Acyl-CoA synthetase catalyzes the synthesis of diadenosine hexaphosphate (Ap6A).

The synthesis of diadenosine hexaphosphate (Ap6A), a potent vasoconstrictor, is catalyzed by acyl-CoA synthetase from Pseudomonas fragi. In a first step AMP is transferred from ATP to tetrapolyphosphate (P4) originating adenosine pentaphosphate (p5A) which, subsequently, is the acceptor of another AMP moiety from ATP generating diadenosine hexaphosphate (Ap6A). Diadenosine pentaphosphate (Ap5A) and diadenosine tetraphosphate (Ap4A) were also synthesized in the course of the reaction. In view of the variety of biological effects described for these compounds the potential capacity of synthesis of diadenosine polyphosphates by the mammalian acyl-CoA synthetases may be relevant.     

Subcellular Distribution Studies of Diadenosine Polyphosphates—Ap4A and Ap5A—in Bovine Adrenal Medulla: Presence in Chromaffin Granules

Diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) have been identified in bovine adrenal medullary tissue using an HPLC method. The values obtained were 0.1 +/- 0.05 mumol/g of tissue for both compounds. The subcellular fraction where Ap4A and Ap5A were present in the highest concentration was chromaffin granules: 32 nmol/mg of protein for both compounds (approximately 6 mM intragranularly). This value was 30 times higher than in the cytosolic fraction. Enzymatic degradation of Ap4A and Ap5A, isolated from chromaffin granules, with phosphodiesterase produces AMP as the final product. The Ap4A and Ap5A obtained from this tissue were potent inhibitors of adenosine kinase. Their Ki values relative to adenosine were 0.3 and 2 microM for Ap4A and Ap5A, respectively. The cytosolic fraction also contains enzymatic activities that degrade Ap4A as well as Ap5A. These activities were measured by an HPLC method; the observed Km values were 10.5 +/- 0.5 and 13 +/- 1 microM for Ap4A and Ap5A, respectively.     

The IalA invasion gene of Bartonella bacilliformis encodes a (de)nucleoside polyphosphate hydrolase of the MutT motif family and has homologs in other invasive bacteria

The product of the ialA invasion gene of Bartonella bacilliformis has been expressed as a thioredoxin fusion protein. It is a (di)nucleoside polyphosphate hydrolase of the MutT motif protein family with strong sequence similarity to plant diadenosine tetraphosphate hydrolases. It hydrolyses nucleoside and dinucleoside polyphosphates with four or more phosphate groups, always producing an NTP as one product. Diadenosine tetraphosphate (Ap4A) is the preferred substrate with a Km of 10 microM and a kcat of 3.0 s-1. It is inhibited by Ca2+ and F- (Ki = 30 microM). Hydrolysis of Ap4A in H218O yielded [18O]AMP as the only labelled product. In terms of sequence, reaction mechanism and properties, IalA is very similar to eukaryotic Ap4A hydrolases and unlike previously described bacterial Ap4A hydrolases. Homologs are present in the genomes of other invasive pathogens. They may function to reduce stress-induced dinucleotide levels during invasion and so enhance pathogen survival.     

Diadenosine tetraphosphatase from human leukemia cells. Purification to homogeneity and partial characterization

Diadenosine tetraphosphatase, an enzyme splitting diadenosine tetraphosphate to AMP and ATP, has been purified to apparent homogeneity from a permanent cell line derived from a leukemic child. The purification procedure consisted of fractionation by ammonium sulfate precipitation, followed by Sephacryl 200 and DEAE-cellulose chromatography, and finally a differential membrane filtration. The enzyme is a single polypeptide chain of Mr = 17,500 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. The apparent molecular weight of the native enzyme was calculated as 20,000 from gel filtration data. The apparent Km for Ap4A was 0.5 microM as determined by two independent kinetic assays. None of the following compounds were substrates of the enzyme: diadenosine triphosphate, NAD, nucleoside 5'-phosphates (AMP, ATP, GDP, GTP, and UTP). The enzyme had optimal activity in the presence of 1 mM Mg2+, showing no activity in the presence of EDTA.     

