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Metabolism by the rabbit of intravenously administered adenine.

A study was made of the metabolism by the rabbit of adenine administered intravenously at a dose of 35 mg/kg with 100 micronCi of 8-14C-adenine. The infused adenine was removed from the blood in two phases, first by diffusion into the tissues and second by metabolic reactions throughout the body. The adenine equilibrated within a few seconds equally between plasma and red blood cells and between them and kidney, liver, duodenum, lung and heart. Diffusion into skeletal muscle was much slower and into brain slowest. The more gradual disappearance of adenine from blood, and from the rest of the body, with a half-life of about 20 minutes and with complete removal by two hours, was predominantly along three pathways, leading to, after four hours: 1) 74 per cent in adenine nucleotide (mostly AMP, ADP, and (ATP); 2) 12 per cent as unchanged adenine in the urine; and 3) 11 per cent as a mixture in almost equal parts of 8-oxyadenine and 2,8-dioxyadenine in the urine. Conversion of adenine to adenine nucleotide, probably by initial reaction with phosphoribosyl pyrophosphate and adenine-phosphoribosyltransferase, was at widely different rates in the organs with duodenum, kidney, liver, and lung high and heart, red blood cell, skeletal muscle and brain relatively low. Sites of formation of the two oxyadenines, probably by action of xanthine oxidase, were not determined.

Adenine↗
PubMed2003

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.

Adenine↗
PubMed2004

Evidence for the functional expression and pharmacological characterization of adenine receptors in native cells and tissues.

An orphan G protein-coupled receptor from rat has recently been discovered to be activated by the nucleobase adenine (Proc Natl Acad Sci USA 99:8573-8578, 2002). In the present study, we show for the first time that the adenine receptor is expressed in membrane preparations of native tissues and cell lines in high density, including rat brain cortex, rat brain striatum, and the mouse neuroblastoma x rat glioma hybrid cell line NG108-15. Saturation analysis with [3H]adenine at rat brain cortical membranes exhibited a single high-affinity binding site with a KD value of 27.2 nM, and a binding capacity of 2.28 pmol/mg of protein. Kinetic studies revealed unusual binding kinetics of [3H]adenine with rapid association and slow dissociation. A series of compounds were investigated in [3H]adenine competition experiments at rat brain cortex. Only minor substitution of the adenine structure was tolerated, the most potent compounds of the present series being 2-fluoroadenine (Ki value of 620 nM), 8-thioadenine (Ki value of 2.77 microM), N6-methyladenine (Ki value of 3.64 microM), and 7-methyladenine (Ki value of 4.13 microM), all of which were partial agonists (40-60% intrinsic activity). Adenine dose dependently inhibited forskolin-stimulated adenylate cyclase in membrane preparations of NG108-15 cells as well as in intact cells, showing that the receptor is functional in NG108-15 cells. Reverse transcriptase-polymerase chain reaction experiments followed by sequencing indicate that the NG108-15 cells express the murine ortholog of the adenine receptor. Moreover, preliminary radioligand binding studies with [3H]adenine at membranes of human astrocytoma 1321N1 cells suggest that a human ortholog of the rat adenine receptor exists.

Adenine↗
PubMed1991

Activation of particulate guanylate cyclase by Escherichia coli heat-stable enterotoxin is regulated by adenine nucleotides.

Guanylate cyclase is regulated by adenine nucleotides in membranes of intestinal mucosal cells. Basal guanylate cyclase was activated about twofold by adenine nucleotides. Activation was specific for adenine, as compared with the pyrimidine nucleotides UTP and CTP. In addition, enzyme activation was obtained in the presence of saturating concentrations of GTP, the substrate for guanylate cyclase. The most potent adenine nucleotide was the nonhydrolyzable analog of ATP, adenosine 5'-O-(3-thiotriphosphate). Adenine nucleotide activation was specific for the particulate form of guanylate cyclase, as compared with the soluble form. Also, adenine nucleotides potentiated the activation of guanylate cyclase by the heat-stable enterotoxin produced by Escherichia coli. Indeed, enzyme activation by adenine nucleotides and toxin was greater than the sum of individual activations by these agents. Adenine nucleotides regulate guanylate cyclase by increasing the maximum velocity of the enzyme without altering its affinity for substrate or its cooperativity. In addition to stimulating guanylate cyclase, adenine nucleotides decreased the specific binding of the heat-stable enterotoxin to receptors in intestinal membranes. The coordinated regulation of the toxin-receptor interaction and guanylate cyclase activity by a process utilizing nonhydrolyzable analogs of a purine nucleotide is similar to the mechanisms involved in the hormone regulation of adenylate cyclase by guanine nucleotide-binding proteins. These data suggest that an adenine nucleotide-dependent protein may couple the toxin-receptor interaction to the regulation of particulate guanylate cyclase in intestinal membranes.

