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PubMed1983

[Separation of natural ribonucleoside triphosphates and their determination in an acid-soluble cell fraction by anion exchange high performance liquid chromatography].

An anion-exchange HPLC procedure for CTP, UTP, ATP and GTP determination in the acid-soluble fraction of cells is described. Ribonucleoside triphosphates are separated on LiChrosorb AN isocratically with NH4H2PO4-acetonitrile. The dependence of the separation efficiency on the salt and acetonitrile concentrations and pH was analyzed and the optimal conditions were chosen. The range, wherein the linearity between the ribonucleoside triphosphate amount and the area of the corresponding peak is observed, was defined and the regression equations were derived. The CTP, UTP, ATP and GTP content in the ovarian cancer cells CaOv in culture was found to be 418 +/- 32, 1122 +/- 21, 9262 +/- 442 and 1036 +/- 49 pmole/10(6) cells, respectively. After 2 hr incubation with 6-mercaptopurine (10(-4) M) the level of ATP and GTP is reduced by 55%, and after 24 hr incubation--by 73% for ATP and 85% for GTP. At the same time the UTP and CTP content is decreased by 12-31%.

Adenosine Triphosphate↗
PubMed1994

Hydrolytic cleavage of purine ribonucleosides by extracts of Aspergillus terricola.

Cell-free extracts of nitrate-grown Aspergillus terricola could catalyze the hydrolytic cleavage of the N-glycosidic bond of adenosine, guanosine and inosine optimally at pH 4 and 50 degrees C. Incubation of the extracts at 60 degrees C for 60 minutes caused about 86%, 67% and 54% loss of activity respectively. The similarities between the pH or the temperatures profiles indicate that the hydrolytic cleavage of these purine ribonucleosides might be effected by one hydrolase. The results obtained indicate the absence of evidence for the involvement of an SH group(s) in the catalytic site. CoSO4 and CuSO4 showed a remarkable inhibiting effect on enzyme activity. The apparent Km values of the ribonucleoside hydrolase for adenosine, guanosine and inosine were calculated from Lineweaver--Burk plots and found to be 20, 22.2 and 10 x 10(-3)M respectively.

Adenosine↗
PubMed2000

Survey of the proton affinities of adenine, cytosine, thymine and uracil dideoxyribonucleosides, deoxyribonucleosides and ribonucleosides.

The kinetic method was applied to the determination of the proton affinities (PAs) of modified deoxy- and dideoxyribonucleosides. A correlation between the measured PAs and the replacement of one of the three hydroxyl groups of the ribose unit is presented. A PA scale was obtained which shows that the replacement of the primary or of one or both secondary hydroxyl groups of a ribonucleoside with a hydrogen atom induces the lowering or the enhancement of the nucleoside PA, respectively. The scale extends over a very narrow range of approximately 2 kcal mol(-1), thus demonstrating the sensitivity of the kinetic method in the evaluation of small differences in thermodynamic parameters.

Adenine Nucleotides↗
PubMed1999

The regulation of ribonucleoside diphosphate reductase by the tumor promoter orotic acid in normal rat liver in vivo.

Our earlier studies have shown that in normal hepatocytes, orotic acid (OA) inhibits DNA synthesis induced by several growth factors in vitro and after two-thirds partial hepatectomy (PH) in vivo. As in the normal liver OA induces an imbalance in nucleotide pools (specifically, an increase in uridine nucleotides, including deoxyuridine nucleotides, and a decrease in adenosine nucleotides, including ATP) and creation of this imbalance is crucial for the mitoinhibitory effects of OA, we hypothesized that ribonucleoside diphosphate reductase (RNR), a key enzyme in DNA synthesis that is regulated by nucleotide/deoxynucleotide levels, might be one of the targets for the inhibition of DNA synthesis by OA. To test this hypothesis, we subjected male Fischer 344 rats (130-150 g) to two-thirds PH in the absence or in the presence of OA (a 300-mg tablet of OA methyl ester implanted intraperitoneally at the time of two-thirds PH). The rats were killed at different times later, and their livers were processed for analysis of levels of RNR enzyme activity, protein, and mRNA transcripts. The results obtained indicated that treatment with OA resulted in a near-100% inhibition of RNR induced by two-thirds PH in rat liver, as monitored by enzyme activity and protein level. Furthermore, this inhibition was paralleled by a decrease in the mRNA transcripts for both the M1 and M2 subunits of RNR. Nuclear run-off assays indicated that this decrease in the levels of mRNA transcripts could not be attributed to an effect on transcription. However, administration of OA 20 h after two-thirds PH, when RNR mRNA transcripts were maximally induced, resulted in increased degradation of the RNR M1 and M2 subunits. Taken together, these results indicate that OA treatment decreases RNR levels induced by two-thirds PH, at the levels of enzyme activity, protein, and mRNA transcripts, and the decreased levels of mRNA transcripts appeared to be due to increased degradation of the transcripts.

