Role of adenine deaminase in purine salvage and nitrogen metabolism and characterization of the ade gene in Bacillus subtilis.

The isolation of mutants defective in adenine metabolism in Bacillus subtilis has provided a tool that has made it possible to investigate the role of adenine deaminase in adenine metabolism in growing cells. Adenine deaminase is the only enzyme that can deaminate adenine compounds in B. subtilis, a reaction which is important for adenine utilization as a purine and also as a nitrogen source. The uptake of adenine is strictly coupled to its further metabolism. Salvaging of adenine is inhibited by the stringent response to amino acid starvation, while the deamination of adenine is not. The level of adenine deaminase was reduced when exogenous guanosine served as the purine source and when glutamine served as the nitrogen source. The enzyme level was essentially the same whether ammonia or purines served as the nitrogen source. Reduced levels were seen on poor carbon sources. The ade gene was cloned, and the nucleotide sequence and mRNA analyses revealed a single-gene operon encoding a 65-kDa protein. By transductional crosses, we have located the ade gene to 130 degrees on the chromosomal map.     

Carboxyatractyloside-insensitive influx and efflux of adenine nucleotides in rat liver mitochondria

Unidirectional transport (influx and efflux) of adenine nucleotides in rat liver mitochondria was examined using carboxyatractyloside to inhibit rapid exchange of matrix and external adenine nucleotides via the adenine nucleotide translocase. Influx of adenine nucleotides was concentration-dependent. ATP was the preferred substrate with a Km of 2.67 mM and V of the preferred substrate with a Km of 2.67 mM and V of 8.33 nmol/min/mg of protein. For ADP, the Km was 14.7 mM and V was 10.8 nmol/min/mg of protein. Efflux of adenine nucleotides was also concentration-dependent, varying directly as a function of the matrix adenine nucleotide pool size. Any increase in the influx of adenine nucleotides was coupled to an increase in efflux. However, as the external ATP concentration was increased, influx was stimulated to a much greater extent than was efflux. This imbalance suggested that under certain conditions adenine nucleotide movement might be coupled to the movement of an alternate anion such as phosphate. Adenine nucleotide efflux increased as the external phosphate concentration was varied from 0.5 to 4 mM. Also, increasing the external phosphate concentration caused adenine nucleotide influx to decrease, suggesting competition. In the absence of external adenines and phosphate, no efflux occurred. Both adenine nucleotide influx and efflux were depressed if Mg2+ was omitted. Adenine nucleotide efflux in the presence of external phosphate was inhibited much less by lack of Mg2+ than was efflux in the presence of external ATP. This evidence supports a model in which either adenine nucleotides (probably with Mg2+) or phosphate can move across the mitochondrial membrane on a single carrier. Net adenine nucleotide movements can occur when adenine nucleotide movement is coupled to the movement of phosphate in the opposite direction.     

Stimulation of Growth of an Adenine-requiring Yeast by Purine Analogues

An adenine-requiring yeast grew in an adenine-free medium after considerably long lag period (approximately 170 hr). Supplement of adenine to the medium resulted in a marked reduction of the lag period and in a diauxic growth. Although the growth rate was much faster, a similar diauxic growth was observed in the medium containing sufficient amount of adenine. In both cases, the primary growth occurred as a result of fermentative metabolism of glucose, and after exhaustion of glucose, the secondary growth started at the expense of accumulated ethanol. When cells obtained from the adenine-deficient medium were analyzed for total adenine compounds, approximately six times as much adenine was detected as that amount of adenine added to the medium. Therefore, the yeast has no block in the biochemical sequences leading to adenine biosynthesis but has a defect in the control mechanism of adenine biosynthesis. Adenine appears to initiate adenine biosynthesis under adenine deficient conditions. In order to understand the initiation mechanism by adenine, the effect of several purine analogues on growth was examined. Among the agents tested, 6-thioguanine or 8-azaadenine shortened the lag time of growth in the adenine-free medium. Furthermore, when small amount of adenine was supplied to the medium, these compounds stimulated the primary growth without affecting the secondary growth. On the contrary, 8-azaguanine or 8-azaxanthine markedly stimulated the secondary growth without affecting the primary growth. Thus, two distinct trigger mechanisms in adenine biosynthesis were proposed.     

