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.     

A diadenosine 5',5'-P1P4 tetraphosphate (Ap4A) hydrolase from Arabidopsis thaliana that is activated preferentially by Mn2+ ions

Asymmetrical diadenosine 5',5'-P(1)P(4) tetraphosphate (Ap(4)A) hydrolases are key enzymes controlling the in vivo concentration of Ap(4)A--an important signaling molecule involved in regulation of DNA replication and repair, signaling in stress response and apoptosis. Sequence homologies indicate that the genome of the model plant Arabidopsis thaliana contains at least three open reading frames encoding presumptive Ap(4)A hydrolases: At1g30110, At3g10620, and At5g06340. In this work we present efficient overexpression and detailed biochemical characteristics of the AtNUDX25 protein encoded by the At1g30110 gene. Aided by the determination of the binding constants of Mn(Ap(4)A) and Mg(Ap(4)A) complexes using isothermal titration calorimetry (ITC) we show that AtNUDX25 preferentially hydrolyzes Ap(4)A in the form of a Mn(2+) complex.     

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.     

The g5R (D250) gene of African swine fever virus encodes a Nudix hydrolase that preferentially degrades diphosphoinositol polyphosphates.

The African swine fever virus (ASFV) g5R gene encodes a protein containing a Nudix hydrolase motif which in terms of sequence appears most closely related to the mammalian diadenosine tetraphosphate (Ap4A) hydrolases. However, purified recombinant g5R protein (g5Rp) showed a much wider range of nucleotide substrate specificity compared to eukaryotic Ap4A hydrolases, having highest activity with GTP, followed by adenosine 5'-pentaphosphate (p5A) and dGTP. Diadenosine and diguanosine nucleotides were substrates, but the enzyme showed no activity with cap analogues such as 7mGp3A. In common with eukaryotic diadenosine hexaphosphate (Ap6A) hydrolases, which prefer higher-order polyphosphates as substrates, g5Rp also hydrolyzes the diphosphoinositol polyphosphates PP-InsP5 and [PP]2-InsP4. A comparison of the kinetics of substrate utilization showed that the k(cat)/K(m) ratio for PP-InsP5 is 60-fold higher than that for GTP, which allows classification of g5R as a novel diphosphoinositol polyphosphate phosphohydrolase (DIPP). Unlike mammalian DIPP, g5Rp appeared to preferentially remove the 5-beta-phosphate from both PP-InsP5 and [PP]2-InsP4. ASFV infection led to a reduction in the levels of PP-InsP5, ATP and GTP by ca. 50% at late times postinfection. The measured intracellular concentrations of these compounds were comparable to the respective K(m) values of g5Rp, suggesting that one or all of these may be substrates for g5Rp during ASFV infection. Transfection of ASFV-infected Vero cells with a plasmid encoding epitope-tagged g5Rp suggested localization of this protein in the rough endoplasmic reticulum. These results suggest a possible role for g5Rp in regulating a stage of viral morphogenesis involving diphosphoinositol polyphosphate-mediated membrane trafficking.     

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.     

Regulation of dinucleoside polyphosphate pools by the YgdP and ApaH hydrolases is essential for the ability of Salmonella enterica serovar typhimurium to invade cultured mammalian cells

The ygdP and apaH genes of Salmonella enterica serovar Typhimurium (S. Typhimurium) encode two unrelated dinucleoside polyphosphate (NpnN) hydrolases. For example, YgdP cleaves diadenosine tetraphosphate (Ap4A) producing AMP and ATP, while ApaH cleaves Ap4A producing 2ADP. Disruption of ygdP, apaH individually, and disruption of both genes together reduced intracellular invasion of human HEp-2 epithelial cells by S. Typhimurium by 9-, 250-, and 3000-fold, respectively. Adhesion of the mutants was also greatly reduced compared with the wild type. Invasive capacity of both single mutants was restored by transcomplementation with the ygdP gene, suggesting that loss of invasion was due to increased intracellular NpnN. The normal level of 3 microM adenylated NpnN (ApnN) was increased 1.5-, 3.5-, and 10-fold in the ygdP, apaH and double mutants, respectively. Expression of the putative ptsP virulence gene downstream of ygdP was not affected in the ygdP mutant. Analysis of 19 metabolic enzyme activities and the ability to use a range of carbohydrate carbon sources revealed a number of differences between the mutants and wild type. The increase in intracellular NpnN in the mutants appears to cause changes in gene expression that limit the ability of S. Typhimurium to adhere to and invade mammalian cells.     

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.     

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.     

