Diguanosinetetraphosphatase from rat liver: Acitivity on diadenosine tetraphosphate and inhibition by adenosine tetraphosphate.

The hydrolysis of diadenosine tetraphosphate, a compound previously described by others to occur in liver at concentrations of around 0.1 mu M, is carried out by a specific enzyme. This enzyme has been partially purified from rat liver extracts, and the following properties have been found. The Km value for diadenosine tetraphosphate is 2 mu M; the products of hydrolysis are ATP and AMP; the Km value for diguanosine tetraphosphate is 2 mu M; none of the following substances were substrates of the enzyme: diadenosine triphosphate, diguanosine di and triphosphates, adenosine tetraphosphate, ATP, ADP, NAD+, NADP+ and bis-p-nitrophenylphosphate. Cyclic AMP was not an inhibitor of the reaction. The enzyme requires Mg2+ ions, is maximally active at a pH value of approximately 8, and has a molecular weight of 22000 as estimated by filtration on Sephadex G-100. The activation energy of the reaction was of 10250 cal times mol-1 (42886 J times mol-1). Particularly striking is the inhibition by adenosine tetraphosphate (Ki equals 48 nM) and guanosine tetraphosphate (Ki equals 14 nM). Other nucleotides tested were also competitive inhibitors with Ki values in the 10--100 mu M range.     

Diadenosine tetraphosphate splitting by rat liver extracts

A splitting activity on diadenosine tetraphosphate, a compound reported by others to occur in liver at a concentration of 10^?7 – 10^?8M, has been found in rat liver extracts. One of the products of the cleavage is ATP. A Km of 6 μmolar has been found. This low Km favors the view that the activity here described may act on this substrate $\text{in vivo}$.     

The role of bound thiamine pyrophosphate in the synthesis of thiamine triphosphate in rat liver.

Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with gamma-labelled [32P]ATP, thiamine [32P]triphosphate was formed whereas the incubation of thiamine [32P]pyrophosphate with ATP did not produce thiamine [32P]triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca2+, cyclic GMP and sulfhydryl reagents.     

Purification of tubulin from rat pancreas

Diadenosine triphosphate (Ap₃A) has been identified and quantified in human platelets using a coupled enzymatic assay specific for Ap₃A, after fractionation of acidic extracts with high-performence liquid chromatography. Upon thrombin-induced aggregation, Ap₃A is released together with the homologue diadenosine tetraphosphate (Ap₄A).Extracts of human platelets do also contain enzymatic activities that degrade diadenosine tetraphosphate as well as diadenosine triphosphate. These enzymes, however, are not released during thrombin-induced aggregation of the platelets.     

Location of dinucleoside triphosphatase in the matrix space of rat liver mitochondria.

The submitochondrial location of dinucleoside triphosphatase (EC 3.6.1.29), previously shown to be in part associated with mitochondria, has been studied in rat liver. The precipitability and latency of activity in organelle suspensions, and the profile of solubilization by digitonin, were like those of the matrix space marker glutamate dehydrogenase, and differed from those of other submitochondrial fractions. This, and the synthesis of diadenosine polyphosphates by mitochondrial aminoacyl-tRNA synthetases, suggest the occurrence of a pathway for the intramitochondrial turnover of diadenosine 5',5'-P1,P3-triphosphate (Ap3A).     

Hydrolysis of bis(5''-nucleosidyl) polyphosphates by Escherichia coli 5''-nucleotidase.

