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PubMed1978

Stimulation of myocardial adenine nucleotide biosynthesis by pentoses and pentitols.

In rats, pentoses and pentitols, intravenously injected in a single dose of 100 mg/kg, induced a considerable enhancement of the available pool of 5-phosphoribosyl-1-pyrophosphate and of the rate of adenine nucleotide biosynthesis in the heart, but not in liver and kidney. De novo synthesis of adenine nucleotides not detectable in skeletal muscle of normal rats became measurable after application of ribose. The stimulatory effect of isoproterenol on myocardial adenine nucleotide biosynthesis could be further potentiated by ribose and xylitol, but not by glucose. The isoproterenol-induced decrease of cardiac adenine nucleotide concentrations could be almost completely prevented by repeated administrations of ribose. Thus, pentoses and pentitols in combination with beta-receptor stimulation markedly and quite specifically enhance adenine nucleotide biosynthesis in the rat heart. The results indicate that the increase in the available pool of 5-phosphoribosyl-1-pyrophosphate is an important factor for the enhancement of cardiac adenine nucleotide biosynthesis. Moreover, the availability of 5-phosphoribosyl-1-pyrophosphate and the rate of de novo synthesis of adenine nucleotides in the heart seem to be limited by the flow through the hexose monophosphate shunt.

Adenine Nucleotides↗
PubMed1978

Role of adenine in pathogenesis of Mycoplasma pneumoniae infections of tracheal epithelium.

Adenine sulfate and several related compounds were evaluated for their ability to retard the cytotoxic effect which normally accompanies M. pneumoniae infections of hamster tracheal explants. Adenine sulfate, at the 0.01 mM level, was found to exert a significant protective effect. Little or no ciliostasis or loss of cell viability was detectable when organ cultures were infected with 10(7) CFU of virulent M. pneumoniae in the presence of the adenine supplement. Mycoplasmas grew in broth and on plastic surfaces in the presence of adenine, and no significant diminution of growth rate or cell yield was detectable. Organisms adhered to the tracheal epithelial surface regardless of the presence or absence of adenine. When explants were incubated in the presence of 14C-(8)-adenine, rinsed, and then infected with M. pneumoniae, the adenine label could be recovered from the mycoplasmas 20 h after the infection. These data are compatible with the known nucleic acid requirement of mycoplasmas and with a model which ascribes a role for purine/pyrimidine competition and/or depletion in the infective process.

Adenine↗
PubMed1980

Radioactive labeling of the adenine nucleotide pool of cells as a method to distinguish among intracellular compartments. Studies on human platelets.

The study of adenine nucleotide metabolism is complicated by cellular nucleotide compartmentalization. In platelets, we have been able to use radioactive labeling with adenine to measure, precisely and accurately, changes in the cytoplasmic/mitochondrial pool of adenine nucleotides inspite of the fact that 60% of adenine nucleotides are present in amine-storing granules. High performance liquid chromatography was used to measure the concentrations of ATP, ADP, AMP and IMP from [14C]adenine-labeled platelets under conditions where the granule pool was absent. The radioactive measurements were directly proportional to the chemical measurements. Ethanol-insoluble, actin-bound ADP also had the same specific radioactivity as other metabolic adenine nucleotides. Since this pool can be directly separated from the bulk of cellular nucleotides, its specific radioactivity can be easily measured and used to calculate the concentration of each cytoplasmic adenine nucleotide from its measured radioactivity. These methods may be applicable to other cells.

Adenine Nucleotides↗
PubMed1991

Catabolism of adenine nucleotides in suspension-cultured plant cells.

Profiles of the catabolism of adenine nucleotides in cultured plant cells were investigated. Adenine nucleotides, prelabelled by incubation of suspension-cultured Catharantus roseus cells with [8-14C]adenosine, were catabolized rapidly and most of the radioactivity appeared in 14CO2. Allantoin and allantoic acid, intermediates of the oxidative catabolic pathway of purines, were temporarily labelled. When the cells, prelabelled with [8-14C]adenosine, were incubated with high concentrations of adenosine, the rate of catabolism of adenine nucleotides increased. The results suggest that the relative rate of catabolism of adenine nucleotides is strongly dependent on the concentration of adenine nucleotides in the cells. Studies using allopurinol, coformycin and tiazofurin, inhibitors of enzymes involved in purine metabolism, suggest that participation of AMP deaminase and xanthine oxidoreductase in the catabolism of adenine nucleotides in plant cells. AMP deaminase was found in extracts from C. roseus cells and its activity increased significantly in the presence of ATP. In contrast, no adenosine deaminase or adenine deaminase activity was detected. Qualitative differences in the catabolic activity of AMP were observed between suspension-cultured cells from different species of plants.