Animals↗
PubMed1982

Biological markers and small cell carcinoma of the lung: a clinical evaluation of urinary ribonucleosides.

Five minor base ribonucleosides, primarily degradation products of transfer ribonucleic acid (tRNA), were evaluated as potential biological markers for patients with small cell carcinoma of the lung. The urinary concentration for pseudouridine, 1-methyladenosine, 1-methylinosine, N2-methylguanosine, and N2,N2-dimethylguanosine was determined by means of reversed-phase high performance liquid chromatography and quantitatively expressed as a function of creatinine excretion. Comparisons were made with carcinoembryonic antigen (CEA) plasma levels. The total frequency of elevated values for the five nucleosides in pretreatment urine samples was directly related to stage of disease with 24/60 (40%) determinations increased in 12 patients with limited disease and 69/85 (81%) in 17 patients with extensive disease. For these same patients, CEA levels were elevated respectively in 2/11 (18%) of the former and 9/17 (53%) of the latter group. The frequency and degree of elevation of the nucleoside/creatinine ratios in pretreatment samples from patients with extensive disease was correlated directly with increasing number of metastatic sites. Of the five nucleosides, the mean number elevated was two for limited disease, 3-4 for extensive disease with one metastatic site, 4 for two or three, and 5 for four or more sites of metastases. Based on a summation of pretreatment nucleoside/creatinine ratios, a discriminant for survival was derived giving curves separating patients (P = 0.086) similar to the discriminant based on stage of disease. Although discordant results were noted, an overall correlation of 75% agreement with clinical assessment was estimated in response categories when monitoring changes associated with therapy.

Antineoplastic Agents↗
PubMed1981

Aminoimidazole carboxamide ribonucleoside toxicity: a model for study of pyrimidine starvation.

Aminoimidazole carboxamide ribonucleoside (AIC-R), a purine precursor, has biphasic effects on the growth of Chinese hamster fibroblasts. At 200 microM AIC-R cell growth is almost completely arrested, while at 50 and 700 microM AIC-R cell growth is comparable to that observed in the absence of nucleoside. The growth inhibition produced by AIC-R is the consequence of inhibition of the orotate phosphoribosyltransferase-orotidylic decarboxylase (OPRT-ODC) reactions, as evidenced by a 87% reduction in the intracellular concentrations of UTP and CTP, accumulation of orotate in the medium, and restoration of normal growth by inclusion of 100 microM uridine in the medium. Inhibition of pyrimidine nucleotide synthesis at 200 microM AIC-R is associated with an 82% reduction in the intracellular concentration of PP-ribose-P and a 150% increase in the concentration of purine nucleotides. Restoration of cell growth to a normal rate at 700 microM AIC-R--a condition under which PP-ribose-P remains depressed and purine nucleotide concentrations are also depressed (40% of control)--and absence of toxicity at 50 microM AIC-R--a condition under which purine nucleotide concentrations are increased by 150% and PP-ribose-P concentration is normal--suggest that the inhibition of OPRT-ODC observed at 200 microM AIC-R is caused by the combination of the reduction in PP-ribose-P and increase in purine nucleotides. These studies provide a better understanding of the control of the OPRT-ODC reactions in the cell and provide additional insight into the basis of pyrimidine starvation induced by purine nucleosides.

Aminoimidazole Carboxamide↗
PubMed1983

Cell cycle regulation of ribonucleoside diphosphate reductase activity in permeable mouse L cells and in extracts.