Fluoride is a strong and specific inhibitor of (asymmetrical) Ap4A hydrolases

Fluoride acts as a noncompetitive, strong inhibitor of (asymmetrical) Ap4A hydrolases (EC 3.6.1.17). The Ki values estimated for the enzymes isolated from seeds of some higher plants (yellow lupin, sunflower and marrow) are in the range of 2-3 microM and I50 for the hydrolase from a mammalian tissue (beef liver) is 20 microM. The anion, up to 25 mM, does not affect the following other enzymes which are able to degrade the bis(5'-nucleosidyl)-oligophosphates: Escherichia coli (symmetrical) Ap4A hydrolase (EC 3.6.1.41), yeast Ap4A phosphorylase (EC 2.7.7.53), yellow lupin Ap3A hydrolase (EC 3.6.1.29) and phosphodiesterase (EC 3.1.4.1). None of halogenic anions but fluoride affects the activity of (asymmetrical) Ap4A hydrolases. Usefulness of the fluoride effect for the in vivo studies on the Ap4A metabolism is shortly discussed.     

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.     

Structure and substrate-binding mechanism of human Ap4A hydrolase

Asymmetric diadenosine 5',5'-P(1),P(4)-tetraphosphate (Ap(4)A) hydrolases play a major role in maintaining homeostasis by cleaving the metabolite diadenosine tetraphosphate (Ap(4)A) back into ATP and AMP. The NMR solution structures of the 17-kDa human asymmetric Ap(4)A hydrolase have been solved in both the presence and absence of the product ATP. The adenine moiety of the nucleotide predominantly binds in a ring stacking arrangement equivalent to that observed in the x-ray structure of the homologue from Caenorhabditis elegans. The binding site is, however, markedly divergent to that observed in the plant/pathogenic bacteria class of enzymes, opening avenues for the exploration of specific therapeutics. Binding of ATP induces substantial conformational and dynamic changes that were not observed in the C. elegans structure. In contrast to the C. elegans homologue, important side chains that play a major role in substrate binding do not have to reorient to accommodate the ligand. This may have important implications in the mechanism of substrate recognition in this class of enzymes.     

Catabolism of bis(5'-nucleosidyl) oligophosphates in Escherichia coli: metal requirements and substrate specificity of homogeneous diadenosine-5',5'-P1,P4-tetraphosphate pyrophosphohydrolase

Diadenosine-5',5'-P1,P4-tetraphosphate pyrophosphohydrolase (diadenosinetetraphosphatase) from Escherichia coli strain EM20031 has been purified 5000-fold from 4 kg of wet cells. It produces 2.4 mg of homogeneous enzyme with a yield of 3.1%. The enzyme activity in the reaction of ADP production from Ap4A is 250 s-1 [37 degrees C, 50 mM tris(hydroxymethyl)aminomethane, pH 7.8, 50 microM Ap4A, 0.5 microM ethylenediaminetetraacetic acid (EDTA), and 50 microM CoCl2]. The enzyme is a single polypeptide chain of Mr 33K, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and high-performance gel permeation chromatography. Dinucleoside polyphosphates are substrates provided they contain more than two phosphates (Ap4A, Ap4G, Ap4C, Gp4G, Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, Ap5A, Ap6A, and dAp4dA are substrates; Ap2A, NAD, and NADP are not). Among the products, a nucleoside diphosphate is always formed. ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, and dTTP are not substrates; Ap4 is. Addition of Co2+ (50 microM) to the reaction buffer containing 0.5 microM EDTA strongly stimulates Ap4A hydrolysis (stimulation 2500-fold). With 50 microM MnCl2, the stimulation is 900-fold. Ca2+, Fe2+, and Mg2+ have no effect. The Km for Ap4A is 22 microM with Co2+ and 12 microM with Mn2+. The added metals have similar effects on the hydrolysis of Ap3A into ADP + AMP. However, in the latter case, the stimulation by Co2+ is small, and the maximum stimulation brought by Mn2+ is 9 times that brought by Co2+. Exposure of the enzyme to Zn2+ (5 microM), prior to the assay or within the reaction mixture containing Co2+, causes a marked inhibition of Ap4A hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diadenosine tetraphosphate binding protein from human HeLa cells: purification and characterization.