Adenine Nucleotides↗
PubMed1996

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.

Adenine↗
PubMed2006

Nicotinamide adenine dinucleotide staining immediately following radio frequency ablation of renal tumors-is a positive stain synonymous with ablative failure?

PURPOSE: Nicotinamide adenine dinucleotide diaphorase staining is arguably the standard for assessing tissue viability following radio frequency ablation, yet the accuracy of this test is questionable. Thus, it is imperative to examine the reliability of nicotinamide adenine dinucleotide to predict ablation success. To evaluate the observation that immediate nicotinamide adenine dinucleotide staining may not be clinically reliable, we compared results from immediate post-ablation biopsies of renal tumors to radiological and clinical followup. MATERIALS AND METHODS: Laparoscopic radio frequency ablation was performed in 9 patients and 10 tumors using a temperature modulated radio frequency system. Cold cup biopsies were taken immediately following ablation, and processed for nicotinamide adenine dinucleotide and hematoxylin and eosin staining. Patients were then followed using contrast enhanced computerized tomography at regular intervals. RESULTS: Median tumor size was 2.3 cm. Hematoxylin and eosin stain diagnosed 8 renal cell carcinomas and 2 angiomyolipomas. A quarter of the renal cell carcinomas and both angiomyolipomas stained positive for scattered nicotinamide adenine dinucleotide diaphorase activity immediately after RFA. Mean followup for the nicotinamide adenine dinucleotide positive tumors was 28.5 months (range 24 to 30) and for the nicotinamide adenine dinucleotide negative tumors was 25 months (range 18 to 30). There was no evidence of local tumor recurrence in any patient. CONCLUSIONS: Four lesions had nicotinamide adenine dinucleotide diaphorase activity on post-ablation biopsy suggesting retained viable tissue, yet there has been no recurrence during an average 2-year followup. While negative nicotinamide adenine dinucleotide staining is consistent with nonviability, these results suggest that false-positive staining can occur immediately following RFA, making the predictive value of positive nicotinamide adenine dinucleotide diaphorase staining unclear.

Adult↗
PubMed1998

Effect of the extramitochondrial adenine nucleotide pool size on oxidative phosphorylation in isolated rat liver mitochondria.

The effect of the concentration of extramitochondrial adenine nucleotides on oxidative phosphorylation was studied in isolated rat liver mitochondria. Mitochondria were incubated with succinate and hexokinase or creatine kinase at constant or varying extramitochondrial adenine nucleotide concentrations ranging over 0.3-5 mM. As parameters of oxidative phosphorylation, rate of respiration, membrane potential as well as intra- and extra-mitochondrial adenine nucleotide concentrations were determined. Below a threshold concentration of extramitochondrial adenine nucleotides of 2 mM, the free Gibb's energy for the adenine nucleotide transport increased but the extramitochondrial ATP/ADP ratio decreased at intermediate rates of respiration with decreasing extramitochondrial adenine nucleotide concentrations. In this range the rate of respiration was dependent on the extramitochondrial ADP concentration. No effect of the extramitochondrial adenine nucleotide concentration on the relationships between the rate of respiration and the membrane potential, the intramitochondrial adenine nucleotide pool and the intramitochondrial ATP/ADP ratio was found. This suggests that the decline of extramitochondrial ATP due to adenine nucleotide degradation and the limitation of adenine nucleotide transport may be of importance in the postischemic phase as nucleotide resynthesis and reorganization of physiological ion distribution are ATP consuming processes.

Adenosine Diphosphate↗
PubMed1985

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.

Adenine↗
PubMed1982

Mechanism of adenine toxicity in Escherichia coli.