Diadenosine tetraphosphate activates cytosol 5'-nucleotidase

The rate of hydrolysis of IMP (0.5 mM) by cytosol 5'-nucleotidase from Artemia embryos was increased up to 7-fold by concentrations of around 10 microM diadenosine tetraphosphate (Ap4A). Half maximal activation of the enzyme was accomplished with 5 microM Ap4A. The Km (S 0.5) values of the nucleotidase for IMP, GMP, AMP, XMP and CMP decreased about 10 fold in the presence of 10 microM Ap4A. Maximum velocity of the enzyme was not affected by Ap4A. ATP had been previously described as an activator of the enzyme. However, comparatively with Ap4A, concentrations of ATP two orders of magnitude higher are needed to elicit similar effects on the enzyme. Preliminary results indicate that Ap4A is also an activator of the cytosol 5'-nucleotidase from rat liver.     

Mechanism of activation of phenylalanine and synthesis of P1, P4-bis(5'-adenosyl) tetraphosphate by yeast phenylalanyl-tRNA synthetase

The activation of L-phenylalanine by yeast phenylalanyl-tRNA synthetase using adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate is shown to proceed with inversion of configuration at P alpha of ATP. This observation taken together with the lack of positional isotope exchange when adenosine 5'-[beta,beta-18O2]triphosphate is incubated with the enzyme in the absence of phenylalanine and in the presence of the competitive inhibitor phenylalaninol indicates that activation of phenylalanine occurs by a direct "in-line" adenylyl-transfer reaction. In the presence of Zn2+, yeast phenylalanyl-tRNA synthetase also catalyzes the phenylalanine-dependent hydrolysis of ATP to AMP and the synthesis of P1,P4-bis(5'-adenosyl) tetraphosphate (Ap4A). With adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate, the formation of AMP and Ap4A is shown to occur with inversion and retention of configuration, respectively. It is concluded that phenylalanyl adenylate is an intermediate in both processes, Zn2+ promoting AMP formation by hydrolytic cleavage of the C-O bond and Ap4A formation by displacement at phosphorus of phenylalanine by ATP.     

Early increase in diadenosine tetraphosphate in regenerating rat liver

Diadenosine tetraphosphate (Ap4A), a candidate for a signal molecule in the induction of DNA synthesis, was measured in regenerating livers of young adult rats at 12 and 24 h and of older rats at 24 h after partial hepatectomy. dATP and dTTP levels, which indicate the degree of proliferation in the livers, were determined by high-performance liquid chromatography and an enzymatic method, respectively. The Ap4A levels were increased in the beginning of DNA synthesis. In young rats the levels were about 140% of those of unoperated rats and in older rats about 300%. This increase was considerably smaller than that found in another study comprising two regenerating rat livers excised 20 h after partial hepatectomy, but still supports the hypothesis that Ap4A might take part in the onset of proliferation. The greater Ap4A increase in older rats may suggest a possible need for a stronger triggering mechanism to start proliferation in aged tissue. However, the experiments do not prove a function for Ap4A in the induction of DNA synthesis and it cannot be excluded that Ap4A is a product of an independent reaction.     

Synthesis of dinucleoside polyphosphates catalyzed by firefly luciferase.