AMP Deaminase↗
PubMed1996

Adenine nucleotide metabolism in primary rat neuronal cultures.

The metabolism of adenine nucleotides (AdRN) has been studied previously in whole brains, brain slices and brain extracts, containing mixed populations of neurons and glia. The availability of primary neuronal cultures enables us to study these pathways in almost pure neuronal preparations. The aim of the present study was to characterize the relative importance of the pathways of AdRN metabolism in the neurons. The metabolic fate of (8-14C) adenine and of AdRN prelabeled with (8-14C)adenine were studied in immature and mature primary rat neuronal cultures. Specific inhibitors were used to clarify the various metabolic fluxes, which were evaluated based on the time-related changes in the distribution of label (the cellular nucleotide content did not change during incubation). The turnover rate of AdRN was found to reflect mainly conversion of label to acid insoluble derivatives (AID) and partly degradation to hypoxanthine. The turnover was faster in the immature neurons. The combined addition of 2'-deoxycoformycin (2'-dCF) and of 5'-amino-5'-deoxyadenosine, inhibiting adenosine metabolism, resulted in both cultures in enhanced loss of label from AdRN, mainly to adenosine and adenine. This finding indicates the activity of the futile cycle AMP-->adenosine-->AMP. In both cultures, in the presence of these inhibitors, the ratio (hypoxanthine + inosine)/(adenine + adenosine) was 1.1, indicating that the fluxes through AMP deamination and AMP dephosphorylation are about equal. Addition of L-alanosine, inhibiting the conversion of IMP to AMP, resulted in both cultures, but especially in the mature neurons, in enhanced loss of label from AdRN to hypoxanthine and inosine. This finding indicates the functioning of the adenine nucleotide cycle (AMP-->IMP-->adenylosuccinic acid-->AMP). Under conditions of enhanced degradation of ATP (induced by iodoacetate and antimycin A), addition of 2'-dCF resulted in the immature cultures in lowering the ratio (hypoxanthine + inosine + IMP)/(adenine + adenosine) to 0.62, indicating a shift in favor of AMP dephosphorylation.

Adenine Nucleotides↗
PubMed2003

Nucleobase adenine as a trophic factor acting on Purkinje cells.

We examined the influence of nucleobases, nucleosides, nucleotides, and their analogs on rat cerebellar Purkinje cells in primary culture and found that the number of cultured Purkinje cells was greatly increased by the nucleobase adenine. Purkinje cells were cultured for 13 days in vitro in the presence of various reagents, and the resulting cell numbers were counted. As a result, the nucleobase adenine was most effective at increasing the number of Purkinje cells among the reagents tested. In the cultures supplemented with adenine in millimolar concentrations (1-2 mM), the number of Purkinje cells was increased by up to 30 times the number of Purkinje cells in the control. Adenine had no affect on the number of granule cells, and it reduced the number of astrocytes, both of which were cocultured in cerebellar primary cultures. Stimulation of purinoceptors by adenosine and adenosine 5'-triphosphate (ATP) did not result in an increase in the number of Purkinje cells. Furthermore, the adenine effect on Purkinje cells was not related to PKA, as determined with the use of a PKA inhibitor. Our findings suggest that adenine exerts neurotrophic effects that have not been described to date; in particular, the present study demonstrated that adenine increases the number of Purkinje cells by an unknown mechanism.

Adenine↗
PubMed2004

Complexing properties of nucleic-acid constituents adenine and guanine complexes.

