Simultaneous determination of guanine nucleotides in rat brain by high-performance liquid chromatography with dual-electrochemical detection

A new, sensitive, and specific assay method for guanine nucleotides using high-performance liquid chromatography with dual-electrochemical detection was developed. GTP, GDP, GMP, and cyclic GMP were separated with reversed-phase "ion-pair" chromatography and detected by a dual-electrochemical detector. Only guanine nucleotides among all purine and pyrimidine nucleotides responded to the electrochemical detector at 0.95 V. The peak heights for these guanine nucleotides were linear at concentrations between 0.5 pmol and 1 nmol. The regional distribution of these guanine nucleotides in the rat brain was studied by this new assay method.     

Seasonal changes of nucleotides in mussel (Mytilus galloprovincialis) mantle tissue

Seasonal variations of nucleotides in Mytilus galloprovincialis mantle tissue were analyzed. Separation and quantification was achieved by reversed-phase high-performance liquid chromatography. Total nucleotides show a pronounced seasonal variation with maximum and minimum values in autumn and spring, respectively. Adenine nucleotides accounted for the major part in spring and summer, guanosine and cytidine nucleotides in winter; uridine nucleotides were relatively constant throughout the year. Their inverse variation suggests inter-conversion among them and the maintenance of the potential cell energy in winter by other triphosphate nucleotides different from ATP. These results reflect environmental and nutritional conditions, and also the reserves and gametogenic cycles taking place in M. galloprovincialis mantle tissue.     

Free nucleotides in bromodeoxyuridine-treated neuroblastoma cells

Free nucleotides in bromodeoxyuridine-treated neuroblastoma cells were investigated. The separation and purification of nucleotides were carried out by anion exchange and paper chromatography. After bromodeoxyuridine treatment, the distribution of free nucleotides with the same base, when referred to total nucleotides, shifted toward a distribution pattern closer to that of mature neuronal cells. There was an enhancement of adenylic nucleotides and a decrease of uridylic and cytidylic nucleotides. The guanylic nucleotides displayed no changes. This nucleotide distribution is in agreement with other biochemical data from differentiated neuroblastoma cells.Dr. Ciesielski-Treska is stagiaire de Recherche à l'INSERMDr. Wintzerith is Chargée de Recherche au CNRSThis paper is part of the Doctorat d'Etat thesis of A. Dierich.     

Preservation of red blood cells with purines and nucleosides. III. Synthesis of adenine, guanine, and hypoxanthine nucleotides

Purine nucleotides of fresh human red cells and of red cells during storage at 4 degrees and 25 degrees C with additions of adenine, guanine, guanosine and inosine were estimated by HPLC. Six nucleotides were found in red cells: ATP, ADP, AMP, GTP, GDP, and IMP. The adenine nucleotides represented 92 per cent of the total purine nucleotides, guanine nucleotides 7 per cent and IMP less than 1 per cent. In red cells stored with adenine the total concentration of purine nucleotides increased to 125 per cent of the normal value. An adenine-free but guanine and guanine + inosine containing medium caused a decrease of the concentration of purine nucleotides by 10 to 20 per cent. When red cells were stored without adding guanine or guanosine the content of the guanine nucleotides decreased from 0.32 to 0.17 mumol/g Hb due to the decrease in the GTP content, but the GDP concentration increased slightly. In CPD-AG blood, however, the concentration of guanine nucleotides increased considerably up to 0.6 mumol/g Hb. IMP was estimated in all investigated stored red cells. In CPD-A and in CPD-AG blood 0.4 mumol/g Hb were produced during 3 weeks of storage, but twice of that in CPD-AI blood. The principles of the synthesis and the degradation of purine nucleotides in stored red cells are discussed in detail.     

Variations of the mononucleotide and short oligonucleotide distributions in the genomes of various organisms.

We calculated the variation coefficients of the mononucleotide and short oligonucleotide distributions in over 1700 long genomic sequences originating from six organisms to demonstrate that the human and Escherichia coli genomic sequences were the least and the most uniform, respectively. The most non-random genomic distributions were exhibited by the four canonical nucleotides, followed by the strong and weak nucleotides, while the distributions of purine or pyrimidine nucleotides and especially the distributions of (A+C) and (G+T) were significantly more uniform even in the human genome. In the human and mouse genomes, the highest coefficients of variation were further observed with the oligonucleotides where CG was combined with the strong nucleotides while its combination with the weak nucleotides significantly decreased the variation which, however, was still very high. High variation was also exhibited by the remaining oligonucleotides composed exclusively of the strong nucleotides or those containing only weak nucleotides. On the other hand, the distributions of oligonucleotides containing similar and especially the same numbers of the strong and weak nucleotides, but no CG or TA dinucleotide, were the most uniform. The information following from the present analysis will be useful not only in the identification of important genomic regions but also in computer simulations of the genomic nucleotide sequences in order to trace and reproduce the pathways of genome evolution.     