Two enzymatic activities that split diadenosine triphosphate have been reported in Escherichia coli: a specific Mg-dependent bis(5'-adenosyl) triphosphatase (EC 3.6.1.29) and the bis(5'-adenosyl) tetraphosphatase (EC 3.6.1.41). In addition to the activities of these two enzymes, a different enzyme activity that hydrolyzes dinucleoside polyphosphates is described. After purification and study of its molecular and kinetic properties, we concluded that it corresponded to the 5'-nucleotidase (EC 3.1.3.5) that has been described in E. coli. The enzyme was purified from sonic extracts and osmotic shock fluid. From sonic extracts, two isoforms were isolated by chromatography on ion-exchange Mono Q columns; they had a molecular mass of about 100 kilodaltons (kDa). From the osmotic shock fluid, a unique form of 52 kDa was recovered. Mild heating transformed the 100-kDa isoform to a 52-kDa form, with an increase in activity of about threefold. The existence of a 5'-nucleotidase inhibitor described previously, which associates with the enzyme and is not liberated in the osmotic shock fluid, may have been responsible for these results. The kinetic properties and substrate specificities of both forms (52 and 100 kDa) were almost identical. The enzyme, which is known to hydrolyze AMP and uridine-(5')-diphospho-(1)-alpha-D-glucose, but not adenosine-(5')-diphospho-(1)-alpha-D-glucose, was also able to split adenosine-(5')-diphospho-(5)-beta-D-ribose, ribose-5-phosphate, and dinucleoside polyphosphates [diadenosine 5',5'-P1,P2-diphosphate,diadenosine 5',5'-P1,P3-triphosphate, diadenosine 5',5'-P1,P4-tetraphosphate, and bis(5'-guanosyl) triphosphate]. The effects of divalent cations and pH on the rate of the reaction with different substrates were studied.     

Presence of diadenosine polyphosphates in microdialysis samples from rat cerebellum in vivo: effect of mild hyperammonemia on their receptors

Diadenosine triphosphate (Ap₃A), diadenosine tetraphosphate (Ap₄A), and diadenosine pentaphosphate (Ap₅A) have been identified in microdialysis samples from the cerebellum of conscious freely moving rats, under basal conditions, by means of a high-performance liquid chromatography method. The occurrence of Ap₃A in the cerebellar microdyalisates is noteworthy, as the presence of this compound in the interstitial medium in neural tissues has not been previously described. The concentrations measured for the diadenosine polyphosphates in the cerebellar dialysate were (in nanomolar) 10.5 ± 2.9, 5.4 ± 1.2, and 5.8 ± 1.3 for Ap₃A, Ap₄A, and Ap₅A, respectively. These concentrations are in the range that allows the activation of the presynaptic dinucleotide receptor in nerve terminals. However, a possible interaction of these dinucleotides with other purinergic receptors cannot be ruled out, as rat cerebellum expresses a variety of P2X or P2Y receptors susceptible to be activated by diadenosine polyphosphates, such as the P2X1-4, P2Y₁, P2Y₂, P2Y₄, and P2Y₁₂ receptors, as demonstrated by quantitative real-time PCR. Also, the ecto-nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP3, able to hydrolyze the diadenosine polyphosphates and terminate their extracellular actions, are expressed in the rat cerebellum. All these evidences contribute to reinforce the role of diadenosine polyphosphates as signaling molecules in the central nervous system. Finally, we have analyzed the possible differences in the concentration of diadenosine polyphosphates in the cerebellar extracellular medium and changes in the expression levels of their receptors and hydrolyzing enzymes in an animal model of moderate hyperammonemia.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-013-9382-3) contains supplementary material, which is available to authorized users.     

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%).     

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.     

Synthesis of biopterin from dihydroneopterin triphosphate by rat tissues

High performance liquid chromatography procedure for the analysis of pterins of biopterin synthesis from dihydroneopterin triphosphate via sepiapterin in rat tissues has been described. Sepiapterin-synthesizing enzyme 1, which catalyzes in the presence of Mg²⁺ the conversion of dihydroneopterin triphosphate to an intermediate designated compound X was assayed by determining pterin which is formed from compound X under acidic conditions. Sepiapterin- and biopterin-synthesizing activity were also assayed by determining sepiapterin and biopterin, respectively. Analytical results revealed the presence of these activities in most rat tissues examined and high levels were found in kidney, pineal gland and liver. Activities were also detectable in peripheral erythrocytes.     