Cobalt, nickel and copper complexes of adenine and guanine, as nucleic-acid constituents, were prepared. The adenine and guanine complexes are of tetrahedral and octahedral geometries, respectively. All are of high spin nature. The nickel complexes are of 2:1 metal:ligand ratio with Ni...Ni direct interaction in the guanine complex. The coordination bonds of adenine metal complexes are calculated and follow the order: Cu(II)-adenine < Ni(II)-adenine < Co(I)-adenine. The Cu(II)-adenine complex is the stronger following the softness of the copper, while that of guanine is less covalent. The copper complexes are with stronger axial field. The differential thermal analysis (DTA) and TGA of the complexes pointed to their stability. The mechanism of the thermal decomposition is detected. The thermodynamic parameters of the dissociation steps are evaluated. The complexes are of semi-conducting behaviour for their technical applications. Empirical equations are deduced between the electrical conducting and the energy of activation of the complexes.

Adenine↗
PubMed1988

Chemical probing of adenine residues within the secondary structure of rabbit 18S ribosomal RNA.

The location of unpaired adenine residues within the secondary structure of rabbit 18S ribosomal RNA was determined by chemical probing. Naked 18S rRNA was first prepared by digestion of purified 40S subunits with matrix-bound proteinase K in sodium dodecyl sulfate, thereby omitting the use of nucleic acid denaturants. Adenines within naked 18S rRNA were chemically probed by using either diethyl pyrocarbonate or dimethyl sulfate, which specifically react with unpaired nucleotides [Peattie, D. A., & Gilbert, W. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 4679-4682]. Adenine modification sites were identified by polyacrylamide sequencing gel electrophoresis either upon aniline-induced strand scission of 32P-end-labeled intact and fragmented rRNA or by primer extension using sequence-specific DNA oligomers with reverse transcriptase. The data indicate good agreement between the general pattern of adenine reactivity and the location of unpaired regions in 18S rRNA determined by comparative sequence analysis [Chan, Y.-L., Gutell, R., Noller, H. F., & Wool, I. G. (1984) J. Biol. Chem. 259, 224-230]. The overall reactivity of adenine residues toward single-strand-specific chemical probes was, also, similar for both rabbit and Escherichia coli small rRNA. The number of strongly reactive adenines appearing within phylogenetically determined helical segments, however, was greater in rabbit 18S rRNA than for E. coli 16S rRNA. Some of these adenines were found clustered in specific helices. Such differences suggest a greater irregularity of many of the helical elements within mammalian 18S rRNA, as compared with prokaryotic 16S rRNA. These helical irregularities could be important for protein association and also may represent biologically relevant flexible regions of the molecule.

Adenine↗
PubMed1989

ESR and fluorescence studies on the adenine binding site of lectins using a spin-labeled analogue.

The techniques of electron spin resonance (ESR) and fluorescence spectroscopy have been used to study the interaction of a spin-labeled analogue of adenine, N6-(2,2,6,6-tetramethyl-1-oxypiperidin-4-yl)adenine (I), with several plant lectins. While most adenine derivatives enhanced lectin-induced fluorescence of 1,8-anilinonaphthalenesulfonic acid by binding to a separate, adenine-specific site [Roberts, D.D., & Goldstein, I.J. (1982) J. Biol. Chem. 257, 11274-11277], the spin label I caused a decrease in this fluorescence with certain lectins. ESR showed the ligand to interact strongly with lectins from lima bean (Phaseolus lunatus), Dolichos biflorus, and Phaseolus vulgaris (PHA); however, no binding was observed with Griffonia simplicifolia isolectins A4 and B4, soybean agglutinin, or Amphicarpaea bracteata lectins. The spin label was highly immobilized by each of these proteins (2T magnitude of = 68 G). Apparent affinities of the spin label for the lectins decreased in the order lima bean lectin greater than PHA erythroagglutinin greater than PHA leukoagglutinin greater than D. biflorus. Spin-labeled adenine appeared to bind specifically to the adenine binding site of D. biflorus and PHA leukoagglutinin, as demonstrated by total abolition of the ESR spectrum of bound spin label by adenine. PHA erythroagglutinin and lima bean lectin bound the analogue with apparent dissociation constants of 5 X 10(-5) and 3.2 X 10(-5) M, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗
PubMed1996

An intracellular adenine nucleotide binding site inhibits guanyly cyclase C by a guanine nucleotide-dependent mechanism.