Ribonucleoside diphosphate reductase (EC1.17.4.1) was previously characterized in exponentially growing mouse L cells selectively permeabilized to small molecules by treatment with dextran sulfate (Kucera and Paulus, 1982b). This characterization has now been extended to cells in specific phases of the cell cycle and in transition between cell cycle phases, with activity studied both in situ (permeabilized cells) and in cell extracts. Cells at various stages in the cell cycle were obtained by unit-gravity sedimentation employing a commercially available reorienting chamber device, by G1 arrest induced by isoleucine limitation, and by metaphase arrest induced by Colcemid. G1 cells from both cycling and noncycling populations had negligible levels of ribonucleotide reductase activity as measured by CDP reduction both in situ and in extracts. When G1 arrested cells were allowed to progress to S phase, ribonucleotide reductase activity increased in parallel with [3H]thymidine incorporation into DNA. Ribonucleotide reductase activity in extracts increased at a somewhat greater rate than in situ activity. S phase ribonucleotide reductase activity measured in situ resembled the previously characterized activity in exponentially growing cells with respect to an absolute dependence on ATP or its analogs as positive allosteric effector, sensitivity to the negative allosteric effector dATP, and low susceptibility to stimulation by NADPH, dithiothreitol, and FeCl3. Disruption of permeabilized cells caused reductase activity to become highly dependent on the presence of both dithiothreitol and FeCl3. As synchronized cultures progressed from S into G2/M phase, no significant change in ribonucleotide reductase activity was seen. On the other hand, when cells that had been arrested in metaphase by Colcemid were allowed to resume cell cycle traversal by removing the drug, in situ ribonucleotide reductase activity decreased by 75% within 2.5 h. This decrease seemed to be a late mitotic event, since it was not correlated with the percentage of cells entering G1 phase. The cause of a subsequent slight increase of in situ ribonucleotide reductase activity is not clear. Parallel measurements of ribonucleotide reductase activity in cell extracts indicated also an initial decline accompanied by increasing dependence on added dithiols and FeCl3, followed by complete activity loss. Our results suggest a cell cycle pattern of ribonucleotide reductase activity that involves negligible levels in G1 phase, a progressive increase of activity upon entry into S phase paralleling overall DNA synthesis, continued retention of significant ribonucleotide reductase activity well into the metaphase period of mitosis, and a very rapid decline in activity during the later phases of mitosis. The periods of increase and decrease of ribonucleotide reductase activity were accompanied by modulation of the properties of the enzyme as indicated by differential changes in enzyme activity measured in situ and in extracts.

Animals↗
PubMed1995

High-performance liquid chromatographic purification, optimization of the assay, and properties of ribonucleoside diphosphate reductase from rabbit bone marrow.

Partial purification of ribonucleoside diphosphate reductase from rabbit bone marrow was achieved by size exclusion HPLC of the crude homogenate. This step, requiring < 15 min, led to 9- to 13-fold purification of the reductase and removal of 64% of the contaminating kinase/phosphatase activities, which in the crude extract degrade > 95% of substrate CDP when reductase is assayed. A systematic study was conducted to evaluate the influence of contaminating kinase/phosphatase activities on CDP concentration during the reductase-catalyzed reaction with either ATP or its kinase-inhibiting analog, 5'-adenylylimidodiphosphate (AMP-PNP), as the allosteric effector. Our studies demonstrated that in the presence of ATP, CDP levels fell instantly to < 24% but thereafter remained fairly constant due to recycling via CTP. In contrast, in the presence of AMP-PNP, CDP levels decreased continuously. The Km values of the reductase for CDP determined in the presence of ATP were significantly higher than those in the presence of AMP-PNP. Furthermore, we also found that the concentration of the ultimate electron donor dithiothreitol (DTT) required for optimum activity of the reductase varies significantly with the level of purity of the reductase preparation. Interestingly, DTT is an inhibitor of the reductase above the optimum concentration. This purification method and the optimized assay together with the understanding of the fate of CDP in partially purified preparations should find application in studies with reductases from other eukaryotic sources.

Adenosine Triphosphate↗
PubMed1984

Requirement of protein synthesis for the induction of ribonucleoside diphosphate reductase mRNA in Escherichia coli.

An RNA-DNA hybridization assay was used to quantitate the ribonucleoside diphosphate reductase mRNA synthesis (nrd mRNA) to show that gene expression was dependent on protein synthesis. The increased nrd mRNA synthesis induced by inhibition of DNA synthesis was eliminated by simultaneous inhibition of protein synthesis. It was further found that protein synthesis is required not only initially but continuously during DNA inhibition for increased expression of nrd mRNA synthesis.

DNA Replication↗
PubMed1976

Nucleoside auxotrophy in Drosophila: an autosomal locus yielding mutants supplementable by purine and pyrimidine ribonucleosides.

Two allelic auxotrophic mutants at a locus close to the bw locus (2-104.5) of Drosophila melanogaster are described. The mutants respond to dietary ribonucleosides (uridine, cytidine, adenosine, guanosine and inosine) but less well to bases or pyrimidine precursors. This phenotype is unique to these mutants. We suggest that the mutants are defective in phosphoribosyl pyrophosphate biosynthesis.

Adenosine↗
PubMed1991

Pyrimidine base and ribonucleoside utilization by the Pseudomonas alcaligenes group.

