Metabolism of 1-methyladenosine

1-[methyl-8-14C] Adenosine was synthesized and its metabolic fate was determined in intact rat. It was found that approximately 57% of 1-[methyl-8-14C] adenosine administered iv was excreted unchanged in the urine and 33% of the excreted radioactivity in the urine was associated with the major metabolite 1-methyl-hypoxanthine and about 4.5% was associated with 1-methylinosine. Very little adenosine or N6-methyladenosine was formed. It is concluded that 1-methyladenosine is initially deaminated by adenosine deaminase to 1-methylinosine which is then cleaved by nucleoside phosphorylase to 1-methylhypoxanthine.     

Proton magnetic resonance study of complex formation between N6-methyladenosine and polyuridylic acid

The interaction between N₆-methyladenosine and polyuridylic acid in D₂O solution at neutral pD has been studied as a function of temperature and N₆-methyladenosine concentration by proton magnetic resonance spectroscopy. A rigid double-stranded 1:1 complex is formed below ～10°C, involving hydrogen-bonded N₆-methyladenine:uracil base-pairing and stacking of the adenine bases. This complex is less stable than the 1:2 complex formed between adenosine and polyU, and involves a more rapid exchange of the monomer between free and polymer-bound environments.     

Effect of Disulfide-Reducing Agents on Activity of 1-Methyladenosine Ribohydrolase in Starfish Follicle Cells

Production of 1-methyladenine (1-MeAde) in isolated starfish follicle cells incubated with 1-methyladenosine was elevated by disulfide-reducing agents, such as dithiothreitol, cysteine, and homocysteine. More than 0.05 mM cysteine and homocysteine brought about a six-fold increase of 1-MeAde production in comparison with controls without disulfide-reducing agents. On the other hand, iodoacetamide, which is a membrane-permeable SH-blocking agent, decreased the 1-methyladenosine-induced 1-MeAde production. A forty percent decrease of 1-MeAde production was obtained with 0.5 mM iodoacetamide. Iodoacetamide inhibited also 1-MeAde production induced by gonad-stimulating substance (GSS). But a membrane-impermeable SH-blocking agent, N-carboxyphenylmaleimide, failed to inhibit both 1-methyladenosine-induced and GSS-induced 1-MeAde production. Furthermore, partially purified 1-methyladenosine ribohydrolase was activated by dithiothreitol, 2-mercaptoethanol, and cysteine derivatives. An approximately ten-fold activation was obtained with these disulfide-reducing agents at concentrations of more than 5 mM. On the contrary, though the SH-blocking agents inhibited 1-methyladenosine ribohydrolase, the inhibition was relieved by the disulfide-reducing agents. These results suggest that the reduced form of SH-group in 1-methyladenosine ribohydrolase plays an important role for enzymatic activation.     

A simple method of the preparation of 2′-O-Methyladenosine Methylation of adenosine with methyl iodide in anhydrous alkaline medium

A simple and effective method of the methylation on the 2′-O position of adenosine is described. Adenosine is treated with CH₃I in an anhydrous alkaline medium at 0°C for 4 h. The major products of this reaction are monomethylated adenosine at either the 2′-O or 3′-O position (total of 64%) and the side products are dimethylated adenosine (2′,3′-O-dimethyladenosi, 21%, and N⁶-2′-O-dimethyladenosine, 11%). The ratio of 2′-O- and 3′-O-methyladenosine has been found to be 8 to 1. Therefore, this reaction preferentially favors the synthesis of 2′-O-methyladenosine. The monomethylated adenosine is isolated from reaction mixture by a silica gel column chromatography. Then the pure 2′-O-methyladenosine can be separated by crystallization in ethanol from the mixture of 2′-O and 3′-O-methylated isomers. The overall yield of 2′-O-methyladenosine is 42%.     

Modified nucleosides of Bacillus subtilis transfer ribonucleic acids.

An analysis of the kinds and amounts of minor nucleosides of transfer ribonucleic acids (tRNA's) from Bacillus subtilis 168 trpC2 is presented. Identification and quantitation were accomplished using ion exclusion chromatography, thin-layer and paper chromatography, and ultraviolet absorption properties. Nucleosides and their amount in moles per 80 residues are as follows: guanosine (25.7), cytidine (22.0), adenosine (15.2), uridine (13.1), 5-methyluridine (0.98), pseudouridine (1.54), 1-methyladenosine (0.15), N6-methyladenosine (0.01), 7-methyladenosine (0.10), 2-methyladenosine (0.03), 7-methylguanosine (0.20), N2-methylguanosine (0.14), 1-methylguanosine (0.14), a methylated pyrimidine (0.17), a methylated derivative of N6-(delta 2-isopentenyl)adenosine (0.02), ribose methylated nucleosides (0.02), 4-thiouridine (0.12), 2-thio-5-(N-methylaminomethyl) (0.09), and an unknown thionucleoside (0.12). Although the composition is similar to that of Escherichia coli in the proportion of major nucleosides, the content of pseudouridine and 5-methyluridine, and the degree of base and ribose methylation, the composition is more similar to that of the tRNA's of yeast and higher organisms in its lower degree of thiolation, the presence of significant amounts of 1-methyladenosine, and the low levels of 2-methyladenosine and 6-methyladenosine. Therefore, the nucleoside composition of B. subtilis presents some different aspects from those usually given as characteristic for bacterial tRNA's. It is not known whether these differences are due to variation between bacterial species in general or related to the process of differentiation.     