Some thoughts on the evolutionary basis for the prominent role of ATP and ADP in cellular energy metabolism

The predominance of the adenosine triphosphate/adenosine diphosphate (ATP/ADP) couple in cellular phosphorylation reactions, including those that form the basis for cellular energy metabolism, cannot be explained on thermodynamic grounds since a variety of "high energy phosphate" compounds (including ADP itself) found in the cell would, based on thermodynamic considerations, be at least as effective as ATP in serving as a phosphoryl donor. How then did present-day organisms come to rely on the ATP/ADP couple as the principal mediator of phosphorylation reactions? The early appearance of adenine compounds in the prebiotic environment is suggested by experiments indicating that, relative to other purine or pyridimine compounds, adenine derivatives are preferentially synthesized under simulated prebiotic conditions (Ponnamperuma et al., 1963). In addition to the roles of adenine nucleotides in phosphorylation reactions, other adenine derivatives (e.g. Coenzyme A, flavin adenine dinucleotide, puridine nucleotides) are employed in a variety of metabolic roles. The principal function of the adenine moiety in these latter cases is in the binding of these derivatives to the relevant enzyme. The capability for binding of the adenine moiety appears to have arisen early in evolution and been exploited in a multitude of contexts, a suggestion consistent with observed similarities between the binding sites of several enzymes employing adenine derivatives as substrate. The early availability of suitable adenine compounds in the biosphere and development of complementary binding sites on cellular proteins, coupled with the expected advantages in having a limited number of metabolites as central mediators of endergonic and exergonic metabolism could readily have led to the observed pre-eminence of adenine nucleotides in cellular energy metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

The contribution of adenine nucleotide loss to ischemia-induced impairment of rat kidney cortex mitochondria.

Adenine nucleotides and respiration were assayed with rat kidney mitochondria depleted of adenine nucleotides by pyrophosphate treatment and by normothermic ischemia, respectively, with the aim of identifying net uptake of ATP as well as elucidating the contribution of adenine nucleotide loss to the ischemic impairment of oxidative phosphorylation. Treatment of rat kidney mitochondria with pyrophosphate caused a loss of adenine nucleotides as well as a decrease of state 3 respiration. After incubation of pyrophosphate-treated mitochondria with ATP, Mg2+ and phosphate, the content of adenine nucleotides increased. We propose that kidney mitochondria possess a mechanism for net uptake of ATP. Restoration of a normal content of matrix adenine nucleotides was related to full recovery of the rate of state 3 respiration. A hyperbolic relationship between the matrix content of adenine nucleotides and the rate of state 3 respiration was observed. Mitochondria isolated from kidneys exposed to normothermic ischemia were characterized by a decrease in the content of adenine nucleotides as well as in state 3 respiration. Incubation of ischemic mitochondria with ATP, Mg2+ and phosphate restored the content of adenine nucleotides to values measured in freshly-isolated mitochondria. State 3 respiration of ischemic mitochondria reloaded with ATP recovered only partially. The rate of state 3 respiration increased by ATP-reloading approached that of uncoupler-stimulated respiration measured with ischemic mitochondria. These findings suggest that the decrease of matrix adenine nucleotides contributes to the impairment of ischemic mitochondria as well as underlining the occurrence of additional molecular changes of respiratory chain limiting the oxidative phosphorylation.     

Inhibition of purine utilization by adenine in Alcaligenes eutrophus H16

With 0.5% substrate present in mineral medium, cells of Alcaligenes eutrophus H 16 were able to grow heterotrophically at the expense of guanine, hypoxanthine and xanthine, but not of adenine as sole sources of carbon and nitrogen. An increase in cell counts, however, was observed at lower adenine concentrations (0.1%). Similarly, adenine was only respired if present at low concentrations. Higher amounts of adenine were inhibitory to the utilization of adenine, guanine, hypoxanthine, xanthine, allantoin and glyoxylate, but not to that of fructose or glycerate. The adenine-dependent inhibition of adenine utilization was not overcome by the addition of thiamine, uridine or cytidine. The enzyme glyoxylate carboligase, usually formed in presence of metabolisable purines and of allantoin, was synthesized only at low adenine concentrations. Higher amounts were inhibitory even with allantoin present as additional substrate. According to these resutls, the utilization of purine derivatives and of allantoin as sources of carbon and energy is repressed by adenine in cells of A. eutrophus H 16.     