The ubiquitous dinucleotide P1,P4-di(adenosine-5') tetraphosphate (Ap4A) has been proposed to be involved in DNA replication and cell proliferation, DNA repair, platelet aggregation, and vascular tonus. A protein binding specifically to Ap4A is associated with a multiprotein form of DNA polymerase alpha (pol alpha 2) in HeLa cells. The Ap4A binding protein from HeLa cells has been purified to homogeneity starting from pol alpha 2 complex. The Ap4A binding protein is hydrophobic and is resolved from the pol alpha 2 complex by hydrophobic interaction chromatography on butyl-Sepharose and subsequently purified to homogeneity by chromatography on Mono-Q and Superose-12 FPLC columns. The Ap4A binding activity elutes as a single symmetrical peak upon gel filtration with a molecular mass of 200 kDa. Upon polyacrylamide gel electrophoresis under nondenaturing conditions, the purified protein migrates as a single protein of 200 kDa. Upon electrophoresis under denaturing conditions, the binding activity is resolved into two polypeptides of 45 and 22 kDa, designated as A1 and A2, respectively. A1 and A2 can be cross-linked using the homobifunctional cross-linking agent disuccinimidyl suberate. The cross-linked protein migrates as a single protein of 210 kDa on polyacrylamide gels under denaturing conditions, suggesting that these two polypeptides are subunits of a single protein. The purified protein binds Ap4A efficiently, and by Scatchard analysis, we have determined a dissociation constant of 0.25 microM, indicating high affinity of Ap4A binding protein to its ligand. ATP is not required for the binding activity. The nonionic detergent Triton X-100 is necessary for stabilizing the purified protein. Amino acid composition analysis indicates that A1 and A2 are distinct.     

Catabolism of bis(5'-nucleosidyl) tetraphosphates in Saccharomyces cerevisiae.

Bis(5'-adenosyl) tetraphosphate (Ap4A) phosphorylase II (P. Plateau, M. Fromant, J. M. Schmitter, J. M. Buhler, and S. Blanquet, J. Bacteriol. 171:6437-6445, 1989) was obtained in a homogeneous form through a 40,000-fold purification, starting from a Saccharomyces cerevisiae strain devoid of Ap4A phosphorylase I activity. The former enzyme behaves as a 36.8K monomer. As with Ap4A phosphorylase I, the addition of divalent cations is required for the expression of activity. Mn2+, Mg2+, and Ca2+ sustain phosphorolysis by the two enzymes, whereas Co2+ and Cd2+ stimulate only phosphorylase II activity. All bis(5'-nucleosidyl) tetraphosphates assayed (Ap4A, Ap4C, Ap4G, Ap4U, Gp4G, and Gp4U) are substrates of the two enzymes. However, Ap4A phosphorylase II shows a marked preference for A-containing substrates. The two enzymes catalyze adenosine 5'-phosphosulfate phosphorolysis or an exchange reaction between Pi and the beta-phosphate of any nucleoside diphosphate. They can also produce Ap4A at the expense of ATP and ADP. The gene (APA2) encoding Ap4A phosphorylase II was isolated and sequenced. The deduced amino acid sequence shares 60% identity with that of Ap4A phosphorylase I. Disruption of APA2 and/or APA1 shows that none of these genes is essential for the viability of Saccharomyces cerevisiae. The concentrations of all bis(5'-nucleosidyl) tetraphosphates are increased in an apa1 apa2 double mutant, as compared with the parental wild-type strain. The factor of increase is 5 to 50 times, depending on the nucleotide. This observation supports the conclusion that, in vivo, Ap4A phosphorylase II, like Ap4A phosphorylase I, participates in the catabolism rather than the synthesis of the bis(5'-nucleosidyl) tetraphosphates.     

Studies on some specific Ap4A-degrading enzymes with the use of various methylene analogues of P1P4-bis-(5'',5''''''-adenosyl) tetraphosphate.

Six new methylenephosphonate analogues of P1P4-bis-(5',5'-adenosyl) tetraphosphate, Ap4A, having P2-P3 carbon bridges CF2, CCl2 and CH2CH2 or P1-P2 and P3-P4 carbon bridges CF2, CCl2 and CH2CH2 in the tetraphosphate chain, were examined as substrates or inhibitors for two specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow-lupin seeds and (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from Escherichia coli. All analogues in which the central oxygen atom was replaced by a stable carbon bridge were hydrolysed by the asymmetrical hydrolase (CF2 greater than CCl2 greater than O greater than CHBr greater than CH2 greater than CH2CH2). As expected, these analogues were not hydrolysed by the symmetrical hydrolase, which was also unable to act on analogues having P1-P2 and P3-P4 carbon bridges.     

Structure and mechanism of MT-ADPRase,a nudix hydrolase from Mycobacterium tuberculosis

Nudix hydrolases are a family of proteins that contain the characteristic sequence GX(5)EX(7)REUXEEXG(I/L/V), the Nudix box. They catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives such as ADP-ribose, Ap(n)A (3 相似文献   

Ectoenzymatic breakdown of diadenosine polyphosphates by Xenopus laevis oocytes.