Guanylyl cyclase C (GCC), the receptor for the Escherichia coli heat-stable enterotoxin (ST), is inhibited by 2-substituted adenine nucleotides in an allosteric fashion. In confluent cultures of Caco-2 intestinal epithelial cells, extracellular 2-methylthioadenosine triphosphate (2MeSATP) had no effect on basal or ST-stimulated cyclic GMP (cGMP) accumulation. However, this nucleotide inhibited cGMP accumulation in digitonin-permeabilized Caco-2 human colon carcinoma cells, demonstrating that allosteric inhibition of GCC by adenine nucleotides is mediated by an intracellular adenine nucleotide binding site rather than purinergic receptors. The role of guanine nucleotides in the regulation of GCC by adenine nucleotides was examined. Increasing GTP concentrations from 5 to 100 microM increased the potency of 2MeSATP inhibition of GCC 20-fold, with a shift in the Ki from 447 to 22 microM, respectively. Also, the hydrolysis-resistant analogue, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), supported 2MeSATP inhibition of GCC with a potency which was 10-fold greater than GTP. In addition, GTP alone, in the absence of adenine nucleotides and at concentrations greater than 1 mM, inhibited GCC through a mechanism convergent with 2MeSATP. Guanine nucleotides supported adenine nucleotide inhibition of GCC at low concentrations and directly inhibited this enzyme at high concentrations when these studies were conducted with receptors expressed in Caco-2 cells, native rat intestine, or cloned rat GCC heterologously expressed in 293 monkey kidney cells. These observations demonstrate that adenine nucleotide inhibition of GCC is mediated through an intracellular mechanism which is guanine nucleotide-dependent.

Adenine Nucleotides↗
PubMed2003

Theoretical study of cisplatin binding to purine bases: why does cisplatin prefer guanine over adenine?

The thermodynamics and kinetics for the monofunctional binding of the antitumor drug cisplatin, cis-diamminedichloroplatinum(II), to a purine base site of DNA were studied computationally using guanine and adenine as model reactants. A dominating preference for initial attack at the N7-position of guanine is established experimentally, which is a crucial first step for the formation of a 1,2-intrastrand cross-link of adjacent guanine bases that leads to bending and unwinding of DNA. These structural distortions are proposed ultimately to be responsible for the anticancer activity of cisplatin. Utilizing density functional theory in combination with a continuum solvation model, we developed a concept for the initial Pt-N7 bond formation to atomic detail. In good agreement with experiments that suggested DeltaG++ = approximately 23 kcal/mol for the monofunctional platination of guanine, our model gives DeltaG++ = 24.6 kcal/mol for guanine, whereas 30.2 kcal/mol is computed when adenine is used. This result predicts that guanine is 3-4 orders of magnitude more reactive toward cisplatin than adenine. A detailed energy decomposition and molecular orbital analysis was conducted to explain the different barrier heights. Two effects are equally important to give the preference for guanine over adenine: First, the transition state is characterized by a strong hydrogen bond between the ammine-hydrogen of cisplatin and the O=C6 moiety of guanine in addition to a stronger electrostatic interaction between the two reacting fragments. When adenine binds, only a weak hydrogen bond forms between the chloride ligand of cisplatin and the H(2)N-C6 group of adenine. Second, a significantly stronger molecular orbital interaction is identified for guanine compared to adenine. A detailed MO analysis is presented to provide an intuitive view into the different electronic features governing the character of the Pt-N7 bond in platinated purine bases.

Adenine↗
PubMed1997

Adenine adducts with diepoxybutane: isolation and analysis in exposed calf thymus DNA.