Pyrimidine base and ribonucleoside utilization was investigated in the two type strains of the Pseudomonas alcaligenes group. As sole sources of nitrogen, the pyrimidine bases uracil, thymine and cytosine as well as the dihydropyrimidine bases dihydrouracil and dihydrothymine supported the growth of Pseudomonas pseudoalcaligenes ATCC 17440 but neither these bases nor pyrimidine nucleosides supported Pseudomonas alcaligens ATCC 14909 growth. Ribose, deoxyribose, pyrimidine and dihydropyrimidine bases as well as pyrimidine nucleosides failed to be utilized by either P. pseudoalcaligenes or P. alcaligenes as sole carbon sources. The activities of the pyrimidine salvage enzymes nucleoside hydrolase, cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase were detected in cell-free extracts of P. pseudoalcaligenes and P. alcaligenes. In P. pseudoalcaligenes, the levels of cytosine deaminase, dihydropyrimidine dehydrogenase and dihydropyrimidinase could be affected by the nitrogen source present in the culture medium.

Amidohydrolases↗
PubMed1983

Determination of ribonucleosides, deoxyribonucleosides, and purine and pyrimidine bases in adult rabbit cerebrospinal fluid and plasma.

Purine and pyrimidine base and nucleoside levels were measured in adult rabbit cisternal CSF and plasma by reversed-phase high-performance liquid chromatography. The concentrations of bases, nucleosides, and nucleoside phosphates were similar in plasma and CSF except for the adenosine phosphates and uracil which were higher in the plasma. In plasma and CSF, adenosine levels were low (0.12 microM) and guanosine, deoxyadenosine, deoxyguanosine, and deoxyinosine were not detectable (less than 0.1 microM); inosine and xanthine concentrations were 1-2 microM and hypoxanthine concentrations were approximately 5 microM; uridine (approximately 8 microM), cytidine (2-3 microM), and thymidine, deoxyuridine, and deoxycytidine (0.5-1.4 microM) were easily detectable. In both plasma and CSF, guanine, and thymine were undetectable (less than 0.1 microM), adenine and cytosine were less than 0.2 microM, but uracil was present (greater than 1 microM). Adenosine, inosine, and guanosine phosphates were also detectable at low concentrations in CSF and plasma. These results are consistent with the hypothesis that purine deoxyribonucleosides are synthesized in situ in the adult rabbit brain. In contrast, pyrimidine deoxyribonucleosides and ribonucleosides, and purine and pyrimidine bases are available in the CSF for use by the brain.

Adenine↗
PubMed1998

Effect of IciA protein on the expression of the nrd gene encoding ribonucleoside diphosphate reductase in E. coli.

The E. coli nrd operon contains the genes encoding the two subunits of ribonucleoside diphosphate reductase. We found that the IciA protein binds specifically to the AT-rich upstream region of nrd promoter. In vivo overexpression of IciA increases the expression of nrd gene by four- to five-fold, suggesting that IciA functions as a transcriptional activator for the nrd gene.

Bacterial Proteins↗
PubMed1983

Rapid incorporation of label from ribonucleoside disphosphates into DNA by a cell-free high molecular weight fraction from animal cell nuclei.

A readily sedimentable nuclear fraction from Chinese hamster embryo fibroblast (CHEF/18) cells catalyzes incorporation of 14C-rCDP into DNA. Data indicated that this incorporation is made possible by the conversion of rCDP into a small and functionally compartmentalized, rather than a large and freely diffusible, pool of dCTP. This catalytically active sedimentable fraction from S phase CHEF/18 cells or actively replicating calf thymus cells contains nascent and template DNA, and numerous enzymes required for DNA biosynthesis including ribonucleoside diphosphate reductase, thymidylate synthetase, dihydrofolate reductase, DNA methylase, topoisomerase and DNA polymerase. We have named this catalytically active macromolecule the replitase. The replitase fraction contained spherical particles with a diameter of approximately 24 to 30 nm and had an estimated molecular weight on the order of 5 X 10(6).

Animals↗
PubMed1994

Bacteriophage T4 ribonucleoside diphosphate reductase: on the defect causing decreased formation of the beta 93(2) subunit encoded by the nrdB93 mutant gene.

