of Bdellovibrio bacteriovorus on (2-14C)uracil-labeled Escherichia coli approximately 50% of the radioactivity is incorporated by the bdellovibrio and most of the remainder is released as free nucleic acid bases. Kinetic studies showed that 50 and 30S ribosomal particles and 23 and 16S ribosomal ribonucleic acid (RNA) of E. coli are almost completely degraded by the first 90 min in a 210- to 240-min bdellovibrio developmental cycle. Synthesis of bdellovibrio ribosomal RNA was first detected after 90 min. The specific activity and the ratio of radioactivity in the bases of the synthesized bdellovibrio RNA was essentially the same as those of the substrate E. coli. The total radioactivity of the bdellovibrio deoxyribonucleic acid (DNA) exceeded that in the DNA of the substrate E. coli cell, and the ratio of radioactivity of cytosine to thymine residues differed. Intraperiplasmic growth of B. bacteriovorus in the presence of added nucleoside monophosphates (singly or in combination) significantly decreased the uptake of radioactivity from (2-14C)uracil-labeled E. coli; nucleosides or nucleic acid bases did not. It is concluded that the RNA of the substrate cell, in the form of nucleoside monophosphates, is the major or exclusive precursor of the bdellovirbrio RNA and also serves as a precursor for some of the bdellovibrio DNA.     

RNase PH catalyzes a synthetic reaction, the addition of nucleotides to the 3' end of RNA.

Escherichia coli RNase PH is a phosphate-dependent exoribonuclease that has been implicated in the 3' processing of tRNA precursors. It degrades RNA chains in a phosphorolytic manner releasing nucleoside diphosphates as products. Here we show that RNase PH also catalyzes a synthetic reaction, the addition of nucleotides to the 3' termini of RNA molecules. The synthetic activity co-purifies with RNase PH throughout an extensive enrichment indicating that it is due to the same enzyme. The synthetic activity can incorporate all nucleoside diphosphates, but not triphosphates, and is strongly inhibited by Pi, but not PPi. Various RNA molecules stimulate nucleotide incorporation, and with tRNA the 3' end of the molecule serves a primer function. RNA chains as long as 40 residues can be synthesized in this system. As with polynucleotide phosphorylase, the synthetic activity of RNase PH apparently represents the reversal of the degradative reaction.     

Synthesis of Polynucleotides by Microorganisms

Polynucleotides could be synthesized from nucleoside diphosphates by microorganisms belonging to genera Pseudotnonas, Serratia, Xatuhonwnas, Proteus, Aerobacter, Bacillus, and Brevibacterium. These strains were rich in polynucleotide phosphorylase easily extractable from cells and poor in both nuclease and nucleoside-diphosphate-degrading enzymes. Polynucleotide phosphorylase was effectively extracted from the bacterial cells, that had been once soaked in saturated saline solution, with hypotonic solution. Synthesis of polynucleotides was observed not only when the substrates were incubated with polynucleotide phosphorylase preparation isolated from the bacterial cells, but also when the substrates were added directly to the bacterial cultures.     

Effect of nucleoside 5′-di- and 5′-tri-phosphates on pancreatic ribonuclease activity

1. ADP, ATP and GDP inhibited the phosphotransferase activity, the release of cyclic nucleotides from RNA, of ribonuclease. No significant inhibition was elicited by pyrimidine 5'-nucleoside diphosphates, CDP and UDP. 2. Inhibition by ADP, AMP, adenosine, adenine, NAD and NADP was insignificant at the concentrations tested. Small inhibition was observed with high concentrations of AMP and only when soluble RNA was the substrate. 3. Inhibition by ADP was found to be ;uncompetitive'. 4. Results seem to indicate that at least for optimum inhibition the polyphosphate of the purine nucleoside is essential. They further suggest that the inhibitor acts by combining with the enzyme only when the enzyme is bound to the substrate.     