1,3-Butadiene (BD) is a high-volume industrial chemical and a common environmental pollutant. Although BD is classified as a "probable human carcinogen", only limited evidence is available for its tumorigenic effects in occupationally exposed populations. Animal studies show a surprisingly high sensitivity of mice to the carcinogenic effects of BD compared to rats (approximately 10(3)-fold), making interspecies extrapolations difficult. Identification and quantitation of specific BD-induced DNA adducts are important for improving our understanding of the mechanisms of BD biological effects and for explaining the observed species differences. Covalent binding of BD to DNA is probably due to its two epoxy metabolites: 3,4-epoxy-1-butene (EB) and 1,2:3,4-diepoxybutane (DEB). Both EB and DEB are direct mutagens producing frameshift and point mutations at both A:T and G:C base pairs. DEB is 100 times more mutagenic than EB and is found in quantity only in tissues of the most sensitive species (mouse). This has led to the suggestion that the higher sensitivity of mice to BD could be due to greater exposure to DEB. The present work was initiated in order to isolate and structurally characterize DEB-induced adenine adducts. The adducts were formed by reacting DEB with free adenine (Ade), 2'-deoxyadenosine (2'-dAdo), and calf thymus DNA followed by HPLC separation and analysis of the products by UV spectrophotometry, electrospray ionization mass spectrometry, and nuclear magnetic resonance. The adenine reaction resulted in three products which were identified as N-3-, N-7-, and N-9-(2'-hydroxy-3',4'-epoxybut-1'-yl)adenine. These adducts underwent acid-catalyzed hydrolysis to their corresponding (2',3',4'-trihydroxybut-1'-yl)adenines upon heating or storage. The 2'-dAdo reaction with DEB followed by acid hydrolysis yielded a single adduct, N6-(2',3',4'-trihydroxybut-1'-yl)adenine (N6-DEB-Ade). N-3-DEB-Ade and N6-DEB-Ade were also found in hydrolysates of calf thymus DNA exposed to DEB. The amounts of N-3-DEB-Ade (13/10(3) normal Ade) and N6-DEB-Ade (5/10(3) normal Ade) were slightly lower than those of the corresponding EB-induced adducts in similar experiments, suggesting comparable reactivity of the two epoxy metabolites of BD toward adenine in DNA. The findings of this study provide a basis for future analyses of BD-induced adenyl DNA adducts in vitro and in vivo.

Adenine↗
PubMed1998

Polyclonal antibodies to adenine N1-oxide: characterization and use for the measurement of DNA damage.

Adenine N1-oxide is a DNA lesion whose formation involves the specific oxidation of the adenine base by hydrogen peroxide under nonradical conditions. The damage may be measured using a HPLC/32P-postlabeling method, which however cannot be used for routine analysis. We propose herein as an alternative an immunological assay which allows a rapid evaluation of the level of adenine N1-oxide in DNA exposed to oxidative stress. Two polyclonal antibodies were raised using two different strategies for the coupling of the hapten to the protein. The first approach is based on the universal method of Erlanger and Beiser, whereas the preparation of the second antigen involves the conjugation of a morpholino derivative of adenosine N1-oxide to the carrier protein. The affinity and the specificity of those antibodies were determined by competitive enzyme-linked immunosorbent assay. The antibody obtained by the traditional method shows some cross-reactivity with normal nucleotides, whereas for the other antiserum, the selectivity was found to be higher. Therefore, this polyclonal antibody was used to quantify the level of adenine N1-oxide in calf thymus DNA oxidized either by m-chloroperbenzoic acid or by hydrogen peroxide. The detection limit of the assay is four residues of adenine N1-oxide per 10(6) normal bases. The level of adenine N1-oxide in nonmodified DNA was lower than the detection limit of the assay, whereas in mCPB- and H2O2-modified DNA, it could be up to 14 and 0.7 adenine N1-oxide residues per 10(4) normal bases, respectively.

Adenine↗
PubMed2000

Accelerated degradation of adenine nucleotide in erythrocytes of patients with chronic renal failure.