Modulation of adenine nucleoside excretion and incorporation in adenosine deaminase deficient human lymphoma cells

The availability of a human lymphoma cell line deficient in adenosine deaminase, adenosine kinase and methylthioadenosine phosphorylase enabled us to compare the effects of nucleoside transport inhibitors on the excretion of endogenously generated adenosine, deoxyadenosine and 5'-methylthioadenosine. The nucleoside transport inhibitors nitrobenzylthioinosine and dipyridamole blocked the efflux of adenosine, but not deoxyadenosine or 5'-methylthioadenosine. The inhibitors also prevented the uptake of exogenous adenosine, but not deoxyadenosine or 5'-methylthioadenosine, by human lymphoblasts. The results show (i) that the transport inhibitors modify adenine nucleoside efflux and influx similarly, and (ii) that the effects of the compounds on the excretion and uptake of these three physiologically important adenine nucleosides are distinctly different.     

Control of substrate specificity by a single active site residue of the KsgA methyltransferase

The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and N(6)-methyladenosine as substrates to catalyze formation of the final product N(6),N(6)-dimethyladenosine. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of S-adenosyl-L-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the N(6)-methyladenosine intermediate to produce N(6),N(6)-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of N(6)-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA's multiple catalytic steps.     

Regulatory functions of cyclic adenosine 3',5'-monophosphate in 1-methyladenine production by starfish follicle cells

The biosynthesis of 1-methyladenine (1-MeAde) in follicle cells of the starfish, Asterina pectinifera, occurred in response to a gonad-stimulating substance (GSS). Simultaneously with 1-MeAde production, the intracellular cAMP level immediately increased following the administration of GSS. This level in follicle cells markedly depended on GSS concentration. Although 1-MeAde production was also induced by 1-methyladenosine, it caused no increase in cAMP content. It thus appears that the effect of GSS on starfish follicle cells results in the receptor-mediated formation of cAMP.     

Urinary excretion of modified purines and nucleosides in immunodeficient children

Studies have been carried out using an XAD-4 resin and ion-exchange chromatography for determination of urinary purines and nucleosides in seven children with severe combined immunodeficiency and in six normal children. These studies have included analyses for five methylated purines or nucleosides produced by catabolism of nucleic acids. The following compounds have been quantitatively determined: 1-methyladenosine, 1-methylinosine, 1-methylguanosine, 1-methylguanine, 3-methylcytidine, adenosine, methylthioadenosine sulfoxide, cytidine, and deoxycytidine. 1-Methyladenosine and 1-methylinosine were most consistently elevated in the urine of immunodeficient children. Methylthioadenosine sulfoxide was very markedly increased in urine of two of the immunodeficient children while more moderate increases were noted with a number of other nucleosides. The germ-free child with severe combined immunodeficiency showed consistently lower excretion levels of these compounds when compared to normal children.     

Interaction of metal ions with nucleic acids. Interaction of copper(II) with adenosine and its derivatives.

The interaction of copper(II) with adenosine, 2'-deoxyadenosine, 1-methyladenosine, 7-deazaadenosine and AMP was studied by spectroscopic and magnetochemical methods. In non-aqueous medium, copper(II) interacts with adenosine and AMP at N-7 and N-1, and with 1-methyladenosine at N-7 and N-3. The copper ion is not bound to the NH2 group. In aqueous solution, copper(II) interacts both with N-7 and N-1 of adenosine, and in AMP additionally with the phosphate group. The interaction of copper(II) with the heterocyclic part, but not withthe phosphate group, is dependent on the extent of protonation of the molecular. A crystalline AMP-copper(II) complex [Cu(C10H12N5O7P).(H2O)2] was obtained; the phosphate group and probably N-7 are involved in the complex formation.     

Xanthine oxidase and adenosine deaminase in commercial bovine spleen phosphodiesterase preparations.

