Hydrogen Bond Equilibrium Constants for the Complexes of Benzotriazole and some Nucleoside Derivatives. A Near Infrared Study

Abstract

Approximate hydrogen bond association constants were determined by near infrared spectroscopy for pairs formed by benzotriazole and a number of nucleoside derivatives.

The molecule of benzotriazole forms, in chloroform, hydrogen bonds with inosine, uridine and adenosine derivatives.

The order of decreasing association constants for complexes formed by benzotriazole and uridine or inosine derivatives is the opposite of the one observed for pairs formed by adenosine and uridine or inosine derivatives.     

Determination of the activation energy for pseudorotation of the furanose ring in nucleosides by 13-C nuclear-magnetic-resonance relaxation.

The activation energies for the pseudorotation of the furanose ring in adenosine, guanosine, inosine and xanthosine dissolved in liquid deuteroammonia have been determined by analysis of the longitudinal relaxation rates of the single tertiary carbons between +40 degrees C and minus 60 degrees C. For the purine ribosides the average activation energy was found to be 4.7 plus or minus 0.5 kcal x mol-1 (20 plus or minus 2 kJ x mol-1). For the pyrimidine nucleosides cytidine and uridine the respective activation energy should be higher since it could not be determined by 13-C relaxation measurements. This result can be explained by the formation of a hydrogen bond between the 5'-hydroxymethyl group and the base. In adenosine, guanosine, inosine and xanthosine the relaxation rates of C(5') are smaller than all others thus excluding the formation of a hydrogen bond between the purine base and the 5'-hydroxymethyl group of a strength comparable to the one suggested for cytidine and uridine.     

Clay Catalysis of Oligonucleotide Formation: Kinetics of the Reaction of the 5'-Phosphorimidazolides of Nucleotides with the Non-Basic Heterocycles Uracil and Hypoxanthine

The montmorillonite clay catalyzed condensation of activated monocleotides to oligomers of RNA is a possible first step in the formation of the proposed RNA world. The rate constants for the condensation of the phosphorimidazolide of adenosine were measured previously and these studies have been extended to the phosphorimidazolides of inosine and uridine in the present work to determine if substitution of neutral heterocycles for the basic adenine ring changes the reaction rate or regioselectivity. The oligomerization reactions of the 5'-phosphoromidazolides of uridine (ImpU) and inosine (ImpI) on montmorillonite yield oligo(U)s and oligo(I)s as long as heptamers. The rate constants for oligonucleotide formation were determined by measuring the rates of formation of the oligomers by HPLC. Both the apparent rate constants in the reaction mixture and the rate constants on the clay surface were calculated using the partition coefficients of the oligomers between the aqueous and clay phases. The rate constants for trimer formation are much greater than those dimer synthesis but there was little difference in the rate constants for the formation of trimers and higher oligomers. The overall rates of oligomerization of the phosphorimidazolides of purine and pyrimidine nucleosides in the presence of montmorillonite clay are the same suggesting that RNA formed on the primitive Earth could have contained a variety of heterocyclic bases. The rate constants for oligomerization of pyrimidine nucleotides on the clay surface are significantly higher than those of purine nucleotides since the pyrimidine nucleotides bind less strongly to the clay than do the purine nucleotides. The differences in the binding is probably due to Van der Waals interactions between the purine bases and the clay surface. Differences in the basicity of the heterocyclic ring in the nucleotide have little effect on the oligomerization process.     

Leishmania donovani: characteristics of adenosine and inosine transporters in promastigotes of two different strains

The nucleoside transport characteristics of two strains of Leishmania donovani promastigotes were studied. Strain S1, growing in fully defined medium, and strain S2 (MHOM/ET/67/HA3) both transported adenosine and inosine, but only strain S1 transported uridine and thymidine. Competition studies in the presence of 100 microM of unlabeled adenosine, inosine, guanosine, 2'-deoxyadenosine, tubercidin, formycin B, 3'-deoxyinosine as well as uridine, thymidine and cytidine, with either 1 microM [3H]adenosine or [3H]inosine as permeant, were carried out. The inhibition profile with [3H]inosine as permeant was essentially identical in S1 and S2 promastigotes, indicating that the same inosine transporter was present in both strains. However, with [3H] adenosine as permeant, significant differences were noted between the two strains. Thus, only adenosine, 2'-deoxyadenosine, tubercidin, uridine, and thymidine were strongly inhibitory in S1 promastigotes, while essentially all nucleosides tested were effective in S2 promastigotes. This indicates that adenosine transport in S2 promastigotes seems to involve a transporter differing from that described for S1 promastigotes.     