In vitro alteration of the size of the liver mitochondrial adenine nucleotide pool: Correlation with respiratory functions

Mitochondrial respiration was studied as a function of the total adenine nucleotide content of rat liver mitochondria. The adenine nucleotide content was varied by treating isolated mitochondria with pyrophosphate or by incubating pyrophosphate-treated mitochondria with ATP. Mitochondria with at least 4 nmol adenine nucleotides/mg protein maintained at least 80% of the State 3 activity of control mitochondria, which had approximately 10 nmol/mg protein. However, State 3 decreased rapidly once the adenine nucleotide content fell below 4 nmol/mg protein. Between 2 and 4 nmol adenine nucleotides/mg, State 3 was not limited by the maximal capacity of electron flow as measured by the uncoupled respiration. However, at very low adenine nucleotide levels (<2 nmol/mg), the uncoupled rates of respiration were markedly depressed. State 4 was not affected by changes in the mitochondrial adenine nucleotide content. Adenine translocase activity varied in almost direct correlation with changes in the adenine nucleotide content. Therefore, adenine translocase activity was more sensitive than State 3 to changes in total adenine nucleotides over the range of 4 to 10 nmol/mg protein. The results suggest that (i) State 3 is dependent on the level of intramitochondrial adenine nucleotides, particularly in the range below 4 nmol/mg protein, (ii) adenine translocase activity is not rate-limiting for oxidative phosphorylation in mitochondria with the normal complement of adenine nucleotides, however, at low adenine nucleotide levels, depressed State 3 rates may be explained in part by the low rate of ADP translocation, and (iii) a mechanism of net ATP uptake exists in mitochondria with low internal adenine nucleotides.     

Ligand-incorporation site in 5-methylcytosine-detection probe modulating the site of osmium complexation with the target DNA

ICON Probes, short DNA strands containing an adenine linked to a bipyridine ligand, formed an interstrand cross-link with 5-methylcytosine located opposite the modified adenine in the presence of an osmium oxidant. The location of a bipyridine-tethered adenine in the probes varied the selectivity of the reactive base. An ICON probe where the modified adenine was located at the probe center showed a 5-methylcytosine-selective osmium complexation, whereas an ICON probe with the modified adenine at the strand end exhibited high reactivity towards thymine as well as 5-methylcytosine. The modulation of reactive bases by the incorporation of a bipyridine-tethered adenine site made facilitates design of ICON probes for the fluorometric detection of 5-methylcytosine.     

Approaches to the measurement of intracellular adenine and the discrimination between adenine transport and metabolism in L1210 leukemia cells

Incubation of L1210 leukemia cells with 10 μM [³H]adenine in the absence of energy substrate results in a very rapid accumulation of ³H within the cells. By 20 s intracellular adenine is near steady-state; beyond this the rate of accumulation of intracellular ³H reflects nucleotide synthesis, predominantly the rate of ATP accumulation within the cell as determined by liquid chromatography. Adenine incorporation into the nucleotides proceeds via adenine-phosphoribosyl transferase, which is rate-limiting to AMP formation and subsequently the formation of ADP and ATP. Acceleration of this pathway by the addition of glucose and phosphate decreases the intracellular adenine level far below equilibrium as metabolism is increased relative to transport. Assessment of methodology to evaluate intracellular adenine and its metabolites indicates that (i) a 4°C wash removes the major portion of intracellular adenine and (ii) at 4°C, transport of adenine remains rapid and while nucleotide synthesis is decreased, ATP still accumulates within the cell. Hence, measurement of cellular uptake of radioactive label at 4°C after cells are washed free of adenine cannot be used as a measurement of adenine surface binding since this radioactive label represents, at least in part, phosphorylated derivatives of adenine within the cell. Unlabeled adenine and structurally related compounds were found to inhibit [³H]adenine net uptake under conditions where metabolism of adenine was reduced, suggesting that base transport is mediated by a facilitated diffusion mechanism. This is consistent with other studies from this laboratory that demonstrate exchange diffusion between adenine and other bases.     