The mechanism of adenine toxicity in an hpt gpt strain of Escherichia coli that is extremely sensitive to adenine inhibition was investigated. Adenine-resistant derivatives had secondary mutations in adeninephosphoribosyltransferase or the purR repressor. Growth studies with various purine salvage pathway mutants and the ability of guanosine to prevent adenine toxicity indicated that adenine exerts its toxic effects by depleting guanine nucleotide pools. In the presence of adenine, ATP pools increased twofold in wild-type cells and stabilized after 5 min. In contrast, ATP pools continued to rise in hpt gpt cells up to 25 min and increased sevenfold after adenine addition. hpt gpt cells were shown to have higher levels of adeninephosphoribosyltransferase than did the wild-type cells. In response to adenine addition, GTP pools dropped three- to fourfold in all strains tested. Although GTP levels returned to near normal values in wild-type cells after 35 min, no restoration of GTP pools was observed in the hpt gpt strain during this period. Measurements of guanine pools before and after the addition of adenine indicated that guaninephosphoribosyltransferase plays an important role in maintaining GTP pools by converting the free guanine to GMP during guanine nucleotide depletion.

Adenine↗
PubMed1975

Relation of growth and protein synthesis to the adenylate energy charge in an adenine-requiring mutant of Escherichia coli.

When Escherichia coli K-12 strain PC 0294, which is unable to synthesize adenine nucleotides, thereonine, proline, and leucine, was starved for adenine, the concentrations of ATP, ADP, and AMP fell rapidly. The adenylate energy charge was not affected until the adenine nucleotide pool fell to about 30% of its normal value. Similarly, cells in an adenine-limited chemostat grew at intracellular adenine nucleotide pool values as low as 30% of normal, but the energy charge in all cases was approximately 0.90. Incorporation of [14C]leucine into protein in adenine-starved cells continued rapidly as long as the energy charge was in or near its normal range, even when the concentration of ATP was 30% to 10% of its normal value. When glucose was added to cells that had been resuspended in medium lacking both glucose and adenine, the energy charge rose rapidly but the concentration of ATP fell, presumably because of nucleic acid synthesis. The rate of [14C]leucine incorporation into protein rose rapidly while adenine nucleotide concentrations fell, and then declined roughly in parallel with the energy charge. At a given value of energy and of total concentration of adenine nucleotides, the rate of protein synthesis may, of course, vary with the concentrations of precursors and modifiers; thus, the rate cannot be predicted from knowledge only of the energy charge. Our results suggest, however, that the rate of protein synthesis and the capacity for growth are much more sensitive to changes in the value of the energy charge than to changes in the concentration of ATP. Growth occurs when the ATP concentration is reduced to one-third of its normal value, but has not been observed when the energy charge has fallen by as much as 10%.

Adenine↗
PubMed1993

Different relationships between cellular adenosine or 3'-deoxyadenosine phosphorylation and cellular adenine ribonucleotide catabolism may be obtained.

Treatment of BALB/c-3T3 mouse fibroblasts with 3'-deoxyadenosine led to a rapid accumulation of 3'-deoxyadenosine phosphates and the kinetics of this process has been determined. Concomitant with accumulation of these compounds, the adenine ribonucleotide pool was reduced. The kinetics of the two processes suggested that they were tightly coupled. The inhibitory effect of relatively high concentrations of coformycin indicated that IMP was an intermediate in the catabolic pathway. Similar experiments with Ehrlich ascites tumor cells were performed in Ringer-Hepes solution at pH 6.5 or 7.5 and with varying concentrations of orthophosphate. The experiments were performed with cells where ATP was [3H]-labeled. This allowed the determination of the catabolism of adenine ribonucleotides to labeled nucleosides under conditions where added adenosine was phosphorylated. The results showed that at low phosphate concentration (5.8 mM) at pH 6.5 adenosine may be phosphorylated at a rate that was completely balanced to the concomitant catabolism of adenine ribonucleotides; that is, there was apparently a tight kinetic coupling between anabolism of adenosine and catabolism of adenine ribonucleotides. With 3'-deoxyadenosine a corresponding effect was obtained although the apparent coupling between phosphorylation of 3'-deoxyadenosine and catabolism of adenine ribonucleotides was not complete. When experiments were performed at the same pH but at high concentration of phosphate (45 mM) there was in contrast no coupling between the two processes; that is, ATP was present in constant amounts while 3'-deoxyadenosine phosphates accumulated at a high rate. In experiments with adenosine under these conditions there was still some although a relatively limited degree of apparent coupling between phosphorylation of adenosine and catabolism of adenine ribonucleotides. In both lines of cells used and with both adenosine and 3'-deoxyadenosine, the main products of the catabolism of adenine ribonucleotides were inosine and hypoxanthine. With 3'-deoxyadenosine there was in addition (about 20%) formation of xanthosine, suggesting that IMP dehydrogenase had also been activated. These results lead to the suggestion that adenosine (or 3'-deoxyadenosine) may be phosphorylated in two ways. 1) Phosphorylation may depend on an adenosine kinase unrelated to catabolism of adenine ribonucleotides. 2) Phosphorylation may be tightly coupled to catabolism of adenine ribonucleotides. A nucleoside phosphotransferase may catalyze the transfer of a phosphoryl group from IMP to adenosine (or 3'-deoxyadenosine) to form AMP (or 3'-dAMP) and inosine, a process that may be tightly coupled to an AMP deaminase reaction. The IMP formed in the latter reaction may not be released but transferred to the phosphotransferase.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗
PubMed2003