Ectonucleotidases in the kidney

Members of all four families of ectonucleotidases, namely ectonucleoside triphosphate diphosphohydrolases (NTPDases), ectonucleotide pyrophosphatase/phosphodiesterases (NPPs), ecto-5′-nucleotidase and alkaline phosphatases, have been identified in the renal vasculature and/or tubular structures. In rats and mice, NTPDase1, which hydrolyses ATP through to AMP, is prominent throughout most of the renal vasculature and is also present in the thin ascending limb of Henle and medullary collecting duct. NTPDase2 and NTPDase3, which both prefer ATP over ADP as a substrate, are found in most nephron segments beyond the proximal tubule. NPPs catalyse not only the hydrolysis of ATP and ADP, but also of diadenosine polyphosphates. NPP1 has been identified in proximal and distal tubules of the mouse, while NPP3 is expressed in the rat glomerulus and pars recta, but not in more distal segments. Ecto-5′-nucleotidase, which catalyses the conversion of AMP to adenosine, is found in apical membranes of rat proximal convoluted tubule and intercalated cells of the distal nephron, as well as in the peritubular space. Finally, an alkaline phosphatase, which can theoretically catalyse the entire hydrolysis chain from nucleoside triphosphate to nucleoside, has been identified in apical membranes of rat proximal tubules; however, this enzyme exhibits relatively high K _m values for adenine nucleotides. Although information on renal ectonucleotidases is still incomplete, the enzymes’ varied distribution in the vasculature and along the nephron suggests that they can profoundly influence purinoceptor activity through the hydrolysis, and generation, of agonists of the various purinoceptor subtypes. This review provides an update on renal ectonucleotidases and speculates on the functional significance of these enzymes in terms of glomerular and tubular physiology and pathophysiology.     

Effects of diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) on platelet aggregation in unfractionated human blood

The effects on platelet aggregation of diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A), both of which are stored in and released from platelet granules, have been studied in unfractionated human blood using a microscopic platelet-count ratio method. Ap3A at submicromolar concentrations induces platelet aggregation whereas the homologue dinucleotide Ap4A has disaggregating potency. In the concentration range between 10(-7) to 10(-5) M, Ap3A has been found to be as effective as ADP in triggering aggregate formation. These results confirm and essentially extend our recent findings with platelet-rich plasma that Ap3A is able to trigger platelet aggregation by a slow release of ADP from Ap3A which is catalyzed by a plasma hydrolase. Formation of platelet aggregates was also followed kinetically using a turbidometric method which has been developed for this purpose. In contrast to ADP which very rapidly induces a transient state of aggregation, the effect of Ap3A occurs much more slowly but induces the same maximum of aggregation. The duration of the Ap3A stimulus, however, is longer than that of ADP pointing to a potential physiological function of Ap3A as a "masked" source for ADP.     

Cloning, characterisation and crystallisation of a diadenosine 5',5"'-P(1),P(4)-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans.

Asymmetrically cleaving diadenosine 5',5"'-P(1),P(4)-tetraphosphate (Ap4A) hydrolase activity has been detected in extracts of adult Caenorhabditis elegans and the corresponding cDNA amplified and expressed in Escherichia coli. As expected, sequence analysis shows the enzyme to be a member of the Nudix hydrolase family. The purified recombinant enzyme behaves as a typical animal Ap4A hydrolase. It hydrolyses Ap4A with a K(m) of 7 microM and k(cat) of 27 s(-1) producing AMP and ATP as products. It is also active towards other adenosine and diadenosine polyphosphates with four or more phosphate groups, but not diadenosine triphosphate, always generating ATP as one of the products. It is inhibited non-competitively by fluoride (K(i)=25 microM) and competitively by adenosine 5'-tetraphosphate with Ap4A as substrate (K(i)=10 nM). Crystals of diffraction quality with the morphology of rectangular plates were readily obtained and preliminary data collected. These crystals diffract to a minimum d-spacing of 2 A and belong to either space group C222 or C222(1). Phylogenetic analysis of known and putative Ap4A hydrolases of the Nudix family suggests that they fall into two groups comprising plant and Proteobacterial enzymes on the one hand and animal and archaeal enzymes on the other. Complete structural determination of the C. elegans Ap4A hydrolase will help determine the basis of this grouping.     