Hydrogen bond equilibrium constants of some unusual nucleotide base pairs

Approximate hydrogen bond association constants were determined for base pairs formed by an adenine derivative and a number of unusual pyrimidine bases. A series is found in which the H-bond strength in the base-pairs varies. In certain cases the H-bond equilibrium constant is larger than in the adenine-thymine pair. Inosine derivatives seem to have a non-negligible chance of replacing guanosine in the guanosine-cytosine pair. Infrared, near-infrared (overtone) and NMR spectra were used to determine the equilibrium constants.     

Hydrogen Bond Equilibrium Constants of Some Unusual Nucleotide Base Pairs

Abstract

Approximate hydrogen bond association constants were determined for base pairs formed by an adenine derivative and a number of unusual pyrimidine bases. A series is found in which the H-bond strength in the base-pairs varies. In certain cases the H-bond equilibrium constant is larger than in the adenine-thymine pair. Inosine derivatives seem to have a non-negligible chance of replacing guanosine in the guanosine-cytosine pair. Infrared, near-infrared (overtone) and NMR spectra were used to determine the equilibrium constants.     

Transport mechanisms in isolated plasma membranes. Nucleoside processing by membrane vesicles from mouse fibroblast cells grown in defined medium.

Plasma membrane vesicles were isolated from a subline of L929 mouse fibroblasts grown on defined medium in the absence of serum. These vesicles were not significantly contaminated by mitochondria or endoplasmic reticulum. The isolation procedure, a modification of that originally developed by McKeel and Jarett (McKeel, D.W., and Jarett, L. (1970) J. Cell Biol. 44, 417-432) employs mechanical homogenization in isotonic medium followed by differential centrifugation. The resultant plasma membrane vesicles take up radioactivity when exposed to uniformly labeled nucleosides. Two subfractions of the plasma membrane were isolated, distinguished by their differing activity of 5'-nucleotidase and (Na+,K+)-stimulated ATPase, two well known plasma membrane enzyme markers. Uptake of nucleoside radioactivity was extensively studied in one subfraction; it was linear with time and membrane concentration over ranges used for the studies. Apparent Km values for uptake of radioactivity from adenosine, inosine, and uridine were 7.1 +/- 26 muM, respectively. Uptake of radioactivity from all three nucleosides exhibits a broad pH optimum from pH 7 to pH 9, but falls off rapidly at lower pH. N-Ethylmaleimide was an effective inhibitor of uptake of radioactivity from all three nucleosides; uptake of radioactivity from uridine is more sensitive than uptake of radioactivity from the purine nucleosides. Adenosine inhibited uptake of radioactivity from inosine more than from uridine. Inosine inhibited the uptake of radioactivity from adenosine, but uridine did not. Caffeine and 6-methylaminopurine riboside (6-N-methyladenosine differentially inhibit uptake of radioactivity from adenosine and inosine, and thus the vesicles apparently possess seperate transport systems for uptake of radioactivity from purine nucleosides and from uridine.     