Neuroprotective effect of adenine on purkinje cell survival in rat cerebellar primary cultures.

Although adenosine or ATP is known to control various physiological functions in the brain, including synaptic transmission, neuronal cell death, and neurite outgrowth via P1 or P2 purinergic receptors in the nervous system, little is known about the functions of many other purine derivatives. We examined the effects of various purines on survival in the cerebellar cortex of Purkinje cells with large cell bodies and highly branched dendrites, and it was found that some purine and pyrimidine derivatives influence Purkinje cell survival. Treatment with adenine, guanine, guanosine, guanine nucleotides, and uracil nucleotides protected Purkinje cells from cell death in the cerebellar primary cultures. Among the effective compounds, adenine had the most potent survival activities on Purkinje cells. Other adenine-based purines such as adenosine, AMP, ADP, and ATP did not promote Purkinje cell survival. Furthermore, metabolic inhibitors of adenine had no effect on the protective ability of adenine for Purkinje cells, suggesting that adenine itself, not adenine metabolites, maintains Purkinje cell survival. These results suggest that adenine is involved in the control of Purkinje cell survival in cerebellar primary cultures via a novel adenine-dependent mechanism.

Adenine↗
PubMed1993

A DNA dodecamer containing an adenine tract crystallizes in a unique lattice and exhibits a new bend.

The structure of d(CGCGAAAAAACG)/d(CGTTTTTTCGCG) was determined at 2.3 A resolution in order to deduce the local structural features that give rise to DNA bending by adenine tracts. Whereas all previously reported B-DNA dodecamers have crystallized isomorphously (spacegroup P2(1)2(1)2(1) with unit cell dimensions of a = 24.5 A, b = 40.3 A, c = 65.9 A), the duplex reported here crystallizes in a different lattice (spacegroup P2(1)2(1)2 with unit cell dimensions of a = 44.8 A, b = 66.1 A, c = 42.9 A). The DNA exhibits a 30 degree bend in the helix axis that is 180 degrees away from the 20 degree bend exhibited by the adenine tract DNA crystal structures that have been previously determined. This bend is 90 degrees away from the bend predicted for an adenine tract by solution and gel experiments. The adenine tract is straight and bending occurs in the G+C-rich regions. Comparison of the various adenine tract DNA crystal structures reveals that in all cases the adenine tracts have nearly identical structures, even though the overall bends of the helix axes are quite different. This implies that the structure of the adenine tract is robust, at least under the conditions of crystallization. The base-pairs in the adenine tracts exhibit a large propeller twist that leads to the formation of bifurcated hydrogen bonds and a narrow minor groove. In the crystal structure of d(CGCGAAAAAACG)/d(CGTTTTTTCGCG), a minor groove spine of hydration is observed that probably stabilizes the straight structure of the adenine tract. This straight structure of the A-tract is not consistent with the results of fiber diffraction, gel experiments, and NMR studies. Although this may imply that the results of solution experiments need to be reinterpreted, the conditions under which the crystals were grown are different from those under which the solution experiments were done. The possibility remains that the 2-methyl-2,4-pentanediol necessary for crystal growth may facilitate formation of the spine of hydration that stabilizes the straight A-tract, although recent NMR results show the presence of the spine of hydration in aqueous solution. We have also extended our previously reported observation that non-self-complementary DNA structures can exist in the crystal lattice in two orientations. A dodecamer brominated on one strand provides experimental evidence that d(CGCAAAAAAGCG)/d(CGCTTTTTTG CG) is positioned in two orientations in the crystal lattice that are related by a 180 degree rotation around the pseudo-dyad axis of the sequence.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine↗
PubMed1987

Phosphate-induced efflux of adenine nucleotides from rat-heart mitochondria: evaluation of the roles of the phosphate/hydroxyl exchanger and the dicarboxylate carrier.