Recently, we have shown that erythrocytes obtained from patients with chronic renal failure (CRF) exhibited an increased rate of ATP formation from adenine as a substrate. Thus, we concluded that this process was in part responsible for the increase of adenine nucleotide concentration in uremic erythrocytes. There cannot be excluded however, that a decreased rate of adenylate degradation is an additional mechanism responsible for the elevated ATP concentration. To test this hypothesis, in this paper we compared the rate of adenine nucleotide breakdown in the erythrocytes obtained from patients with CRF and from healthy subjects. Using HPLC technique, we evaluated: (1) hypoxanthine production by uremic RBC incubated in incubation medium: (a) pH 7.4 containing 1.2 mM phosphate (which mimics physiological conditions) and (b) pH 7.1 containing 2.4 mM phosphate (which mimics uremic conditions); (2) adenine nucleotide degradation (IMP, inosine, adenosine, hypoxanthine production) by uremic RBC incubated in the presence of iodoacetate (glycolysis inhibitor) and EHNA (adenosine deaminase inhibitor). The erythrocytes of healthy volunteers served as control. The obtained results indicate that adenine nucleotide catabolism measured as a hypoxanthine formation was much faster in erythrocytes of patients with CRF than in the cells of healthy subjects. This phenomenon was observed both in the erythrocytes incubated at pH 7.4 in the medium containing 1.2 mM inorganic phosphate and in the medium which mimics hyperphosphatemia (2.4 mM) and metabolic acidosis (pH 7.1). The experiments with EHNA indicated that adenine nucleotide degradation proceeded via AMP-IMP-Inosine-Hypoxanthine pathway in erythrocytes of both patients with CRF and healthy subjects. Iodoacetate caused a several fold stimulation of adenylate breakdown. Under these conditions: (a) the rate of AMP catabolites (IMP + inosine + adenosine + hypoxanthine) formation was substantially higher in the erythrocytes from patients with CRF; (b) in erythrocytes of healthy subjects degradation of AMP proceeded via IMP and via adenosine essentially at the same rate; (c) in erythrocytes of patients with CRF the rate of AMP degradation via IMP was about 2 fold greater than via adenosine. The results presented in this paper suggest that adenine nucleotide degradation is markedly accelerated in erythrocytes of patients with CRF.

Adenine Nucleotides↗
PubMed1967

Studies on the nature of the regulation by purine nucleotides of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase from Ehrlich ascites-tumour cells.

1. The progress curves of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase activity plotted against 5-phosphoribosyl pyrophosphate concentration were hyperbolic in nature. The inhibition of the former enzyme by AMP and GMP and of the latter enzyme by IMP and GMP showed completely competitive characteristics. 2. The effect of temperature on the reaction of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase was examined. The energy of activation of the former enzyme decreased at temperatures greater than 27 degrees and that of the latter enzyme at temperatures greater than 23 degrees . For each enzyme, the change in the heat of formation of the 5-phosphoribosyl pyrophosphate-enzyme complex at the critical temperature was approximately equal to the change in the energy of activation but was in the opposite direction. The inhibitor constants with both enzymes in the presence of nucleotides varied in different ways with temperature from the Michaelis constants for 5-phosphoribosyl pyrophosphate indicating that different functional groups were involved in binding substrates and inhibitors. 3. ATP was found to stimulate adenine-phosphoribosyltransferase activity at concentrations less than about 250mum and to inhibit the enzyme at concentrations greater than 250mum. The stimulation was unaffected by 5-phosphoribosyl pyrophosphate concentration but the inhibitory effect could be overcome by increasing concentrations of this compound. At low concentrations ATP reversed the inhibition of adenine phosphoribosyltransferase by AMP and GMP to an extent dependent on their concentration. 4. The properties of adenine phosphoribosyltransferase changed markedly on purification. Crude extracts of ascites-tumour cells had Michaelis constants for 5-phosphoribosyl pyrophosphate and adenine 75 and six times as high respectively as those obtained with purified enzyme. ATP had no stimulatory effect on activity of the purified enzyme or on that of crude extracts heated 15min. or longer at 55 degrees . 5. It is suggested that at low concentrations ATP is bound to an ;activator' site which is separate from the substrate binding site of adenine phosphorytransferase and that at high concentrations ATP competes with 5-phosphoribosyl pyrophosphate at the active site of the enzyme.

Adenine Nucleotides↗
PubMed1989

High-pressure-liquid-chromatographic and fluorimetric methods for the determination of adenine released from ribosomes by ricin and gelonin.