Bacteriophage T4 ribonucleoside diphosphate reductase is composed of two proteins, alpha 2 and beta 2, encoded by the nrdA and nrdB genes, respectively. The expression of nrdB is the limiting factor for the assembly of the enzyme. A recently described mutation, nrdB93, may give new insight into the regulation of synthesis of the beta subunit encoded by nrdB. Infection by T4 nrdB93 produced only low concentrations of the beta 2(93) protein. However, a site-specific mutation of phage T4 gene 39, encoding one of the subunits of T4 DNA topoisomerase, phenotypically suppressed the defect. The present work sought to characterize the nature of this defect. The mutation in nrdB93 was a single-base transition (G-->A) resulting in a Gly253-->Asp change. In vivo and in vitro studies provided no evidence of degradation of the beta 2(93) protein. Furthermore, the decrease in beta 2(93) formation was not caused by a delayed onset of transcription, neither by a decreased rate of mRNA formation from the nrdB promoter, nor by a defective intron splicing of the nrdB gene or in the transcription of the terminal segments of the message. These findings are consistent with the concept that the nrdB93 lesion produces a defect at the level of translation.

Amino Acid Sequence↗
PubMed2006

Differentiation of 2'-O- and 3'-O-methylated ribonucleosides by tandem mass spectrometry.

Recent studies revealed that the 3'-terminal nucleotides in plant microRNAs were methylated on the ribose at the 2' or 3' hydroxyl groups. Here we examined the fragmentation of the electrospray-produced [M + H]+ and [M - H]- ions of 2'- and 3'-O-methylated ribonucleosides. It turned out that the predominant fragmentation pathway for the [M + H]+ ions of ribose-methylated nucleosides was the neutral loss of the methylated ribose, which made it impossible to distinguish 2'-O-methylation from 3'-O-methylation by positive-ion MS/MS. However, characteristic fragment ions, resulting from the cleavage through the ribose rings, were produced for the [M - H]- ions of each pair of ribose-methylated nucleosides. In this respect, the neutral loss of a 90-Da fragment (C3H6O3) was observed for 2'-O-methylated cytidine, guanosine and adenosine, but not for their 3'-O-methylated counterparts. On the other hand, the neutral loss of a 60-Da fragment (C2H4O2) was found for 3'-O-methyluridine, but not for 2'-O-methyluridine.

Methylation↗
PubMed2006

α2 isoform-specific activation of 5'adenosine monophosphate-activated protein kinase by 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside at a physiological level activates glucose transport and increases glucose transporter 4 in mouse skeletal muscle.

5'Adenosine monophosphate-activated protein kinase (AMPK) has been implicated in exercise-induced stimulation of glucose metabolism in skeletal muscle. Although skeletal muscle expresses both the alpha1 and alpha2 isoforms of AMPK, the alpha2 isoform is activated predominantly in response to moderate-intensity endurance exercise in human and animal muscles. The purpose of this study was to determine whether activation of alpha2 AMPK plays a role in increasing the rate of glucose transport, promoting glucose transporter 4 (GLUT4) expression, and enhancing insulin sensitivity in skeletal muscle. To selectively activate the alpha2 isoform, we used 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR), which is metabolized in muscle cells and preferentially stimulates the alpha2 isoform. Subcutaneous administration of 250 mg/kg AICAR activated the alpha2 isoform for 90 minutes, but not the alpha1 isoform in hind limb muscles of the C57/B6J mouse. The maximal activation of the alpha2 isoform was observed 30 to 60 minutes after administration of AICAR and was similar to the activation induced by a 30-minute swim in a current pool. The increase in alpha2 activity paralleled the phosphorylation of Thr(172), the essential residue for full kinase activation, and the activity of acetyl-coenzyme A carboxylase beta, a known substrate of AMPK in skeletal muscle. Subcutaneous injection of AICAR rapidly increased, by 30%, the rate of 2-deoxyglucose (2DG) transport into soleus muscle; 2DG transport increased within 30 minutes and remained elevated for 4 hours after administration of AICAR. Repeated intraperitoneal injection of AICAR, 3 times a day for 4 to 7 days, increased soleus GLUT4 protein by 30% concomitant with a significant 20% increase in insulin-stimulated 2DG transport. These data suggest that moderate endurance exercise promotes glucose transport, GLUT4 expression, and insulin sensitivity in skeletal muscle at least partially via activation of the alpha2 isoform of AMPK.

AMP-Activated Protein Kinases↗
PubMed1983

On-line high-performance liquid affinity chromatography-high-performance liquid chromatography analysis of monomeric ribonucleoside compounds in biological fluids.

We describe an on-line multi-dimensional column chromatography system. It consists of a high-performance liquid affinity chromatography perfractionation column for cis-diol compounds and a series-connected reversed-phase high-performance liquid chromatography system for the analysis of ribonucleosides. This on-line procedure allows the rapid and direct analysis of methylated ribonucleic acid catabolites in biological fluids (serum, urine) and therefore might be useful in pathobiochemistry.

Chemical Phenomena↗