Upon the addition of inorganic phosphate, isolated rat-heart mitochondria released endogenous adenine nucleotides. To elucidate the mechanism of this phosphate-induced efflux, we evaluated the relative roles of three inner mitochondrial membrane carriers: the adenine nucleotide translocase, the phosphate/hydroxyl exchanger, and the dicarboxylate carrier. Atractyloside (a specific inhibitor of the adenine nucleotide translocase) prevented this efflux, but did not inhibit mitochondrial swelling. Inhibitors of the phosphate/hydroxyl exchanger (200 microM n-ethylmaleimide and 10 microM mersalyl) did not inhibit phosphate-induced efflux. 200 microM mersalyl (which inhibited both the phosphate/hydroxyl exchanger and the dicarboxylate carrier) inhibited the rate of efflux approx. 65% Phenylsuccinate and 2-n-butylmalonate (inhibitors of the dicarboxylate carrier) partially inhibited phosphate-induced efflux and adenine nucleotide translocase activity. Mersalyl (200 microM) had no effect on adenine nucleotide translocase activity. Partial inhibition of the adenine nucleotide translocase by phenylsuccinate and butylmalonate could not explain the extent of inhibition of phosphate-efflux by these agents. Moreover, the rates of adenine nucleotide efflux in the presence of phenylsuccinate, butylmalonate, or mersalyl correlated well with the ability of these agents to inhibit succinate-supported respiration. We conclude that phosphate-induced efflux of adenine nucleotides from rat heart mitochondria occurs over the adenine nucleotide translocase, and that the site of action of the phosphate is not the phosphate/hydroxyl exchanger, but is likely the dicarboxylate carrier.

Adenine Nucleotides↗
PubMed2000

Adenine protonation in domain B of the hairpin ribozyme.

Protein enzymes often use ionizable side chains, such as histidine, for general acid-base catalysis because the imidazole pK(a) is near neutral pH. RNA enzymes, on the other hand, are comprised of nucleotides which do not have apparent pK(a) values near neutral pH. Nevertheless, it has been recently shown that cytidine and adenine protonation can play an important role in both nucleic acid structure and catalysis. We have employed heteronuclear NMR methods to determine the pK(a) values and time scales of chemical exchanges associated with adenine protonation within the catalytically essential B domain of the hairpin ribozyme. The large, adenine-rich internal loop of the B domain allows us to determine adenine pK(a) values for a variety of non-Watson-Crick base pairs. We find that adenines within the internal loop have pK(a) values ranging from 4.8 to 5.8, significantly higher than the free mononucleotide pK(a) of 3. 5. Adenine protonation results in potential charge stabilization, hydrogen bond formation, and stacking interactions that are expected to stabilize the internal loop structure at low pH. Fast proton exchange times of 10-50 micros were determined for the well-resolved adenines. These results suggest that shifted pK(a) values may be a common feature of adenines in non-Watson-Crick base pairs, and identify two adenines which may participate in hairpin ribozyme active site chemistry.

Adenine↗
PubMed2005

Duplex-promoted platination of adenine-N3 in the minor groove of DNA: challenging a longstanding bioinorganic paradigm.

The interactions of [Pt(en)Cl(ACRAMTU-S)](NO3)2 (PT-ACRAMTU, en = ethane-1,2-diamine, ACRAMTU = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) with adenine in DNA have been studied using a combination of analytical and high-resolution structural methods. For the first time, a cytotoxic platinum(II) complex has been demonstrated to form adducts in the minor groove of DNA through platination of the adenine-N3 endocyclic nitrogen. An acidic depurination assay was developed that allowed the controlled and selective (pH 2, 60 degrees C, 12 h) release of platinum-modified adenine from drug-treated nucleic acid samples. From the digested mixtures, three adducts were isolated by semipreparative reverse phase high-performance liquid chromatography and studied by electrospray ionization mass spectrometry (in-line LC-MS), variable-pH 1H NMR spectroscopy, and, where applicable, X-ray crystallography. The three species were identified as the N7 (A-I), N3 (A-II), and N1 (A-III) linkage isomers of [Pt(en)(ACRAMTU-S)(adenine)]3+ (A). Incubations carried out with the single- and double-stranded model sequences, d(TA)5 and d(TA)15, as well as native DNA indicate that the adduct profiles (A-I:A-II:A-IIIratios) are sensitive to the nature of the nucleic acid template. A-II was found to be a double-strand specific adduct. The crystal structure of this adduct has been determined, providing ultimate evidence for the N3 connectivity of platinum. A-II crystallizes in the triclinic space group P in the form of centrosymmetric dimers, {[Pt(en)(ACRAMTU-S)(adenine-N3)]2}6+. The cations are stabilized by a combination of adenine-adenine base pairing (N6...N1 2.945(5) A) and mutual acridine-adenine base stacking. Tandem mass spectra and 1H chemical shift anomalies indicate that this type of self-association is not merely a crystal packing effect but persists in solution. The monofunctional platination of adenine at its N7, N3, and N1 positions in a significant fraction of adducts breaks a longstanding paradigm in platinum-DNA chemistry, the requirement for nucleophilic attack of guanine-N7 as the principal step in cross-link formation. The biological consequences and potential therapeutic applications of the unique base and groove recognition of PT-ACRAMTU are discussed.