In the presence of ATP, luciferin (LH2), Mg2+ and pyrophosphatase, the firefly (Photinus pyralis) luciferase synthesizes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) through formation of the E-LH2-AMP complex and transfer of AMP to ATP. The maximum rate of the synthesis is observed at pH 5.7. The Km values for luciferin and ATP are 2-3 microM and 4 mM, respectively. The synthesis is strictly dependent upon luciferin and a divalent metal cation. Mg2+ can be substituted with Zn2+, Co2+ or Mn2+, which are about half as active as Mg2+, as well as with Ni2+, Cd2+ or Ca2+, which, at 5 mM concentration, are 12-20-fold less effective than Mg2+. ATP is the best substrate of the above reaction, but it can be substituted with adenosine 5'-tetraphosphate (p4A), dATP, and GTP, and thus the luciferase synthesizes the corresponding homo-dinucleoside polyphosphates:diadenosine 5',5"'-P1,P5-pentaphosphate (Ap5A), dideoxyadenosine 5',5"'-P1,P4-tetraphosphate (dAp4dA) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G). In standard reaction mixtures containing ATP and a different nucleotide (p4A, dATP, adenosine 5'-[alpha,beta-methylene]-triphosphate, (Ap[CH2]pp), (S')-adenosine-5'-[alpha-thio]triphosphate [Sp)ATP[alpha S]) and GTP], luciferase synthesizes, in addition to Ap4A, the corresponding hetero-dinucleoside polyphosphates, Ap5A, adenosine 5',5"'-P1,P4-tetraphosphodeoxyadenosine (Ap4dA), diadenosine 5',5"'-P1,P4-[alpha,beta-methylene] tetraphosphate (Ap[CH2]pppA), (Sp-diadenosine 5',5"'-P1,P4-[alpha-thio]tetraphosphate [Sp)Ap4A[alpha S]) and adenosine-5',5"'-P1,P4-tetraphosphoguanosine (Ap4G), respectively. Adenine nucleotides, with at least a 3-phosphate chain and with an intact alpha-phosphate, are the preferred substrates for the formation of the enzyme-nucleotidyl complex. Nucleotides best accepting AMP from the E-LH2-AMP complex are those which contain at least a 3-phosphate chain and an intact terminal pyrophosphate moiety. ADP or other NDP are poor adenylate acceptors as very little diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) or adenosine-5',5"'-P1,P3-triphosphonucleosides (Ap3N) are formed. In the presence of NTP (excepting ATP), luciferase is able to split Ap4A, transferring the resulting adenylate to NTP, to form hetero-dinucleoside polyphosphates. In the presence of PPi, luciferase is also able to split Ap4A, yielding ATP. The cleavage of Ap4A in the presence of Pi or ADP takes place at a very low rate. The synthesis of dinucleoside polyphosphates, catalyzed by firefly luciferase, is compared with that catalyzed by aminoacyl-tRNA synthetases and Ap4A phosphorylase.     

Ap4A is not an efficient Zn(II) binding agent. A concerted potentiometric, calorimetric and NMR study

Diadenosine 5',5'-P(1)P(4) tetraphosphate (Ap(4)A) has been considered as an intracellular partner for Zn(II). We applied potentiometry, ITC and NMR to study protonation equilibria of Ap(4)A and Zn(II) complexation by this dinucleotide. The values of binding constants obtained by these three techniques under various experimental conditions coherently demonstrated that Ap(4)A binds Zn(II) weakly, with an apparent binding constant of ca. 10(4) at neutral pH. Such a low stability of Zn(II) complexes with Ap(4)A excludes a possibility for interactions between these two agents in vivo.     

Diadenosine tetraphosphate (Ap4A) levels in HL-60 cells during differentiation into granulocytes and monocytes

1. Diadenosine tetraphosphate (Ap4A) levels were determined in HL-60 cells differentiating into granulocytes or monocytes after treatment for 0-7 days with retinoic acid (RA) or 4-beta-phorbol-12-myristate-13-acetate (PMA) respectively. 2. The levels increased significantly compared to untreated control cells within 2 days and then declined again. 3. In RA treated cells the levels finally decreased far below those of untreated HL-60 cells and became equal to those found in human granulocytes. 4. PMA treatment had no effect on Ap4A levels in human granulocytes. 5. A possible interaction between Ap4A and ADP-ribosyl transferase is discussed.     

Characterization and quantification of diadenosine hexaphosphate in chromaffin cells: Granular storage and secretagogue-induced release

The presence of diadenosine hexaphosphate (Ap6A) in chromaffin cells is described. The characterization of Ap6A has been accomplished by HPLC techniques, using three different elution conditions, rechromatography, and coelution with standards. Treatment with phosphodiesterase from Crotalus durissus produced AMP and adenosine pentaphosphate. The HPLC techniques described allowed the quantification of Ap6A in the picomole range. Chromaffin granules store Ap6A in a quantity of 48.5 +/- 9.7 nmol/mg protein, with a molar ratio ATP/Ap6A of 27. In chromaffin cells the Ap6A value was 1.46 +/- 0.32 nmol/10(6) cells. Diadenosine hexaphosphate was released from chromaffin cells by the action of carbachol and a value of 64 +/- 15 pmol/10(6) cells was obtained, which represents 4-5% of the total cellular content.     