Regulation of cyclosporin A sensitive mitochondrial permeability transition by the redox state of pyridine nucleotides

The mechanisms involved in the induction of cyclosporine A sensitive mitochondrial swelling by oxidative stress were investigated in isolated guinea pig liver mitochondria. The aim of our study was to investigate, if swelling is inevitably associated with the oxidation of pyridine nucleotides, and if the oxidized pyridine nucleotides have to be hydrolysed for the induction of mitochondrial swelling. Quantitative measurement of oxidized pyridine nucleotides was performed with HPLC. Mitochondrial swelling was recorded by monitoring the decrease in light scattering of the mitochondrial suspension. Reduction and oxidation of pyridine nucleotides were followed by monitoring the changes of the autofluorescence signal of reduced pyridine nucleotides. Qualitative measurement of mitochondrial membrane potential was performed with the fluorescence indicator rhodamine 123. Neither t-butyl hydroperoxide nor the dissipation of the mitochondrial inner membrane potential with FCCP (carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone) induced the opening of the membrane permeability transition pore, unless an extensive oxidation of mitochondrial pyridine nucleotides took place. Mitochondrial swelling induced by our experimental conditions was always sensitive to cyclosporine A and accompanied by a cyclosporine A sensitive release of inner mitochondrial pyridine nucleotides without pyridine nucleotide hydrolysis. Not the cycling of calcium across the mitochondrial inner membrane but the accumulation of calcium inside the mitochondria was a prerequisite for mitochondrial swelling. The mitochondrial membrane permeability transition is neither caused nor accompanied by the hydrolysis of mitochondrial pyridine nucleotides.     

Sequence requirements in the catalytic core of the "10-23" DNA enzyme

A systematic mutagenesis study of the "10-23" DNA enzyme was performed to analyze the sequence requirements of its catalytic domain. Therefore, each of the 15 core nucleotides was substituted separately by the remaining three naturally occurring nucleotides. Changes at the borders of the catalytic domain led to a dramatic loss of enzymatic activity, whereas several nucleotides in between could be exchanged without severe effects. Thymidine at position 8 had the lowest degree of conservation and its substitution by any of the other three nucleotides caused only a minor loss of activity. In addition to the standard nucleotides (adenosine, guanosine, thymidine, or cytidine) modified nucleotides were used to gain further information about the role of individual functional groups. Again, thymidine at position 8 as well as some other nucleotides could be substituted by inosine without severe effects on the catalytic activity. For two positions, additional experiments with 2-aminopurine and deoxypurine, respectively, were performed to obtain information about the specific role of functional groups. In addition to sequence-function relationships of the DNA enzyme, this study provides information about suitable sites to introduce modified nucleotides for further functional studies or for internal stabilization of the DNA enzyme against endonucleolytic attack.     

Membrane Biochemistry : by E. Sim Chapman and Hall; London, 1982

Adenine, cytidine and guanosine nucleotides were supplied to cultures of Rhodopseudomonas capsulata under aerobic heterotrophic and phototrophic growth conditions. Aerobic growth is not affected by exogenous nucleotides (up to 10 mM) whereas phototrophic growth is strongly inhibited by adenine but not by guanosine or cytidine nucleotides. During phototrophic growth there is an inverse relationship between the concentration of exogenous adenine nucleotides and photopigment synthesis. There are no statistically significant differences between the inhibitory effect of AMP, ADP and ATP on the growth rate and bacteriochlorophyll synthesis since adenine nucleotides are incorporated into the cell as AMP by means of the phosphoribosyl transferase system.     