Adenine↗
PubMed2004

Adenine-containing DNA-DNA cross-links of antitumor nitrogen mustards.

Nitrogen mustards (NMs) are useful chemotherapeutic agents in the treatment of lymphoma, leukemia, multiple myeloma, and ovarian carcinoma. The antitumor activity of NMs has been attributed to their ability to cross-link the twin strands of DNA. The resulting bifunctional lesions, if not repaired, can inhibit DNA replication and transcription, eventually leading to cell cycle arrest, apoptosis, and the inhibition of tumor growth. The predominant bifunctional DNA lesions of NM have been reported to involve the distal guanine bases in the opposite strands of 5'-GNC sequences. In the present work, the formation of guanine-adenine and adenine-adenine adducts of N,N-bis(2-chloroethyl)methylamine (mechlorethamine) in double-stranded DNA is demonstrated. Guanine-adenine cross-links of mechlorethamine were identified as N-(2-[N3-adenyl]ethyl)-N-(2-[N7-guanyl]ethyl)methylamine (N3A-N7G-EMA), N-(2-[N1-adenyl]ethyl)-N-(2-[N7-guanyl]ethyl)methylamine, and N-(2-[N(6)-adenyl]ethyl)-N-(2-[N7-guanyl]ethyl)methylamine. All three adducts were produced interstrand, while N3A-N7G-EMA was the dominant intrastrand G-A cross-link. The prevalent adenine-adenine mechlorethamine lesions have the structure of N,N-bis(2-[N3-adenyl]ethyl)methylamine (bis-N3A-EMA). DNA-derived lesions have the same HPLC retention times, UV spectra, and MS/MS fragmentation patterns as the authentic standards prepared independently. bis-N3A-EMA lesions were produced in a concentration-dependent manner in calf thymus DNA treated with increasing amounts of mechlorethamine. Furthermore, HPLC-ESI-MS/MS analysis was used to demonstrate the formation of analogous N3-N3 adenine lesions in DNA treated with aromatic nitrogen mustards, N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid and L-phenylalanine mustard. The presence of cross-linked adenine-adenine lesions may explain the enhanced cytotoxicity and mutagenicity of NMs in cells deficient in N3-alkyladenine glycosylase.

Adenine↗
PubMed1981

Adenine, the precursor of nucleic acids in intestinal cells unable to synthesize purines de novo.

The capacity of rat intestinal epithelial cells for de novo purine synthesis and reutilization was studied in vitro with isolated cells and in vivo in functionally hepatectomized rats. De novo purine synthesis and purine reutilization were measured as the rate of incorporation of [14C]-glycine or [14C]-adenine, respectively, into the adenine and guanine pools of the intestinal cells. Isolated intestinal epithelial cells incubated with labeled purine or glycine incorporated only labeled adenine. Labeled glycine, guanine, hypoxanthine or xanthine were not incorporated into cellular adenine or guanine. Labeled glycine, given intravenously to functionally hepatectomized rats, was not incorporated into the adenine and guanine of intestinal villus or crypt cells. Labeled glycine was readily incorporated into hepatic adenine and guanine pools of a sham-operated rat. The failure of glycine to be incorporated into intestinal cells in vitro or in vivo demonstrated that these cells lack de novo purine synthesis. Since adenine was readily incorporated into the adenine pool of these cells, we believe that adenine, which is synthesized in the liver or supplied in the diet, was an important precursor for the nucleic acid synthesis in this study.

Adenine↗