Xenopus laevis oocytes exhibit ectoenzymatic activity able to hydrolytically cleave extracellular diadenosine polyphosphates (Ap(n)A). The basic properties of this ectoenzyme were investigated using as substrates di-(1,N(6)-ethenoadenosine) 5',5"'-P(1),P(4)-tetraphospate [epsilon-(Ap(4)A)] and di-(1,N(6)-ethenoadenosine) 5',5"'-P(1),P(5)-pentaphospate [epsilon-(Ap(5)A)], fluorogenic derivatives of Ap(4)A and Ap(5)A, respectively. epsilon-(Ap(4)A) and epsilon-(Ap(5)A) are hydrolysed by folliculated oocytes according to hyperbolic kinetics with K(m) values of 13.4 and 12.0 microM and Vmax values of 4.8 and 5.5 pmol per oocyte per min, respectively. The ectoenzyme is activated by Ca(2+) and Mg(2+), reaches maximal activity at pH 8--9 and is inhibited by suramin. Defolliculated oocytes also hydrolyse both substrates with similar K(m) values but V(max) values are approximately doubled with respect to folliculated controls. Chromatographic analysis indicates that extracellular epsilon-(Ap(4)A) and epsilon-(Ap(5)A) are first cleaved into 1,N(6)-ethenoAMP (epsilon-AMP) + 1,N(6)-ethenoATP (epsilon-ATP) and epsilon-AMP + 1,N(6)-ethenoadenosine tetraphosphate (epsilon-Ap(4)), respectively, which are catabolized to 1,N(6)-ethenoadenosine (epsilon-Ado) as the end product by folliculated oocytes. Denuded oocytes, however, show a drastically reduced rate of epsilon-Ado production, epsilon-AMP being the main end-product of extracellular epsilon-(Ap(n)A) catabolism. Results indicate that, whereas the Ap(n)A-cleaving ectoenzyme appears to be located mainly in the oocyte, ectoenzymes involved in the dephosphorylation of mononucleotide moieties are located mainly in the follicular cell layer.     

Dinucleotide polyphosphates contribute to purinergic signalling via inhibition of adenylate kinase activity

Dinucleoside polyphosphates are well described as direct vasoconstrictors and as mediators with strong proliferative properties, however, less is known about their effects on nucleotide-converting pathways. Therefore, the present study investigates the effects of Ap(4)A (diadenosine tetraphosphate), Up(4)A (uridine adenosine tetraphosphate) and Ap(5)A (diadenosine pentaphosphate) and the non-selective P2 antagonist suramin on human serum and endothelial nucleotide-converting enzymes. Human serum and HUVECs (human umbilical vein endothelial cells) were pretreated with various concentrations of dinucleotide polyphosphates and suramin. Adenylate kinase and NDP kinase activities were then quantified radiochemically by TLC analysis of the ATP-induced conversion of [(3)H]AMP and [(3)H]ADP into [(3)H]ADP/ATP and [(3)H]ATP respectively. Endothelial NTPDase (nucleoside triphosphate diphosphohydrolase) activity was additionally determined using [(3)H]ADP and [(3)H]ATP as preferred substrates. Dinucleoside polyphosphates and suramin have an inhibitory effect on the serum adenylate kinase [pIC(50) values (-log IC(50)): Ap(4)A, 4.67+/-0.03; Up(4)A, 3.70+/-0.10; Ap(5)A, 6.31+/-0.03; suramin, 3.74+/-0.07], as well as on endothelial adenylate kinase (pIC(50) values: Ap(4)A, 4.17+/-0.07; Up(4)A, 2.94+/-0.02; Ap(5)A, 5.97+/-0.04; suramin, 4.23+/-0.07), but no significant effects on serum NDP kinase, emphasizing the selectivity of these inhibitors. Furthermore, Ap(4)A, Up(4)A, Ap(5)A and suramin progressively inhibited the rates of [(3)H]ADP (pIC(50) values: Ap(4)A, 3.38+/-0.09; Up(4)A, 2.78+/-0.06; Ap(5)A, 4.42+/-0.11; suramin, 4.10+/-0.07) and [(3)H]ATP (pIC(50) values: Ap(4)A, 3.06+/-0.06; Ap(5)A, 3.05+/-0.12; suramin, 4.14+/-0.05) hydrolyses by cultured HUVECs. Up(4)A has no significant effect on the endothelial NTPDase activity. Although the half-lives for Ap(4)A, Up(4)A and Ap(5)A in serum are comparable with the incubation times of the assays used in the present study, secondary effects of the dinucleotide metabolites are not prominent for these inhibitory effects, since the concentration of metabolites formed are relatively insignificant compared with the 800 mumol/l ATP added as a phosphate donor in the adenylate kinase and NDP kinase assays. This comparative competitive study suggests that Ap(4)A and Ap(5)A contribute to the purinergic responses via inhibition of adenylate-kinase-mediated conversion of endogenous ADP, whereas Up(4)A most likely mediates its vasoregulatory effects via direct binding-mediated mechanisms.     

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.