Passive and carrier-mediated permeation of different nucleosides through the reconstituted nucleoside transporter

When reconstituted into proteoliposomes, the human erythrocyte nucleoside transporter catalysed nitrobenzylthioguanosine (NBTGR)-sensitive zero-trans influx of three different nucleosides at broadly similar rates (inosine, uridine greater than adenosine). However, proteoliposomes also exhibited high rates of NBTGR-insensitive uptake of adenosine, making this nucleoside unsuitable for reconstitution studies. Equivalent high rates of adenosine influx were observed in protein-free liposomes, establishing that this permeability pathway represents simple diffusion of nucleoside across the lipid bilayer. In contrast to adenosine, inosine and uridine exhibited acceptable rates of NBTGR-insensitive uptake. Of the two, inosine is the more attractive permeant for reconstitution experiments, having a 2.5-fold lower basal membrane permeability. Studies of nucleoside transport specificity in reconstituted membrane vesicles should take account of the widely different passive permeabilities of different nucleosides.     

Characterization of inosine–uridine nucleoside hydrolase (RihC) from Escherichia coli

A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480 μM. No activity was exhibited against 2′-OH and 3′-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5′-OH group or with the 2′-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.     

Sustained depolarisation induces changes in the extracellular concentrations of purine and pyrimidine nucleosides in the rat thalamus

ATP and adenosine are well-known neuroactive compounds. Other nucleotides and nucleosides may also be involved in different brain functions. This paper reports on extracellular concentrations of nucleobases and nucleosides (uracil, hypoxanthine, xanthine, uridine, 2'-deoxycytidine, 2'-deoxyuridine, inosine, guanosine, thymidine, adenosine) in response to sustained depolarisation, using in vivo brain microdialysis technique in the rat thalamus. High-potassium solution, the glutamate agonist kainate, and the Na(+)/K(+) ATPase blocker ouabain were applied in the perfusate of microdialysis probes and induced release of various purine and pyrimidine nucleosides. All three types of depolarisation increased the level of hypoxanthine, uridine, inosine, guanosine and adenosine. The levels of measured deoxynucleosides (2'-deoxycytidine, 2'-deoxyuridine and thymidine) decreased or did not change, depending on the type of depolarisation. Kainate-induced changes were TTX insensitive, and ouabain-induced changes for inosine, guanosine, 2'-deoxycytidine and 2'-deoxyuridine were TTX sensitive. In contrast, TTX application without depolarisation decreased the extracellular concentrations of hypoxanthine, uridine, inosine, guanosine and adenosine.Our data suggest that various nucleosides may be released from cells exposed to excessive activity and, thus, support several different lines of research concerning the regulatory roles of nucleosides.     

Post-transcriptional modification of the wobble nucleotide in anticodon-substituted yeast tRNAArgII after microinjection into Xenopus laevis oocytes

An enzymatic procedure for the replacement of the ICG anticodon of yeast tRNAArgII by NCG trinucleotide (N = A, C, G or U) is described. Partial digestion with S1-nuclease and T1-RNAase provides fragments which, when annealed together, form an "anticodon-deprived" yeast tRNAArgII. A novel anticodon, phosphorylated with (32P) label on its 5' terminal residue, is then inserted using T4-RNA ligase. Such "anticodon-substituted" yeast tRNAArgII are microinjected into the cytoplasm of Xenopus laevis oocytes and shown to be able to interact with the anticodon maturation enzymes under in vivo conditions. Our results indicate that when adenosine occurs in the wobble position (A34) in yeast tRNAArgII it is efficiently modified into inosine (I34) while uridine (U34) is transformed into two uridine derivatives, one of which is probably mcm5U. In contrast, when a cytosine (C34) or guanosine (G34) occurs, they are not modified. These results are at variance with those obtained previously under similar conditions with anticodon derivatives of yeast tRNAAsp harbouring A, C, G or U as the first anticodon nucleotide. In this case, guanosine and uridine were modified while adenosine and cytosine were not.     