Effect of diadenosine tetraphosphate microinjection on heat shock protein synthesis in Xenopus laevis oocytes.

Bisnucleosides polyphosphates are thought to be chemical messengers signalling to the cell the onset of various stresses. Diadenosine tri- and tetraphosphates (respectively, Ap3A and Ap4A) accumulate in prokaryotic and eukaryotic cells under heat shock conditions, suggesting they could trigger the synthesis of heat shock proteins (hsps). In this study, Ap4A, Ap3A and, as a control, Ap4 (adenosine tetraphosphate) were injected into Xenopus oocytes. Whereas none of these compounds is able to trigger the synthesis of hsps in the absence of hyperthermic treatment, nuclear microinjection of Ap4A after a mild heat shock specifically enhances the synthesis of the 70-kd hsp, which is involved in the regulation and possibly the termination of the heat shock response. The microinjection of Ap4A prior to the hyperthermic treatment results in a strong inhibition of hsps synthesis (with the exception of the 70-kd hsp) suggesting that Ap4A is involved in the regulation and/or termination of the heat shock response. Ap3A and Ap4 do not induce any detectable modification of hsps expression.     

Assay of diadenosine tetraphosphate hydrolytic enzymes by boronate chromatography

A new procedure was described for assay of diadenosine tetraphosphate (Ap4A) hydrolases based on boronate chromatography. Potential reaction products, AMP, ADP, and ATP, of the hydrolysis of Ap4A were separated from residual substrate by chromatography on a boronate-derivatized cation-exchange resin, Bio-Rex 70. Separation was achieved by changing the concentrations of ethanol and ammonium acetate in the elution buffers. Picomole masses of products were detectable, blank dpm values were less than 0.5% of the total dpm, and auxiliary enzymes were not required. The procedure was specifically described for Ap4A pyrophosphohydrolase from Physarum polycephalum. The assay is generally applicable for dinucleoside polyphosphate hydrolases which hydrolyze other substrates such as Ap3A, Ap5A, Ap6A, and Gp4G. Dinucleotide polyphosphates are readily purified by chromatography on this boronate resin in a volatile buffer. Tes, Tricine, and Tris buffers significantly interfered with the chromatography of ATP.     

Characterization of the bis(5''-nucleosidyl) tetraphosphate pyrophosphohydrolase from encysted embryos of the brine shrimp Artemia.

The P1P4-bis(5'-nucleosidyl) tetraphosphate asymmetrical-pyrophosphohydrolase from encysted embryos of the brine shrimp Artemia has been purified over 11,000-fold to homogeneity. Anion-exchange chromatography resolves two major species with very similar properties. The enzyme is a single polypeptide of Mr 17,600 and is maximally active at pH 8.4 and 2 mM-Mg2+. It is inhibited by Ca2+ (IC50 = 0.9 mM with 2 mM-Mg2+) but not by Zn2+ ions. It preferentially hydrolyses P1P4-bis(5'-nucleosidyl) tetraphosphates, e.g. P1P4-bis(5'-adenosyl) tetraphosphate (Ap4A) (kcat. = 12.7 s-1; Km = 33 microM) and P1P4-bis(5'-guanosyl) tetraphosphate (Gp4G) (kcat. = 6.2 s-1; Km = 5 microM). With adenosine 5'-P1-tetraphospho-P4-5"'-guanosine (Ap4G) as substrate, there is a 4.5-fold preference for AMP and GTP as products and biphasic reaction kinetics are observed giving Km values of 4.7 microM and 34 microM, and corresponding rate constants of 6.5 s-1 and 11.9 s-1. The net rate constant for Ap4G hydrolysis is 7.6 s-1. The enzyme will also hydrolyse nucleotides with more than four phosphate groups, e.g. Ap5G, Ap6A and Gp5G are hydrolysed at 25%, 18% and 10% of the rate of Ap4A respectively. An NTP is always one of the products. Ap2A and Gp2G are not hydrolysed, while Ap3A and Gp3G are very poor substrates. When the enzyme is partially purified from embryos and larvae at different stages of development by sedimentation through a sucrose density gradient, its activity increases 3-fold during the first 12 h of pre-emergence development. This is followed by a slow decline during subsequent larval development. The similarity of this enzyme to other asymmetrical-pyrophosphohydrolases suggests that it did not evolve specifically to degrade the large yolk platelet store of Gp4G which is found in Artemia embryos, but that it probably serves the same general function in bis(5'-nucleosidyl) oligophosphate metabolism as in other cells.     