The high fluorescence of adenine-containing compounds after reaction with chloroacetaldehyde was used to measure the adenine released from rat liver and Artemia salina ribosomes by the action of ricin A chain and gelonin, two ribosome-inactivating proteins (RIPs) that share the same mechanism of action, consisting in the hydrolysis of the N-glycosidic bond of A-4324 of 28 S rRNA. Two methods were employed: (i) h.p.l.c. of the chloroacetaldehyde-reactive material released by RIPs; h.p.l.c. associated with a fluorescence detector allows the identification of adenine and its dosage at quantities as low as 2 ng; (ii) the direct fluorimetric measurement of the material that had reacted with chloroacetaldehyde. The amount of adenine released increases when ribosomes are pretreated in conditions that lead to their dissociation into subunits. Adenine protects ribosomes from the inhibition by ricin A-chain. When ribosomes were incubated with ricin A-chain in the presence of [14C]adenine no incorporation of radioisotope in ribosomes was observed, indicating that neither exchange nor reversal reactions occurred. A binding of [14C]adenine to ricin A chain was not detected by equilibrium dialysis.

Adenine↗
PubMed1997

Reading-frame restoration by transcriptional slippage at long stretches of adenine residues in mammalian cells.

We previously characterized a mutant apoB allele (the apoB86 allele) that produces both a truncated apoB (apoB86) and a full-length apoB100. The mutant allele contained a deletion of a single cytosine in exon 26, creating a stretch of eight consecutive adenines in the -1 reading frame. The altered reading-frame allele was restored, with approximately 10% efficiency, by the transcriptional insertion of an extra adenine into the stretch of eight consecutive adenines, thereby accounting for the synthesis of the full-length apoB100. Here, we demonstrate that this reading-frame restoration does not occur when the long stretch of adenines is interrupted by a cytosine. To assess whether reading-frame restoration is unique to a single site in the apoB gene, the same mutation (eight consecutive adenines in the -1 reading frame) was inserted into another site within the apoB gene. Reading-frame restoration occurred at the second site and was abrogated when the stretch of adenines was interrupted by another base. Of note, a computerized analysis of human cDNA sequences revealed that long stretches of adenines in protein-coding sequences occur at a lower than predicted frequency, suggesting that evolution has selected against these sequences.

Adenine↗
PubMed1999

Identification of the binding site for the extrahelical target base in N6-adenine DNA methyltransferases by photo-cross-linking with duplex oligodeoxyribonucleotides containing 5-iodouracil at the target position.

DNA methyltransferases flip their target bases out of the DNA double helix for catalysis. Base flipping of C5-cytosine DNA methyltransferases was directly observed in the protein-DNA cocrystal structures of M.HhaI and M.HaeIII. Indirect structural evidence for base flipping of N6-adenine and N4-cytosine DNA methyltransferases was obtained by modeling DNA into the three-dimensional structures of M.TaqI and M.PvuII in complex with the cofactor. In addition, biochemical evidence of base flipping was reported for different N6-adenine DNA methyltransferases. As no protein-DNA cocrystal structure for the related N6-adenine and N4-cytosine DNA methyltransferases is available, we used light-induced photochemical cross-linking to identify the binding site of the extrahelical target bases. The N6-adenine DNA methyltransferases M.TaqI and M.CviBIII, which both methylate adenine within the double-stranded 5'-TCGA-3' DNA sequence, were photo-cross-linked to duplex oligodeoxyribonucleotides containing 5-iodouracil at the target position in 50-60% and almost quantitative yield, respectively. Proteolytic fragmentation of the M. CviBIII-DNA complex followed by Edman degradation and electrospray ionization mass spectrometry indicates photo-cross-linking to tyrosine 122. In addition, the mutant methyltransferases M. TaqI/Y108A and M.TaqI/F196A were photo-cross-linked with 6-fold and 2-fold reduced efficiency, respectively, which suggests that tyrosine 108 is the primary site of modification in M.TaqI. Our results indicate a close proximity between the extrahelical target base and tyrosine 122 in M.CviBIII or tyrosine 108 in M.TaqI. As both residues belong to the conserved motif IV ((N/D/S)(P/I)P(Y/F/W)) found in all N6-adenine and N4-cytosine DNA as well as in N6-adenine RNA methyltransferases, a similar spatial relationship between the target bases and the aromatic amino acid residue within motif IV is expected for all these methyltransferases.

Amino Acid Sequence↗