Direct determination of uracil in [32P,uracil-3H]poly(dA.dT) and bisulfite-treated phage PM2 DNA

A simple but effective technique for determining the presence of uracil existing as either A:U base pairs or G:U base pairs in DNA was developed. DNA is degraded to deoxynucleoside 3'-monophosphates by a combination of micrococcal nuclease and spleen phosphodiesterase. The monophosphates are converted to 5'-end-labeled 32P-labeled diphosphates in a reaction catalyzed by T4 polynucleotide kinase. The resultant product is then converted to 5'-end-labeled deoxynucleoside monophosphates by P1 nuclease digestion, which specifically removes 3'-phosphates. Successful separation of labeled dUMP from conventional bases in DNA is achieved by two-dimensional polyethyleneimine chromatography, with its detection determined by autoradiography and liquid scintillation counting. The sensitivity of the technique described can detect a minimum 1 X 10(-16) mol of dUMP in DNA. Additionally, the detection of 5-methylcytosine in placental DNA demonstrates the flexibility of the technique for the analysis of modified bases in DNA.     

Nucleotide analogue inhibitors of purine nucleoside phosphorylase

The diphosphate of the antiherpetic agent acyclovir [9-[(2-hydroxyethoxy)methyl]guanine] has been shown to inhibit purine nucleoside phosphorylase with unique potency (Tuttle, J. V., and Krenitsky, T. A. (1984) J. Biol. Chem. 259, 4065-4069). A major factor contributing to the superior inhibition by this diphosphate over the corresponding mono- and triphosphates is revealed here. Homologues of acyclovir mono- and diphosphate that extend the ethoxy moiety by one to four methylene groups were synthesized. These homologues were evaluated for their ability to inhibit human purine nucleoside phosphorylase. Within the diphosphate series, the Ki values increased progressively with increasing chain length. With the monophosphates, the Ki values reached a minimum with the homologue containing a pentoxy moiety. A plot of chain length versus Ki values for both mono- and diphosphates showed that both series had similar optimal distances between the aminal carbon and the terminal oxygen anion. Monophosphates with optimal positioning were somewhat less potent than diphosphates with similar positioning. Nevertheless, it was clear that a major factor in determining potency of inhibition was the distance of the terminal phosphate from the guanine moiety.     

Germ Tube Growth Inhibitor from Cronartium comandrae Aeciospores

The reduction of nucleic acid by an endogenous polynucleotide phosphorylase and ribonuclease in cells of Brevibacterium JM98A (ATCC 29895) was studied. A simple process was developed for the activation of the endogenous RNA-degrading enzyme(s). RNA degradation was activated by the presence of Pi with 14.2 mumol of ribonucleoside 5'-monophosphate per g of cell mass accumulating extracellularly. The optimum pH for degradation of RNA was 10.5 and the optimum temperature was 55 to 60 degrees C. Enzymatic activity was inhibited by the presence of Ca2+, Zn2+, or Mg2+. Although some of the RNA-degrading enzymatic activity was associated with the ribosomal fraction, most was soluble. Both polynucleotide phosphorylase and ribonuclease activities were identified.     

Metabolic phosphorylation and excretion of some nucleoside analogues in insects

After oral administration to the hemipteran insect Pyrrhocoris apterus L. (Fireburg), the L-enantiomers and certain open-chain analogues of the nucleosides are rapidly converted into the corresponding monophosphates, which are then excreted. This metabolic phosphorylation is almost quantitative; it occurs at the primary as well as at the secondary hydroxylic groups. The process is abolished when the respective nucleoside analogues contain a free carboxylic group. By contrast, the phosphorylating capacity is unaffected by structural variations at the heterocyclic base. This phosphorylation and excretion may represent a part of detoxication mechanism for the above nucleoside analogues.     

Separation of nucleosides and nucleotides by reversed-phase high-performance liquid chromatography with volatile buffers allowing sample recovery

Reversed-phase high-performance liquid chromatography using a C18 column with volatile buffers as the eluant was applied to the separation of a number of nucleosides and nucleotides. Groups of seven nucleosides and five nucleoside monophosphates were separated isocratically employing 0.1 M trimethylammonium acetate and 2% acetonitrile at pH 7.0. Groups of seven nucleoside diphosphates and seven nucleoside triphosphates were separated with 0.1 M triethylammonium bicarbonate and 2% acetonitrile titrated to a pH of 7.1 with acetic acid. The techniques described give resolution and separations comparable to nonvolatile buffers. Moreover, the eluant trimethylammonium acetate or triethylammonium bicarbonate buffer can easily be removed in vacuo from the column effluent, making the technique useful for preparative separations of these compounds. The observed elution pattern of nucleoside phosphates suggests that "paired-ion" chromatography is involved in the separation.