Diadenosine tetraphosphate-gating of recombinant pancreatic ATP-sensitive K(+) channels

Diadenosine tetraphosphate (Ap4A) has been recently discovered in the pancreatic cells where targets ATP-sensitive K⁺ (K_ATP) channels, depolarizes the cell membrane and induces insulin secretion. However, whether Ap4A inhibit pancreatic K_ATP channels by targeting protein channel complex itself was unknown. Therefore, we coexpressed pancreatic KATP channel subunits, Kir6.2 and SUR1, in COS-7 cells and examined the effect of Ap4A on the single channel behavior using the inside-out configuration of the patch-clamp technique. Ap4A inhibited channel opening in a concentration-dependent manner. Analysis of single channels demonstrated that Ap4A did not change intraburst kinetic behavior of K_ATP channels, but rather decreased burst duration and increased between-burst duration. It is concluded that Ap4A antagonizes K_ATP channel opening by targeting channel subunits themselves and by keeping channels longer in closed interburst states.     

Selective degradation of 2'-adenylated diadenosine tri- and tetraphosphates, Ap(3)A and Ap(4)A, by two specific human dinucleoside polyphosphate hydrolases

It is known that the interferon-inducible 2',5'-oligoadenylate synthetase can catalyze the 2'-adenylation of various diadenosine polyphosphates. However, catabolism of those 2'-adenylated compounds has not been investigated so far. This study shows that the mono- and bis-adenylated (or mono- and bis-deoxyadenylated) diadenosine triphosphates are not substrates of the human Fhit (fragile histidine triad) protein, which acts as a typical dinucleoside triphosphate hydrolase (EC 3.6.1.29). In contrast, the diadenosine tetraphosphate counterparts are substrates for the human (asymmetrical) Ap(4)A hydrolase (EC 3.6.1.17). The relative rates of the hydrolysis of 0.15 mM AppppA, (2'-pdA)AppppA, and (2'-pdA)AppppA(2"'-pdA) catalyzed by the latter enzyme were determined as 100:232:38, respectively. The asymmetrical substrate was hydrolyzed to ATP + (2'-pdA)AMP (80%) and to (2'-pdA)ATP + AMP (20%). The human Fhit protein, for which Ap(4)A is a poor substrate, did not degrade the 2'-adenylated diadenosine tetraphosphates either. The preference of the interferon-inducible 2'-5' oligoadenylate synthetase to use Ap(3)A over Ap(4)A as a primer for 2'-adenylation and the difference in the recognition of the 2'-adenylated diadenosine triphosphates versus the 2'-adenylated diadenosine tetraphosphates by the dinucleoside polyphosphate hydrolases described here provide a mechanism by which the ratio of the 2'-adenylated forms of the signalling molecules, Ap(3)A and Ap(4)A, could be regulated in vivo.     

Chromate, molybdate, tungstate and vanadate behave as substrates of yeast diadenosine 5',5'-p1,p4-tetraphosphate alpha, beta-phosphorylase

Diadenosine 5',5'-p1,p4-tetraphosphate (Ap4A) alpha, beta-phosphorylase from yeast Saccharomyces cerevisiae catalyzes two reactions: Ap4A cleavage and nucleoside diphosphate--phosphate (NDP-Pi) exchange. In both reactions phosphate can be substituted by arsenate, chromate, molybdate, tungstate or vanadate. In the presence of each anion, nucleoside 5'-monophosphate (NMP) always accumulates as a product of the reaction. This indicates that an unstable NMP anion is formed as an intermediate.     