Stimulus- and cell-type-specific release of purines in cultured rat forebrain astrocytes and neurons

Adenosine is formed during conditions that deplete ATP, such as ischemia. Adenosine deaminase converts adenosine into inosine, and both adenosine and inosine can be beneficial for postischemic recovery. This study investigated adenosine and inosine release from astrocytes and neurons during chemical hypoxia or oxygen-glucose deprivation. In both cell types, 2-deoxyglucose was the most effective stimulus for depleting cellular ATP and for evoking inosine release; in contrast, oxygen-glucose deprivation evoked the greatest adenosine release. alpha,beta-Methylene ADP, an inhibitor of ecto-5'nucleotidase, significantly reduced adenosine release from astrocytes but not neurons. Dipyridamole, an inhibitor of equilibrative nucleoside transporters, inhibited both adenosine and inosine release from neurons. Erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase, reduced neuronal inosine release evoked by oxygen-glucose deprivation but not by 2-deoxyglucose treatment. These data indicate that (1). astrocytes release adenine nucleotides that are hydrolyzed extracellularly to adenosine, whereas neurons release adenosine per se, (2). inosine is formed intracellularly and released via nucleoside transporters, and (3). inosine is formed by an adenosine deaminase-dependent pathway during oxygen-glucose deprivation but not during 2-deoxyglucose treatment. In summary, the metabolic pathways for adenosine formation and release were cell-type dependent whereas the pathways for inosine formation were stimulus dependent.     

Investigation of Possible Participation of Nucleoside Transport Systems in the Postischemic Release of Purines and Pyrimidines from Cold Stored Liver

The aim of the study was to elucidate the role of nucleoside transport systems in the postischemic release of nucleosides and nucleobases accumulated by the rat liver during cold storage. Livers were preserved for 24 h in Euro-Collins (EC) or in a lactobionate-based solution (LBS) without exogenous adenosine. The rates of release of uric acid, xanthine, hypoxanthine, inosine, adenosine, uridine, and cytidine were monitored during early reperfusion. The greater part of the purines and pyrimidines (up to 80%) was lost in the first 2 min of reperfusion. After storage in EC, uric acid and xanthine formed more than 90% of the total purines released; nucleosides did not exceed 5% of the total. After storage in LBS, hypoxanthine formed more than 80% of purine efflux and the release of inosine and uridine was increased 5-10 times. These changes were shown to be due to the presence of allopurinol in LBS. Dipyridamole (an inhibitor of equilibrative nucleoside transporters) decreased the efflux of uric acid after storage in EC but residual release remained high. Dipyridamole exerted the most pronounced effect on the release of nucleosides (inosine and uridine) from livers stored in LBS. The use of sodium-free media for liver preservation and reperfusion did not alter the rates of purine and pyrimidine release. We conclude that equilibrative nucleoside transporters mediate the postischemic release of nucleosides and also, but to a less degree, of uric acid. Simple diffusion is an important factor in the release of nucleobases. Active Na(+)/nucleoside cotransport does not play an important role in early reperfusion.     

Effects of adenosine and adenine nucleotides on synaptic transmission in the cerebral cortex.

Adenosine and the adenine nucleotides have a potent depressant action on cerebral cortical neurons, including identified corticospinal cells. Other purine and pyrimidine nucleotides were either weakly depressant (inosine and guanosine derivatives) or largely inactive (xanthine, cytidine, thymidine, uridine derivatives). The 5'-triphosphates and to a lesser extent the 5'-diphosphates of all the purine and pyrimidines tested had excitant actions on cortical neurons. Adenosine transport blockers and deaminase inhibitors depressed the firing of cortical neurons and potentiated the depressant actions of adenosine and the adenine nucleotides. Methylxanthines (theophylline, caffeine, and isobutylmethylxanthine) antagonized the depressant effects of adenosine and the adenine nucleotides and enhanced the spontaneous firing rate of cerebral cortical neurons. Intracellular recordings showed that adenosine 5'-monophosphate hyperpolarizes cerebral cortical neurons and suppresses spontaneous and evoked excitatory postsynaptic potentials in the absence of any pronounced alterations in membrane resistance or of the threshold for action potential generation. It is suggested that adenosine depresses spontaneous and evoked activity by inhibiting the release of transmitter from presynaptic nerve terminals. Furthermore, the depressant effects of potentiators and excitant effects of antagonists of adenosine on neuronal firing are consistent with the hypothesis that cortical neurons are subject to control by endogenously released purines.     

Independent blood-brain barrier transport systems for nucleic acid precursors.