Identification of diadenosine triphosphate in Brugia malayi by reverse phase high performance liquid chromatography and MALDI mass spectrometry

The presence of diadenosine oligophosphates (ApnA) in eukaryotic pathogens has been difficult technically to assess and thus is often overlooked. ApnA are a family of intercellular and intracellular signaling molecules and their biological activities differ relative to the number of phosphate moieties. The application of mass spectrometry to differentiate nucleotide phosphates has been limited by the high salt content in tissue extracts, enzymatic reactions or high performance liquid chromatography (HPLC) buffers, as well as the potential for sample loss when processing and desalting small biological samples. To address this problem a simple reverse phase HPLC (RP-HPLC) method using volatile organic buffers at low pH was developed to create elution profiles of adenosine and diadenosine phosphates. To test this method on a eukaryotic pathogen, small intravascular human filarial parasites (Brugia malayi) were extracted in phosphate buffered saline and a nucleotide phosphate profile was visualized by RP-HPLC. A major peak eluting at 10.4 min was analyzed directly by mass spectrometry and this confirmed the presence of significant quantities of diadenosine triphosphate, Ap3A. Application of this simplified RP-HPLC method will facilitate research on the normal and pathophysiological effects of ApnA particularly in situations when analysis of small biological samples is required.     

Diadenosine triphosphate (Ap3A) mediates human platelet aggregation by liberation of ADP

Human platelets store considerable amounts of diadenosine 5′, 5′′′-p¹, p³-triphosphate, which is released together with the homologue diadenosine tetraphosphate (Ap₄A) upon thrombin-induced aggregation (Lüthje, J. & Ogilvie, A. (1983) Biochem. Biophys. Res. Commun. 115, 253–260). We now report that, when added to platelet-rich plasma at 10–20 μM, diadenosine triphosphate gradually induces aggregation. The addition of diadenosine tetraphosphate antagonizes this effect by rapidly disaggregating the platelets. When another physiological but structurally unrelated stimulus, i.e. PAF (Platelet activating factor) is introduced into the system, diadenosine triphosphate drastically enhances and prolongs the aggregatory effect of PAF. Again, Ap₄A is antagonistic in this system. The mechanism of Ap₃A-stimulation can be explained by the slow and continuous liberation of ADP from Ap₃A by the action of a hydrolyzing enzyme which is present in human plasma. Our studies suggest that Ap₃A may be physiologically important in providing a relative long-lived stimulus that can modulate platelet aggregation.     

The role of a guanine nucleotide-binding protein in the activation of rat liver plasma-membrane adenylate cyclase by forskolin.

The effects of the photoreactive GTP analogue GTP-gamma-azidoanilide on rat liver plasma-membrane adenylate cyclase are described. U.v. irradiation in the presence of the analogue abolished activation by any effector or combination of effectors that function via the activatory G protein. Partial protection against this inhibition was given by F- and guanosine 5'-[gamma-thio]triphosphate. It is concluded that GTP-gamma-azidoanilide acts by a light-induced covalent reaction with the G protein. In the dark the effects of the analogue were similar to those of GTP. Irradiation in the presence of GTP-gamma-azidoanilide was found to reduce but not to abolish activation of rat liver plasma membrane adenylate cyclase by forskolin. The activation by forskolin and GTP together were greater than the sum of the individual activations. Forskolin doubled adenylate cyclase activity in the presence of glucagon and guanosine 5'-[beta, gamma-imido]triphosphate, which might be expected to activate to the maximum possible extent via the G protein. It is concluded that there are two components to the forskolin activation, a guanine nucleotide-dependent and a guanine nucleotide-independent component.     