Functional nucleotides of U5 LTR determining substrate specificity of prototype foamy virus integrase

In order to study functional nucleotides in prototype foamy virus (PFV) DNA on specific recognition by PFV integrase (IN), we designed chimeric U5 long terminal repeat (LTR) DNA substrates by exchanging comparative sequences between human immunodeficiency virus type-1 (HIV-1) and PFV U5 LTRs, and investigated the 3'-end processing reactivity using HIV-1 and PFV INs, respectively. HIV-1 IN recognized the nucleotides present in the fifth and sixth positions at the 3'-end of the substrates more specifically than any other nucleotides in the viral DNA. However, PFV IN recognized the eighth and ninth nucleotides as distinctively as the fifth and sixth nucleotides in the reactions. In addition, none of the nucleotides present in the twelfth, sixteenth, seventeenth, eighteenth, nineteenth, and twentieth positions were not differentially recognized by HIV-1 and PFV INs, respectively. Therefore, our results suggest that the functional nucleotides that are specifically recognized by its own IN in the PFV U5 LTR are different from those in the HIV-1 U5 LTR in aspects of the positions and nucleotide sequences. Furthermore, it is proposed that the functional nucleotides related to the specific recognition by retroviral INs are present inside ten nucleotides from the 3'-end of the U5 LTR.     

Minimal DNA duplex requirements for topoisomerase I-mediated cleavage in vitro

The minimal DNA duplex requirements for topoisomerase I-mediated cleavage at a specific binding sequence were determined by analyzing the interaction of the enzyme with sets of DNA substrates varying successively by single nucleotides at the 5'- or 3' end of either strand. Topoisomerase I cleavage experiments showed a minimal region of nine nucleotides on the scissile strand and five nucleotides on the noncleaved strand. On the scissile strand, seven of the nine nucleotides were situated upstream to the cleavage site, while all five nucleotides required on the non-cleaved strand were located to this side. The results suggested that topoisomerase I bound tightly to this region, stabilizing the DNA duplex extensively. On minimal substrates which were partially single-stranded downstream to the cleavage site, cleavage was suicidal, that is, the enzyme was able to cleave the substrates, but unable to perform the final religation.     

Determination of sugar phosphates and nucleotides related to photosynthetic metabolism by high-performance anion-exchange liquid chromatography with fluorometric and ultraviolet detection

A high-performance anion-exchange liquid chromatography system was constructed to identify sugar phosphates and nucleotides involved in photosynthetic metabolism. First sugar phosphates and nucleotides were separated by a gradient elution with boric acid and sodium phosphate, then they were detected by a fluorescence detector (as fluorescent derivatives with arginine) and UV detector, respectively. Eight authentic sugar phosphates and 11 authentic nucleotides could be analyzed using the system. The applicability of the system to the determination of the corresponding sugar phosphates and nucleotides in extracts from only five soybean leaf discs (8.95 cm2) was shown.     

Cytokinin-induced differentiation of human myeloid leukemia HL-60 cells is associated with the formation of nucleotides, but not with incorporation into DNA or RNA

Cytokinins are important purine derivatives that act as hormones to control many processes in plants. Cytokinins such as isopentenyladenine (IPA), kinetin and benzyladenine were very effective at inducing the granulocytic differentiation of human myeloid leukemia HL-60 cells. The metabolism of cytokinins to their nucleotides was closely associated with cytokinin-induced differentiation and growth inhibition. When the cells were incubated with [14C]-benzyladenine, radioactivity was significantly incorporated into RNA and DNA. However, the radioactive nucleotides in RNA or DNA were adenine nucleotides, not benzyladenine nucleotides, suggesting that cytokinins were not incorporated into RNA and DNA. The benzyladenine nucleotides were not stably released into the medium in intact form. Cytokinins effectively induced a phosphorylated (active) form of mitogen-activated protein kinase (MAPK). MAPK activation was necessary for cytokinin-induced differentiation, because PD98059, an inhibitor of MAPK kinase, suppressed the differentiation induced by cytokinins. These results suggest that cytokinin nucleotides themselves play an important role in inducing the differentiation of HL-60 cells.     

Inactivation of rat liver HMG-CoA reductase phosphatases by nucleotides

Incubation of four purified rat liver HMG-CoA reductase phosphatases, with ATP, ADP and AMP caused a concentration-dependent inactivation of enzyme activities. The nucleotides of guanine, cytosine and uracil produced similar effects to those by the nucleotides of adenine for the same number of phosphates present in the molecules. The greater the number of phosphate groups in nucleotides, the higher was the inhibition in reductase phosphatases observed. Preincubation of phosphatases with ATP and subsequent dilution did not diminish the inactivation effect, showing that nucleotides inhibit the enzyme prior to their binding to the substrate. A relationship was observed between those concentrations of nucleotides which produce 50% inactivation and the logarithm stability constant of Mg or Mn salts of nucleotides. ATP-inactivated enzymes were reactivated by Mn++ and to a lesser proportion by Mg++, the conclusion being that HMG-CoA reductase phosphatases have the characteristics of metalloenzymes.     