Effect of adenine metabolites on survival of Drosophila melanogaster of low xanthine dehydrogenase activity.

1. Low xanthine dehydrogenase (LXD) mutant Drosophila melanogaster were fed 0.2% adenine for 7 generations, no adenine for the next 2 generations (relaxed) and 0.2% adenine again for the next 3 generations (rechallenged) to obtain adenine-resistant lines of Drosophila (LXD-adenine). Flies grown without adenine served as LXD-controls. 2. Purines ranked as follows; adenine > adenosine > AMP > inosine > IMP in decreasing order of toxicity to LXD-adenine flies. 3. Addition of ribose to 9N position, or phosphate or carboxy to 6C position of the purine ring alleviated the toxicity. 4. More LXD-adenine offspring survived than did LXD-control offspring rechallenged with adenine.     

Metabolism of exogenous adenine by Acer Pseudoplatanus cells

The cells of Acer pseudoplatanus convert erogenous adenine to various metabolites. The balance between synthesis and degradation of adenine nucleotides has been studied for different adenine concentrations and different periods of incubation. The enzymic pathway mediating the synthesis of adenylic nucleotides from erogenous adenine, and those accounting for the degradation of adenine are discussed, and the deamination of AMP as a possible regulatory mechanism governing the size of the pool of adenylic nucleotides is considered.     

Electrostatic potential of nucleotide-free protein is sufficient for discrimination between adenine and guanine-specific binding sites

Despite sharing many common features, adenine-binding and guanine-binding sites in proteins often show a clear preference for the cognate over the non-cognate ligand. We have analyzed electrostatic potential (ESP) patterns at adenine and guanine-binding sites of a large number of non-redundant proteins where each binding site was first annotated as adenine/guanine-specific or non-specific from a survey of primary literature. We show that more than 90% of ESP variance at the binding sites is accounted for by only two principal component ESP vectors, each aligned to molecular dipoles of adenine and guanine. Projected on these principal component vectors, the adenine/guanine-specific and non-specific binding sites, including adenine-containing dinucleotides, show non-overlapping distributions. Adenine or guanine specificities of the binding sites also show high correlation with the corresponding electrostatic replacement (cognate by non-cognate ligand) energies. High correlation coefficients (0.94 for 35 adenine-binding sites and 1.0 for 20 guanine-binding sites) were obtained when adenine/guanine specificities were predicted using the replacement energies. Our results demonstrate that ligand-free protein ESP is an excellent indicator for discrimination between adenine and guanine-specific binding sites and that ESP of ligand-free protein can be used as a tool to annotate known and putative purine-binding sites in proteins as adenine or guanine-specific.     

Metabolism of adenine nucleotides in thymocyte cytoplasm and subcellular fractions after treatment with concanavalin A, papaverine and adenosine

Studies with rat thymocytes labeled with [14C]adenine and fractionated by digitonin treatment revealed that the cytoplasm of these cells contains about 60% of the total adenine nucleotide pool with a higher ATP/ADP ratio and metabolic activity as compared with the structural components. The incorporation of [14C]adenine and [14C]adenosine into thymocyte adenine nucleotides results in predominant labeling of cytoplasmic ATP, in which the specific radioactivity of this nucleoside triphosphate is two and three times as high as in subcellular structures. Concanavalin A decreases the ATP level in thymocytes without changing its specific radioactivity. This compound does not influence the total content and amount labeled adenine nucleotides in the structural fraction. Papaverine accelerates the catabolism of ATP, mainly in thymocyte cytoplasm and, in a lesser degree, in its structural fraction. In each fraction the papaverine-induced catabolism of ATP is localized in the compartment which is more intensively labeled with [14C]adenine than the whole fractionation ATP pool. Adenosine markedly accelerates adenine nucleotide catabolism in the cytoplasmic and structural fractions of thymocytes; however, only in the first one of them this acceleration is due to ATP elevation. Papaverine and adenosine do not directly influence either the content or specific radioactivity of adenine nucleotides of the structural fraction isolated from [14C]adenine-labeled thymocytes.     

