Identification and characterization of diadenosine 5',5"'-P1,P2 -diphosphate and diadenosine 5',5"'-P1,P3-triphosphate in human myocardial tissue.

We examined whether human cardiac tissue contains diadenosine polyphosphates and investigated their physiological role. Extracts from human cardiac tissue from transplant recipients were fractionated by size exclusion-, affinity-, anion exchange- and reversed-phase chromatography. MALDI-MS analysis of two absorbing fractions revealed molecular masses of 676.2 Da and 756.0 Da. The UV spectra of both fractions were identical to that of adenosine. Postsource decay MALDI mass spectrometry indicated that the molecules with a mass of 676.2 Da and 757.0 Da contained AMP and ATP, respectively. As shown by enzymatic cleavage, both molecules consist of two adenosines interconnected by either two or three phosphates in 5'-positions of the riboses. Two substances can be identified as 5',5"'-P1,P2-diphosphate (Ap2A) and 5',5"'-P1, P3-triphosphate (Ap3A). Ap2A and Ap3A, together with ATP and ADP, are stored in myocardial-specific granules in biologically active concentrations. In the isolated perfused rat heart, Ap2A and Ap3A caused dose-dependent coronary vasodilations. In myocardial preparations, Ap2A and Ap3A attenuated the effect of isoproterenol, exerting a negative inotropic effect. The calcium current of guinea pig ventricular myocytes, stimulated by isoproterenol, was also attenuated by Ap2A and Ap3A. The presence of Ap2A and Ap3A in cardiac-specific granules and the actions of these substances on the myocardium and coronary vessels indicate a role for these substances as endogenous modulators of myocardial functions and coronary perfusion.     

Synthesis and applications of 8-azido photoaffinity analogs of P1,P3-bis(5'-adenosyl)triphosphate and P1,P4-bis(5'-adenosyl)tetraphosphate

32P-labeled photoaffinity analogs of bis(5'-adenosyl)-tetraphosphate and bis(5'-adenosyl)triphosphate which contain a single photoreactive 8-azidoadenosine group distal to the radiolabel have been synthesized from commercially available components using a combination of chemical and enzymatic procedures including a water-soluble carbodiimide. The method is simple, rapid, and produces yields of high specific activity products of around 60%. The analog of bis(5'-adenosyl)-tetraphosphate is very similar to the parent compound in its inhibition of rat liver adenosine kinase and its efficiency as a substrate for the bis(5'-nucleosidyl)tetraphosphate pyrophosphohydrolase from Artemia embryos. In the latter case, ATP and 8-azidoAMP are the preferred products. As would be expected, this analog is a much more effective photoprobe for both adenosine and adenylate kinases than the corresponding analog of bis(5'-adenosyl)triphosphate. Both compounds have been used to photoaffinity label crude extracts of Artemia, Vero cells, and Clostridium acetobutylicum and preferential specific labeling of different polypeptides by each analog has been shown. In extracts of C. acetobutylicum, the labeling of a polypeptide of Mr 48,500 by the bis(5'-adenosyl)tetraphosphate analog was totally dependent on the presence of Co2+ ions. These compounds should therefore prove valuable both for the active site labeling of purified binding proteins and for the detection and identification of new target proteins for these nucleotides.     

Characterization of the interaction of P1,P4-diadenosine 5'-tetraphosphate with luciferase

Adenylated dinucleotides (Ap(n)A) are regulatory molecules that control various cellular processes. A very likely intracellular target for Ap(4)A are enzymes that require ATP as either substrate or modulator. We report the results of new biochemical studies aimed at characterizing the Ap(4)A interaction with firefly luciferase, by using the luminometric and thin layer chromatography techniques. The data presented herein demonstrate that Ap(4)A is a noncompetitive inhibitor for the ATP-induced luminescence. These results together with our previous findings that Ap(4)A is a luciferase substrate [Nucleosides Nucleotides Nucleic Acids 23 (2004) in press.] support the notion that, similar to its interaction with P(2) receptors, Ap(4)A also has a dual interaction with luciferase. Other Ap(n)As (n = 2, 5, and 6) also inhibited the ATP-luciferase interaction. Since Ap(n)As may have similar interactions with other intracellular ATP-requiring enzymes, the study presented herein validates ulterior investigations of the Ap(n)A interaction with such enzymes, and opens the way to a better understanding of their intracellular roles.     