Purine salvage in Entamoeba histolytica

Pulse-labeling of the nucleotide pool in Entamoeba histolytica with radioactive precursors, and subsequent high performance liquid chromatographic (HPLC) analysis of the radiolabeled nucleotides, indicate that E. histolytica is incapable of de novo synthesis of purine nucleotides. Hypoxanthine, inosine and xanthine could not be converted to nucleotides in E. histolytica, which suggests the absence of interconversion between adenine nucleotides and guanine nucleotides through formation of IMP. Adenosine was actively incorporated into nucleotides at an initial rate of 130 pmoles per minute per 10(6) trophozoites. Adenine, guanosine and guanine were also incorporated at much lower rates. The rate of adenine incorporation was enhanced by the presence of guanosine; the rate of guanine incorporation was significantly increased by adenosine. These stimulatory effects suggest that the ribose moiety of adenosine or guanosine can be transferred to another purine base to form a new nucleoside, and that the purine nucleosides are the immediate precursors of E. histolytica nucleotides. HPLC results showed that the radiolabel in adenine was exclusively incorporated into adenine nucleotides and that guanine was found only among guanine nucleotides, whereas the radioactivity associated with the ribose moiety of adenosine or guanosine was distributed among both adenine and guanine nucleotides.     

Purine metabolism in human lymphocytes.

In peripheral human blood lymphocytes the uptake and metabolism of adenine, guanine, and hypoxanthine was investigated. This was achieved by incubation of purified lymphocytes with 14C-purine bases, separation of cells from the incubation medium by a rapid filtration technique, and subsequent separation of the acid soluble material by thin-layer chromatography. No perferential uptake for one of the purine bases was observed. In all cases only traces of 14C-purine bases not added originally and labeled nucleosides could be demonstrated. Approximately 2/3 of adenine and 1/2 of guanine or hypoxanthine were converted to nucleotides. Separation of formed nucleotides showed that adenine and guanine were metabolized mainly to their corresponding nucleotides; hypoxanthine was converted to a considerable amount to adenine nucleotides and only to a small proportion into its own nucleotides. These results demonstrate the predomonance of adenine nucleotide formation in normal human lymphocytes.     

A1 Adenosine Receptor-G Protein Coupling in Bovine Brain Membranes: Effects of Guanine Nucleotides, Salt, and Solubilization

The effects of guanine nucleotides, NaCl, and solubilization on the interaction of antagonists and agonists with the A1 adenosine receptor of bovine brain membranes were studied using the high-affinity antagonist radioligand [3H]xanthine amine congener ([3H]XAC). In membranes, guanine nucleotides and NaCl had no effect on [3H]XAC saturation curves. Using agonist (R)-phenylisopropyladenosine (R-PIA) competition curves versus [3H]XAC, it was demonstrated that agonists could differentiate two affinity states having high and low affinity for agonist and that guanine nucleotides shifted the equilibrium to an all-low-affinity state that was indistinguishable from the low-affinity state in the absence of guanine nucleotides. In contrast, NaCl decreased agonist affinity by a distinctly different mechanism characterized by a parallel rightward shifted agonist curve such that R-PIA still recognized two affinity states albeit of lower affinity than in the absence of salt. R-PIA competition curves in the presence of both guanine nucleotides and salt were still shallow but were shifted far to the right, and two very low affinity states were discerned. On solubilization, guanine nucleotides in a reversible, concentration-dependent manner increased antagonist ([3H]XAC) but not agonist (R-N6-[3H]phenylisopropyladenosine) binding. This was consequent to a change in maximal binding capacity. R-PIA competition curves (versus [3H]XAC) in solubilized preparations demonstrated that agonist could still differentiate two agonist specific affinity states which were modulated by guanine nucleotides. In the presence of guanine nucleotides all the receptors were shifted to a uniform low-affinity state. In contrast, NaCl had no effect on agonist affinity as determined by agonist competition curves in a solubilized receptor preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exchange and hydrolysis of tightly bound nucleotides in normal and photolabelled bovine heart mitochondrial F1-ATPase