Commercial bovine spleen phosphodiesterase preparations contain xanthine oxidase activity; the xanthine oxidase in such preparations mediates the oxidation of a pteridine derivative as well as a standard purine substrate (hypoxanthine). The xanthine oxidase activity in the phosphodiesterase preparations is inhibited strongly by allopurinol (4-hydroxypyrazolo(3,4-d) pyrimidine). The reported ability of phosphodiesterase preparations to catalyze the deamination of adenosine derivatives appears to be due to contamination with a conventional adenosine deaminase in view of the observations that this activity is inhibited by an established inhibitor of adenosine deaminase and that the relative rates of deamination of N¹-methyladenosine and adenosine are similar with both the phosphodiesterase preparation and calf intestine adenosine deaminase.     

EFFECTS OF METHIONINE AND S-ADENOSYLMETHIONINE ON PRODUCTION OF 1-METHYLADENINE IN STARFISH FOLLICLE CELLS

Incubation of follicle cells of the starfish ( Asterias amurensis ) with a gonad-stimulating hormonal peptide (GSS) leads to the production of 1-methyladenine. the trigger of oocyte maturation. Addition of L-methionine to the incubation medium promotes the production of 1-methyladenine. This study shows that S -adenosylmethionine also enhances the production of 1-methyladenine in such an incubation mixture. However, in the absence of GSS, addition of S -adenosylmethionine failed to produce an appreciable amount of 1-methyladenine. When an homogenate of isolated follicle cells was incubated, a certain amount of 1-methyladenine was produced, whether or not GSS was present. Addition of S -adenosylmethionine to the incubation mixture of follicle homogenate enhanced the production of 1-methyladenine. Although addition of adenosine triphosphate (ATP) to such an incubation mixture had little effect in producing 1-methyladenine, it exerted a promoting effect on 1-methyladenine production when S -adenosyl-methionine was present. These results suggest that methionine, through its active form, S -adenosylmethionine, acts as a donor of methyl group in the formation of 1-methyladenine.     

CHEMICAL STRUCTURAL REQUIREMENTS FOR INDUCTION OF OOCYTE MATURATION AND SPAWNING IN STAREISHES

Effects of various adenine derivatives on oocyte maturation and spawning were studied in the starfishes, Marthasterias glacialis, Astropecten aurantiacus, Patiria miniata, Asterina pectinifera and Asterias forbesi . 1-Methyladenine and 1-ethyladenine were very effective in inducing oocyte maturation and spawning, whereas the following related compounds had no effect: adenine, 3-methyladenine, 7-methyl-adenine, 9-methyladenine, 1-methylguanine, 1-methylhypoxanthine, 6-methylpurine, N₆-methyladenine, N₆-
dimethyladenine, N₆-benzyladenine, N₆-furfuryladenine(kinetin), adenosine, 5' -adenylic acid, adenosine 3',5'-cyclic monophosphate, adenosine triphos-phate, inosine, 5'-inocinic acid, guanine, guanosine, 5'-guanylic acid, hypoxanthine, xanthine, xanthosine, 3-methylcytidine and 5-methylcytosine. 1-Methyladenosine induced oocyte maturation and spawning when isolated ovarian fragments were used as assay material; however, it had little effect in inducing maturation of isolated oocytes. Therefore, this compound seems to active only after its decomposition to 1-methyladenine and ribose. The chemical structure responsible for inducing oocyte maturation and spawning in starfishes is proposed: a short alkyl radical such as methyl or ethyl at N₁ site and an imino radical at C₆ site of the purine nucleus.     

Phosphorylase-mediated mobilization of the amino group of adenine in Bacillus cereus

Mobilization of the ribose moiety of purine nucleosides as well as of the amino group of adenine may be realized in Bacillus cereus by the concerted action of three enzymes: adenosine phosphorylase, adenosine deaminase, and purine nucleoside phosphorylase. In this pathway, ribose-1-phosphate and inorganic phosphate act catalytically, being continuously regenerated by purine nucleoside phosphorylase and adenosine phosphorylase, respectively. As a result of such a metabolic pathway, adenine is quantitatively converted into hypoxanthine, thus overcoming the lack of adenase in B. cereus.     

Radioimmunoassay for N6(methylnitroso)adenosine: production and characterization of antibodies

Antibodies specific for N⁶(methylnitroso)adenosine have been produced in rabbits and a sensitive radioimmunoassay was developed. The nitroso group is immunodominant; 50% inhibition of the binding of [³H]N⁶(methylnitroso)adenosine to antibody was obtained with 9.6 pmoles of N⁶(methylnitroso)adenosine and 200 nmoles of N⁶-methyladenosine. Adenosine was essentially inactive. After nitrosation, N⁶(methylnitroso)adenosine can be detected only in those RNA molecules known to contain N⁶-methyladenosine.     

MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex-specific splicing factor

N6-Methyladenosine is a ubiquitous modification identified in the mRNA of numerous eukaryotes, where it is present within both coding and noncoding regions. However, this base modification does not alter the coding capacity, and its biological significance remains unclear. We show that Arabidopsis thaliana mRNA contains N6-methyladenosine at levels similar to those previously reported for animal cells. We further show that inactivation of the Arabidopsis ortholog of the yeast and human mRNA adenosine methylase (MTA) results in failure of the developing embryo to progress past the globular stage. We also demonstrate that the arrested seeds are deficient in mRNAs containing N6-methyladenosine. Expression of MTA is strongly associated with dividing tissues, particularly reproductive organs, shoot meristems, and emerging lateral roots. Finally, we show that MTA interacts in vitro and in vivo with At FIP37, a homolog of the Drosophila protein FEMALE LETHAL2D and of human WILMS' TUMOUR1-ASSOCIATING PROTEIN. The results reported here provide direct evidence for an essential function for N6-methyladenosine in a multicellular eukaryote, and the interaction with At FIP37 suggests possible RNA processing events that might be regulated or altered by this base modification.     

Induction and repression of enzymes involved in exogenous purine compound utilization of Bacillus cereus

5'-Nucleotidase, adenosine phosphorylase, adenosine deaminase and purine nucleoside phosphorylase, four enzymes involved in the utilization of exogenous compounds in Bacillus cereus, were measured in extracts of this organism grown in different conditions. It was found that adenosine deaminase is inducible by addition of adenine derivatives to the growth medium, and purine, nucleoside phosphorylase by metabolizable purine and pyrimidine ribonucleosides. Adenosine deaminase is repressed by inosine, while both enzymes are repressed by glucose. Evidence is presented that during growth of B. cereus in the presence of AMP, the concerted action of 5'-nucleotidase and adenosine phosphorylase, two constitutive enzymes, leads to formation of adenine, and thereby to induction of adenosine deaminase. The ionsine formed would then cause induction of the purine nucleoside phosphorylase and repression of the deaminase. Taken together with our previous findings showing that purine nucleoside phosphorylase of B. cereus acts as a translocase of the ribose moiety of inosine inside the cell (Mura, U., Sgarrella, F. and Ipata, P.L. (1978) J. Biol Chem. 253, 7905-7909), our results provide a clear picture of the molecular events leading to the utilization of the sugar moiety of exogenous AMP, adenosine and inosine as an energy source.     

Adenosine phosphorylase activity in mycoplasma-free growth media for mammalian cells

Mammalian cells have enzymes that deaminate adenosine to inosine, which can readily be phosphorolysed to hypoxanthine. They do not, however, possess enzymes to form adenine by the cleavage of adenosine. For this reason, the release of adenine from adenosine by mammalian cell cultures has usually been interpreted as indicating the presence of mycoplasma, a frequent microbial contaminant that contains high levels of adenosine phosphorylase. We found that some human lymphoblast cultures free of mycoplasma showed high levels of adenosine cleavage and that this activity resulted from adenosine phosphorylase in the bovine serum used as the culture growth supplement. A survey of 13 serum supplements disclosed that fetal bovine serum (six lots) contains the highest adenosine phosphorylase activity, ranging from 9 to 648 nmol adenine produced per hour per ml serum; newborn calf serum (four lots) has much less activity, ranging from 0 to 5 nmol adenine produced per hour per ml serum; and donor horse serum (three lots) contains no detectable activity. These results suggest that mycoplasma tests dependent on the presence of adenosine phosphorylase or other enzyme activities may give false-positives with cultures containing fetal bovine serum supplements.     

Blood glycoprotein from antarctic fish. Possible conformational origin of antifreeze activity

Two purine nucleoside phosphorylases (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) were purified from vegetative Bacillus subtilis cells. One enzyme, inosine-guanosine phosphorylase, showed great similarity to the homologous enzyme of Bacillus cereus. It appeared to be a tetramer of molecular weight 95 000. The other enzyme, adenosine phosphorylase, was specific for adenosine and deoxyadenosine. The molecular weight of the native enzyme was 153 000 +/- 10% and the molecular weight of the subunits was 25 500 +/- 5%. This indicates a hexameric structure. The adenosine phosphorylase was inactivated by 10(-3) M p-chloromercuribenzoate and protected against this inactivation by phosphate, adenosine and ribose 1-phosphate.     

Reactions of adenosine 5′-phosphorimidazolide with adenosine analogs on a polyuridylic acid template: The uniqueness of the 2′-3′-unsubstituted β-ribosyl system

We have studied the reactions between adenosine 5′-phosphorimidazolide and various adenosine analogs on a poly(U) template. The nucleosides were adenosine (I), 2′-deoxyadenosine (II), 3′-deoxyadenosine (III), 2′-O-methyladenosine (IV), 3′-O-methyladenosine (V), 9-β-d-xylofuranosyladenine (VI), and 9-β-d-arabinofuranosyladenine (VII). We find that the various analogs form triple helices with poly(U) which are of comparable stability, but that only the β-riboside takes part in an efficient template-directed condensation.