Metabolism and salvage of adenine and hypoxanthine by myocytes isolated from mature rat heart

Adenine and hypoxanthine can be utilised by cardiac muscle cells as substrates for the synthesis of ATP. A possible therapeutic advantage of these compounds as high-energy precursors is their lack of vasoactive properties. Myocytes isolated from mature rat heart have been used to establish in kinetic detail the capacity of the heart to incorporate adenine, hypoxanthine and ribose into cellular nucleotides. Maximum rates of catalysis by enzymes on the salvage pathways have been established. Whilst the rate of incorporation of adenine into the ATP pool appears to depend upon intracellular concentrations of adenine and phosphoribosylpyrophosphate, for hypoxanthine the pattern is more complex. Hypoxanthine is salvaged at a slow rate compared with adenine, and is incorporated into GTP and IMP as well as into adenine nucleotides. The rate of incorporation of hypoxanthine into both IMP and ATP is accelerated in myocytes incubated with ribose. However, the rate-limiting reaction appears to be that catalysed by adenylosuccinate synthetase, for the rate of ATP synthesis is not accelerated when hypoxanthine concentration is increased from 10 to 50 microM, while the rate of IMP synthesis is more than doubled. Adenine and hypoxanthine phosphoribosyl transferases are present in equal catalytic amounts, but rat cardiac myocytes have very little adenylosuccinate synthetase activity. Exogenous ribose is incorporated into adenine nucleotides in amounts equimolar with adenine or hypoxanthine.     

An Investigation of Prebiotic Purine Synthesis from the Hydrolysis of HCN Polymers

The polymerization of concentrated NH₄CN solutions has been studied at various temperatures and ammonia concentrations. The products of the oligomerization of ammonium cyanide include adenine and guanine, as well as trace amounts of 2,6-diaminopurine. Our results indicate that the adenine yield is not strongly dependent on temperature. Guanine is produced in lower yield. The original studies by Oró and Kimball (1961) showed that the 6 N HCl hydrolysis of the NH₄CN polymerization supernatant greatly increased the adenine yield. However, this hydrolysis also decomposes adenine and other purines. Therefore, we have measured the yields from an NH₄CN polymerization as a function of hydrolysis time, and found that shorter hydrolytic periods give higher yields of adenine.We have also investigated the hydrolysis of the supernatant at pH 8, which is a more reasonable model of primitive oceanic conditions, and found that the adenine yield is comparable to that obtained with acid hydrolysis (approximately 0.1%). The yield of adenine does not decline at longer hydrolysis times because of the greater stability of adenine at pH 8. The insoluble black polymer formed from NH₄CN has been analyzed by both acid and neutral hydrolysis. In both cases adenine yields of approximately 0.05% were obtained. This suggests that the polymer may have been as important a prebiotic source of purines as the usually analyzed supernatant.     

Adenine release is fast in MutY-catalyzed hydrolysis of G:A and 8-Oxo-G:A DNA mismatches

MutY, a DNA repair enzyme, is unusual in that it binds exceedingly tightly to its products after the chemical steps of catalysis. Until now it was not known whether the product being released in the rate-limiting step was DNA, adenine, or both. MutY hydrolyzes adenine from 8-oxo-G:A (OG:A) base pair mismatches as the first step in the base excision repair pathway, as well as from G:A mismatches. The products are adenine and DNA containing an apurinic (AP) site. Tight product binding may have a physiological role in preventing further damage at the OG:AP site. We developed a rate assay using [8-14C]adenine in OG:A or G:A mismatches that distinguishes between adenine hydrolysis and adenine release. [8-14C]Adenine was released quickly from the MutY.AP-DNA.[8-14C]adenine complex, with a rate constant greater than 5 min-1. This was much faster than the rate-limiting step, at 0.006-0.015 min-1. Gel retardation experiments showed that AP-DNA release was very slow, consistent with it being the rate-limiting step. Thus, the kinetic mechanism involves fast adenine release after hydrolysis followed by rate-limiting AP-DNA release. Adenine appears to be buried deep in the protein.DNA interface, but there is enough flexibility or open space for it to dissociate from the MutY.APDNA.adenine complex. These results have implications for the catalytic mechanism of MutY.     