The blood-brain barrier permeability to certain 14-C-labelled purine and pyrimidine compounds was studied by simultaneous injection in conjunction with two reference isotopes into the rat common carotid artery and decapitation 15s later. The amount of 14-C-labelled base or nucleoside remaining in brain was expressed in relation to 3-H2O (a highly diffusible internal standard) and 113m-In-labelled EDTA (an essentially non-diffusible internal standard). Of the 17 compounds tested, measurable, saturable uptakes were established for adenine, adenosine, guanosine, inosine and uridine. Two independent transport systems in the rat blood-brain barrier were defined. One transported adenine (Km equals 0.027 mM) and could be inhibited with hypoxanthine. Adenosine (Km equals 0.018 mM), guanosine, inosine and uridine all cross-inhibit, defining a second independent nucleoside carrier system. Adenosine inhibited [14-D]uridine uptake more effectively than did uridine, suggesting a weaker affinity of uridine for this nucleoside carrier.     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. I. Kinetics of incorporation of nucleosides into nucleotide pools and pool sizes during growth cycle

Results from kinetic studies on the incorporation of ³H-5-uridine and ³H-8-adenosine into the acid-soluble nucleotide poor and nucleic acids by Novikoff hepatoma cells (subline N1S1-67) in suspension culture indicate that the uridine transport reaction is saturated at about 100 μM and that for adenosine at about 10 μM nucleoside in the medium, and that above 100 μM simple diffusion becomes the predominant mode of entry of both nucleosides into the cell. The Km of the transport reactions is approximately 1.3 × 10^?5 M for uridine and 6 × 10^?6 M for adenosine. The incorporation of these nucleosides into both the nucleotide pool and into nucleic acids seems to be limited by the rate of entry of the nucleic acid synthesis from the rate of incorporation of nucleosides. Other complicating factors are a change with time of labeling in the relative proporation of nucleoside incorporated into DNA and into the individual nucleotides of RNA, the splitting of uridine to uracil by th ecells, the deamination of adenosine kto inosine and the subsequent cleavage of inosine to hypoxanthine. Various lines of evidence are presented which indicate that the overall nucleotide pools of the cells are very small under normal growth conditions. During growth in the presence of 200 μM uridine or adenosine, however, the cells continue to convert the nucleosides into intracellular nucleotides much more rapidly than required for nucleic acid synthesis. This results in an accumulation of free uridine and adenosine nucleotides in the cells, the maximum amounts of which are at least equivalent to the amount of these nucleotides in total cellular RNA.     

Identification and characterisation of high affinity nucleoside and nucleobase transporters in Toxoplasma gondii

The protozoan parasite Toxoplasma gondii depends upon salvaging the purines that it requires. We have re-analysed purine transport in T. gondii and identified novel nucleoside and nucleobase transporters. The latter transports hypoxanthine (TgNBT1; K(m)=0.91+/-0.19 microM) and is inhibited by guanine and xanthine: it is the first high affinity nucleobase transporter to be identified in an apicomplexan parasite. The previously reported nucleoside transporter, TgAT1, is low affinity with K(m) values of 105 and 134 microM for adenosine and inosine, respectively. We have now identified a second nucleoside transporter, TgAT2, which is high affinity and inhibited by adenosine, inosine, guanosine, uridine and thymidine (K(m) values 0.28-1.5 microM) as well as cytidine (K(i)=32 microM). TgAT2 also recognises several nucleoside analogues with therapeutic potential. We have investigated the basis for the broad specificity of TgAT2 and found that hydrogen bonds are formed with the 3' and 5' hydroxyl groups and that the base groups are bound through H-bonds with either N3 of the purine ring or N(3)H of the pyrimidine ring, and most probably pi-pi-stacking as well. The identification of these high affinity purine nucleobase and nucleoside transporters reconciles for the first time the low abundance of free nucleosides and nucleobases in the intracellular environment with the efficient purine salvage carried out by T. gondii.     