Guanosine triphosphate cyclohydrolase activity in rat tissues.

The GTP cyclohydrolase activity of rat tissues has been studied by means of the measurement of formic acid release and neopterin synthesis from GTP. After gel filtration of a 45%-satd.-(NH4)2SO4 fraction of liver homogenates, three enzyme fractions were separated and named A1, A2 and A3 according to the order of their elution. Fractions A1 and A3 displayed an 8-formyl-GTP deformylase activity; no proof of cyclized product has yet been established. This activity was heat-labile and required Mg2+ for maximal activity. Fraction A2 displayed a 'neopterin-synthetase' activity, with dihydroneopterin triphosphate and formic acid formed in stochiometric amounts. Fraction A1 isolated from heat-treated homogenates also produced dihydroneopterin triphosphate. Neopterin synthetase activity in fractions A1 and A2 was heat-resistant and inhibited by Mg2+. In liver the A2 fraction represented 70-75% of the neopterin synthetase capacity and was inhibited by reduced pterines (sepiapterin, dihydrobiopterin and tetrahydrobiopterin) and to a lesser extent by reduced forms of folic acid. In kidney and brain, fraction A1 and A3 GTP 8-formylhydrolase activities were found in significant amounts, in contrast with the neopterin synthetase activity, which was low and appeared to be confined to the A1 fraction.     

Ca<Superscript>2+</Superscript> Signalling in Brain Synaptosomes Activated by Dinucleotides

Diadenosine polyphosphates are a family of dinucleotides formed by two adenosines joined by a variable number of phosphates. Diadenosine tetraphosphate, Ap₄A, diadenosine pentaphosphate Ap₅A, and diadenosine hexaphosphate, Ap₆A, are stored in synaptic vesicles and are released upon nerve terminal depolarization. At the extracellular level, diadenosine polyphosphates can stimulate presynaptic dinucleotide receptors. Responses to diadenosine polyphosphates have been described in isolated synaptic terminals (synaptosomes) from several brain areas in different animal species, including man. Dinucleotide receptors are ligand-operated ion channels that allow the influx of cations into the terminals. These cations reach a threshold for N- and P/Q-type voltage-dependent calcium channels, which become activated. The activation of the dinucleotide receptor together with the activation of these calcium channels triggers the release of neurotransmitters. The ability of Ap₅A to promote glutamate, GABA or acetylcholine release has been recently described by the present authors in rat midbrain synaptosomes.     

In human and rat lung membranes [35S]GTPgammaS binding is a tool for pharmacological characterization of G protein-coupled dinucleotide receptors.

The P2Y receptor family is activated by extracellular nucleotides such as ATP and UTP. P2Y receptors regulate physiological functions in numerous cell types. In lung, the P2Y2 receptor subtype plays a role in controlling Cl- and fluid transport. Besides ATP or UTP, also diadenosine tetraphosphate (Ap4A), a stable nucleotide, seems to be of physiological importance. In membrane preparations from human and rat lung we applied several diadenosine polyphosphates to investigate whether they act as agonists for G protein-coupled receptors. We assessed this by determining the stimulation of [35S]GTPgammaS binding. Stimulation of [35S]GTPgammaS binding to G proteins has already been successfully applied to elucidate agonist binding to various G protein-coupled receptors. Ap(n)A (n = 2 to 6) enhanced [35S]GTPgammaS binding similarly in human and rat lung membranes, an indication of the existence of G protein-coupled receptor binding sites specific for diadenosine polyphosphates. Moreover, in both human and rat lung membranes comparable pharmacological properties were found for a diadenosine polyphosphate ([3H]Ap4A) binding site. The affinity for Ap2A, Ap3A, Ap4A, Ap5A, and Ap6A was also comparable. 8-Diazido-Ap4A and ATP were less potent, whereas the pyrimidine nucleotide UTP showed hardly any affinity. Thus, we present evidence that different diadenosine polyphosphates bind to a common G protein-coupled receptor binding site in membranes derived either from human or rat lung.