Establishment and characterization of B cell lines from individuals with various types of adenine phosphoribosyltransferase deficiencies

Patients with 2,8-dihydroxyadenine urolithiasis are either completely or partially deficient in adenine phosphoribosyltransferase activities. Patients with partial enzyme deficiencies, all of whom have been found among Japanese, are homozygotes having a unique mutant adenine phosphoribosyltransferase gene (APRT*J) in double dose (Japanese type deficiency). We have established B-cell lines from heterozygotes and homozygotes of complete and Japanese type adenine phosphoribosyltransferase deficiencies as well as normal individuals. Characterization of the cell lines indicated that all homozygous cells were deficient in adenine phosphoribosyltransferase function while all heterozygous and normal cells had functional adenine phosphoribosyltransferase.     

Inhibitory action of the adenine analog, 4-aminopyrazolo(3,4-d)pyrimidine, in Crithidia fasciculata

The adenine analog 4-aminopyrazolo(3,4-d)pyrimidine inhibits the growth of the kinetoplastid (trypanosomatid) flagellate Crithidia fasciculata. This inhibition is partially overcome only by adenine (of a number of purines tested), with an inhibition index of 0.025. More effective reversal of inhibition is obtained with any of a number of naturally occurring pyrimidine compounds, up to a concentration of 0.18 mM. Higher concentrations of pyrimidines or addition of guanine, as well as adenine and uracil, to the medium increases inhibition. The analog (presumably as the ribonucleotide) was found not to be inhibitory to any enzyme of the pyrimidine biosynthetic pathway that could be tested. It is suggested that the analog competes with adenine for adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7), is converted to a ribonucleotide, and is incorporated into nucleic acid.     

Kinetics and mechanism of protection of adenine from sulphate radical anion by caffeic acid under anoxic conditions

The rates of photo-oxidation of adenine in the presence of peroxydisulphate (PDS) have been determined by measuring the absorbance of adenine at 260.5 nm spectrophotometrically. The rates and the quantum yields (phi) of oxidation of adenine by sulphate radical anion (SO4(-)) have been determined in the presence of different concentrations of caffeic acid. Increase in the concentration of caffeic acid is found to decrease the rate of oxidation of adenine suggesting that caffeic acid acts as an efficient scavenger of SO4(-) and protects adenine from it; SO4(-) competes for adenine as well as for caffeic acid. From competition kinetics, the rate constant of SO4(-) with caffeic acid has been calculated to be 1.24 +/- 0.2 x 10(10) mol(-1)dm(3)s(-1). The quantum yields of photo-oxidation of adenine have been calculated from the rates of oxidation of adenine and the light intensity absorbed by PDS at 254 nm, the wavelength at which PDS is activated to SO4' -. The results of experimentally determined quantum yields (phi exptl) and the quantum yields calculated (phi cl) by assuming that caffeic acid acts only as a scavenger of SO4(-) radicals show that phi exptl values are lower than phi cl values. The phi prime values, which are experimentally found quantum yield values at each caffeic acid concentration and corrected for SO4(-) scavenging by caffeic acid, are also found to be greater than phi exptI values. These observations suggest that the adenine radicals are repaired by caffeic acid, in addition to scavenging of sulphate radical anions.     

Direct electrocatalytic oxidation of adenine and guanine on carbon ionic liquid electrode and the simultaneous determination

A carbon ionic liquid electrode (CILE) was fabricated by using an ionic liquid of N-butylpyridinium hexafluorophosphate (BPPF(6)) as binder and further used for the simultaneous detection of adenine and guanine. The direct electrooxidation behaviors of adenine and guanine were carefully investigated on the CILE. The results indicated that both adenine and guanine showed the increase of the oxidation peak currents with the negative shift of the oxidation peak potentials in contrast to that on the traditional carbon paste electrode (CPE). The electrochemical parameters of adenine and guanine on the CILE were calculated and a new electroanalytical method was established for the detection of adenine and guanine, respectively. The CILE exhibited good behaviors in the simultaneous detection of adenine and guanine with the peak separation as 0.304V. The measurements of thermally denatured single-stranded DNA (ssDNA) were further carried out and the value of (G+C)/(A+T) of ssDNA was calculated as 0.81.