Sugar-free growth of mammalian cells on some ribonucleosides but not on others

It was shown earlier that a variety of vertebrate cells could grow indefinitely in sugar-free medium supplemented with either uridine or cytidine at greater than or equal to 1 mM. In contrast, most purine nucleosides do not support sugar-free growth for one of the following reasons. The generation of ribose-1-P from nucleoside phosphorylase activity is necessary to provide all essential functions of sugar metabolism. Some nucleosides, e.g. xanthosine, did not support growth because they are poor substrates for this enzyme. De novo pyrimidine synthesis was inhibited greater than 80% by adenosine or high concentrations of inosine, e.g. 10 mM, which prevented growth on these nucleosides; in contrast, pyrimidine synthesis was inhibited only marginally on 1 mM inosine or guanosine, but normal growth was only seen on 1 mM inosine, not on guanosine. The inhibition of de novo adenine nucleotide synthesis prevented growth on guanosine, since guanine nucleotides could not be converted to adenine nucleotides. Guanine nucleotides were necessary for this inhibition of purine synthesis, since a mutant blocked in their synthesis grew normally on guanosine. De novo purine synthesis was severely inhibited by adenosine, inosine, or guanosine, but in contrast to guanosine, adenosine and inosine could provide all purine requirements by direct nucleotide conversions.     

Synthesis and electron microscopic investigation of model polyacryloynucleosides

Using the interaction of polyacrylic anhydride with uridine or N-acetyl derivatives of adenosine, cytidine, and guanosine, the water-soluble copolymers, polyacryloylnucleosides, were obtained. The acryloylnucleoside units to acrylic acid units ratio in the copolymer was usually about 1:20. The attaching of nucleosides to the polymer occurs mostly through the 5′-hydroxyl group of the sugar. The prominent feature of all polyacryloylnucleosides obtained is their fibrous structure at an ionic strength 0.2 and neutral pH. At concentrations <200 μg/ml the separate strands with a length of 0.2–1.0 μ and diameter of 30–40 Å are distinguishable. Evidently they are formed by side association of two molecules of copolymer or by folding of one molecule on itself. At higher concentrations branched multistranded structures are formed. In the same conditions polyacrylic acid alone does not form the fibrous structures. Heating of polyacryloylnucleoside solutions at 100°C and fast cooling in ice water, or raising of the pH to 12 turned the stranded structures to coils. After annealing or neutralization the stranded structures reformed. These transformations are similar to those which occur with nucleic acids. The results show that the fibrous structure of the copolymers depends on the hydrogen bonds formed by purine and/or pyrimidine bases.     

NUCLEOTIDE METABOLISM IN RAT BRAIN

Abstract— The uptake, the conversion to nucleotides, and their incorporation into RNA for labelled glycine, aspartate, the free bases and nucleosides of purines and pyrimidines were investigated with cortical slices of rat cerebrum. At the end of a 1-hr incubation time the slice-to-medium ratio of the radioactivities for labelled aspartate, glycine, adenine and adenosine were 34, 26, 20 and 5, respectively, while the slice-to-medium ratios for hypoxanthine, inosine, guanine, guanosine, xanthine, orotate, cytidine, cytosine, uridine, and uracil ranged from 1.3:1 to 2:1. Over 99 per cent of the total radioactivity taken up by the cortical slices was present in the TCA supernatant and 86, 82, 65, 50, 34, 23, 20 and 1.6 per cent of this radioactivity was in the form of nucleotides at the end of a 1-hr incubation with labelled adenine, adenosine, hypoxanthine, inosine, uridine, orotate, cytidine, and glycine, respectively. The incorporation of various radioactive precursors into RNA of cortical slices suggests that nucleotides originating from either de novo synthesis or preformed purine derivatives enter the same nucleotide pool utilized for RNA synthesis. The supernatant fraction from homogenized cerebrum was investigated for the presence of various anabolic and catabolic enzymes associated with nucleotide metabolism. These results were correlated with the data from the RNA incorporation studies, and a possible role for AMP: pyrophosphate phosphoribosyltransferase (adenine phosphoribosyltransferase, I.U.B. 2.4.2.7) to achieve intercellular transfer of AMP is discussed.