Treatment of F1 by threefold fast-column centrifugation or by single ammonium sulphate precipitation followed by fast-column centrifugation resulted in enzyme preparations containing 2.5-2.8 mol of bound nucleotides per mol of F1. Short incubations of such F1 preparations in the presence of relatively low concentrations of [14C]ATP and 2-azido[alpha-32P]ATP (100-250 microM), followed by ammonium sulphate precipitation and fast-column centrifugation, resulted in exchange of about 1 mol of the bound nucleotide per mol of F1 not affecting the total amount of bound nucleotides. Exchange of bound nucleotides with 2-azidoATP, followed by ultraviolet irradiation, results in inhibition of the enzyme activity, full inhibition being obtained (via extrapolation) when 1 mol of 2-nitreno-adenosine 5'-tri- or diphosphate (2-N-AT(D)P) is covalently bound to the presumably catalytic site on the enzyme (Van Dongen, M.B.M., De Geus, J.P., Korver, T., Harton, A.F. and Berden, J.A. (1986) Biochim. Biophys. Acta 850, 359-368). In agreement with this, it was found that incorporated [gamma-32P]ATP was hydrolysed by more than 80%. Newly incorporated, not covalently bound radioactive nucleotides could be rapidly exchanged again by the addition of non-radioactive nucleotides, but a higher concentration of nucleotides was needed to fully exchange the incorporated nucleotide. Also, when F1 was depleted of most of its bound nucleotides by repeated ammonium sulphate precipitation, part of the residual nucleotides was still rapidly exchangeable. The ability of F1 to exchange (and hydrolyse) one of the bound nucleotides was not lost when one catalytic and one non-catalytic binding site were occupied by covalently bound 8-N-ATP. Similar results were obtained with F1 containing 2-nitrenoATP covalently bound to one of the catalytic sites. Also, after photolabelling of up to four binding sites with 8-N[( 2-3H]AT(D)P, part of the two remaining non-covalently bound nucleotides could still be rapidly exchanged. In this case the exchanged nucleotide was also hydrolysed. It is concluded that one of the two bound nucleotides became exchangeable when all four other sites (i.e., two catalytic and two non-catalytic) were occupied with covalently bound nucleotides. The site involved showed catalytic properties suggestive of localisation on a beta-subunit.     

Purine metabolism in trypanosomatids.

Purine nucleotide biosynthesis was studied in culture forms of Trypanosoma cruzi strain Y, Crithidia deanei (a reduviid trypanosomatid with an endosymbiote) and an aposymbiotic strain of C. deanei (obtained by curing C. deanei with chloramphenicol). Trypanosoma cruzi was found to synthesize purine nucleotides only fring incorporated into both adenine and guanine nucleotides. Similar results were obtained with guanine, indicating that this flagellate has a system for the interconversion of purine nucleotides. Crithidia deanei was able to synthesize purine and pyrimidine nucleotides from glycine ("de novo" pathway) and purine nucleotides from adenine and guanine ("salvage" pathway). Adenine was incorporated into both adenine and guanine nucleotides, while guanine was incorporated into guanine nucleotides only, indicating the presence of a metabolic block at the level of GMP reductase. The aposymbiotic C. deanei strain was unable to utilize glycine for the synthesis of purine nucleotides, although glycine was utilized for synthesizing pyrimidine nucleotides. These results suggest that the endosymbiote is implicated in the de novo purine nucleotide pathway of the C. deanei-endosymbiote complex. The incorporation of adenine and guanine by aposymbiotic C. deanei strain followed a pattern similar to that observed for C. deanei.     

31P NMR visibility and characterization of rat liver mitochondrial matrix adenine nucleotides

Compartmentation and NMR visibility of mitochondrial adenine nucleotides were quantitated in isolated rat liver mitochondria respiring on succinate and glutamate in vitro at 8 and 25 degrees C. Intra- and extramitochondrial nucleotides were discriminated by adding the chelator trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA). T1 values of about 0.2-0.3 s for magnesium-bound matrix nucleotides were determined. Adenine nucleotide T1 values were influenced by the ionic environment; only magnesium-free ATP T1's were affected by temperature. Intra- and extramitochondrial adenine nucleotide ratios were varied in ATP-loaded mitochondria with added ATP and phosphate using the mitochondrial inhibitors oligomycin and carboxyatractyloside, and adenine nucleotides were quantitated by using NMR and enzymatic analysis. There was good agreement between matrix ATP concentrations (magnesium-bound ATP) calculated by using NMR and standard biochemical techniques. Although matrix ADP could be detected by NMR, it was difficult to quantitate accurately by NMR. The data indicate that mitochondrial ATP is NMR-visible in isolated mitochondria in vitro.