Synthesis of P1,P4-di(adenosine 5'-) tetraphosphate by leucyl-tRNA synthetase, coupled with ATP regeneration

A simple and practical procedure for the synthesis of P1,P4-di(adenosine 5'-) tetraphosphate from ATP by the catalysis of leucyl-tRNA synthetase from Bacillus stearothermophilus is described. Km for leucine was 6.7 microM and for ATP was 3.3 mM. The reaction yielded not only diadenosine tetraphosphate, but various byproducts such as P1,P3-(diadenosine 5'-) triphosphate, ADP and AMP. By coupling the reaction with an ATP regeneration system by acetate kinase and adenylate kinase with acetylphosphate as a phosphate donor, diadenosine tetraphosphate was prepared as a sole product at a high yield (96%).     

Influence of diadenosine tetraphosphate (Ap4A) on lipid metabolism

Diadenosine polyphosphates (Ap(x)A) are physiologically released and may be partly involved in the pathogenesis of diabetes mellitus. Ap(4)A (diadenosine tetraphosphate) leads to an increase in blood glucose while it decreases insulin levels in plasma. A possible link between Ap(x)A and diabetes mellitus-associated diseases such as insulin resistance and hyperlipidemia (plasma free fatty acids, cholesterol and its biosynthesis, triacylglycerols) has not been investigated yet. Parameters such as free fatty acid and cholesterol content in blood were determined enzymically. The biosynthesis of cholesterol and triacylglycerols was determined in HepG2 cells using the radioactive precursor [(14)C]-acetate and by using gas chromatography. Plasma free fatty acids were significantly decreased 5 and 10 min after an Ap(4)A bolus (0.75 mg kg(-1) b.w.) given to rats. Plasma cholesterol was reduced 5 and 60 min after Ap(4)A administration. LPDS (lipoprotein-deficient serum)-stimulated cholesterol biosynthesis in HepG2 cells was significantly reduced after 1 h incubation with Ap(4)A. Triacylglycerol (TAG) biosynthesis in HepG2 cells was not significantly influenced by Ap(4)A; there was just a tendency for a concentration-dependent decrease in TAG levels. In conclusion Ap(4)A as a diabetogenetic compound is not likely to be responsible for the development of insulin resistance or of hyperlipidemia. Parameters such as free fatty acids, cholesterol and triacylglycerols are not elevated by Ap(4)A, but are even decreased. Ap(4)A seems to be involved in the development of diabetes mellitus by increasing blood glucose and decreasing plasma insulin as shown earlier, but not in diabetes mellitus-associated diseases such as insulin resistance or hyperlipidemia.     

Several dinucleoside polyphosphates are acceptor substrates in the T4 RNA ligase catalyzed reaction.

Several dinucleoside polyphosphates accept cytidine-3', 5'-bisphosphate from the adenylylated donor 5'-adenylylated cytidine 5',3'-bisphosphate in the T4 RNA ligase catalyzed reaction. The 5'-adenylylated cytidine 5',3'-bisphosphate synthesized in a first step, from ATP and cytidine-3',5'-bisphosphate, is used as a substrate to transfer the cytidine-3',5'-bisphosphate residue to the 3'-OH group(s) of diguanosine tetraphosphate (Gp4G) giving rise to Gp4GpCp and pCpGp4GpCp in a ratio of approximately 10 : 1, respectively. The synthesized Gp4GpCp was characterized by treatment with snake venom phosphodiesterase and alkaline phosphatase and analysis (chromatographic position and UV spectra) of the reaction products by HPLC. The apparent Km values measured for Gp4G and 5'-adenylylated cytidine 5',3'-bisphosphate in this reaction were approximately 4 mM and 0.4 mM, respectively. In the presence of 0.5 mM ATP and 0.5 mM cytidine-3',5'-bisphosphate, the relative efficiencies of the following nucleoside(5')oligophospho(5')nucleosides as acceptors of cytidine-3',5'-bisphosphate from 5'-adenylylated cytidine 5', 3'-bisphosphate are indicated in parentheses: Gp4G (100); Gp5G (101); Ap4G (47); Ap4A (39). Gp2G, Gp3G and Xp4X were not substrates of the reaction. Dinucleotides containing two guanines and at least four inner phosphates were the preferred acceptors of cytidine-3', 5'-bisphosphate at their 3'-OH group(s).