Distribution of immunoreactivity for P2X3, P2X5, and P2X6-purinoceptors in mouse retina

Previous findings have shown that P2X-purinoceptor-mediated signaling pathways regulate the release of ACh in the retina. We previously reported the existence of immunoreactivity for P2X1-, P2X2-, P2X4-, and P2X7-purinoceptors in mouse retina and speculated that P2X2 and P2X7-purinoceptors may modulate the activity of cholinergic amacrine cells. In the present study, we used an immunohistochemical technique to examine whether P2X3-, P2X5, and P2X6-purinoceptors are also important for the modulation of cholinergic amacrine cells in mouse retina. Immunoreactivity for P2X3-, P2X5-, and P2X6-purinoceptors was observed in mouse retina. Immunoreactivity for P2X3- purinoceptors was observed in the dendrites of cholinergic amacrine cells. Immunoreactivity for P2X5-purinoceptors existed in the soma of cholinergic amacrine cells. P2X6-purinoceptor immunoreactivity was not colocalized with the cholinergic amacrine cells. We concluded that, among the three P2X-purinoceptors that were examined, P2X3-purinoceptors seem to affect the function of cholinergic amacrine cells in the mouse retina.     

Characterization of P1,P4-diadenosine 5'-tetraphosphate binding on bovine aortic endothelial cells

In recent years it has become increasingly clear that alpha, omega-dinucleotides act as extracellular modulators of various biological processes. P1,P4-diadenosine 5'-tetraphosphate (Ap4A) is the best characterized alpha,omega-dinucleotides and acts as an extracellular signal molecule by inducing the release of nitric oxide (NO) from bovine aortic endothelial cells (BAEC) (R. H. Hilderman, and E. F. Christensen (1998) FEBS Lett. 407, 320-324). However, the characteristics of Ap4A binding to endothelial cells have not been determined. In this report we demonstrate that Ap4A binds to a heterogeneous population of receptors on BAEC. Competition ligand-binding studies using various adenosine dinucleotides, guanosine dinucleotides, adenosine/guanosine dinucleotides, and synthetic P2 purinoceptor agonists and antagonists demonstrate that Ap4A binds to a receptor on BAEC that has a high affinity for some of the adenosine dinucleotides. The apparent IC50 values for Ap4A, Ap2A, and Ap3A are between 12 and 15 microM, while the apparent IC50 values for Ap5A and Ap6A are greater than 500 microM. Evidence is also presented which suggests that this receptor can be classified as a putative P4 purinoceptor. Competition studies also demonstrate that Ap4A binds at a lower affinity to a second class of binding sites.     

Immunoaffinity chromatography of diadenosine 5',5'-P1,P4-tetraphosphate phosphorylase from Saccharomyces cerevisiae

Diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) phosphorylase has been isolated previously using classical protein isolation techniques [A. Guranowski and S. Blanquet (1985) J. Biol. Chem. 260, 3542-3547]. A protein A-Sepharose immunoaffinity column was prepared to simplify the purification procedure. The immunoaffinity column was prepared using specific polyclonal antibodies to Ap4A phosphorylase covalently coupled to protein A-Sepharose with dimethyl pimelimidate by a modification of the procedure of C. Schneider et al. [(1982) J. Biol. Chem. 257, 10,766-10,769]. The specific activity of the immunoaffinity-purified enzyme showed an increase equivalent to the specific activity obtained by chromatography on DEAE-cellulose and hydroxyapatite columns.     

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.     

Specific magnesium-dependent diadenosine 5'',5''''''-P1,P3-triphosphate pyrophosphohydrolase in Escherichia coli.

A specific Mg2+-dependent bis(5'-adenosyl)-triphosphatase (EC 3.6.1.29) was purified 270-fold from Escherichia coli. The enzyme had a strict requirement for Mg2+. Other divalent cations, such as Mn2+, Ca2+, or Co2+, were not effective. The products of the reaction with bis(5'-adenosyl) triphosphate (Ap3A) as the substrate were ADP and AMP in stoichiometric amounts. The Km for Ap3A was 12 +/- 5 microM. Bis(5'-adenosyl) di-, tetra-, and pentaphosphates, NAD+, ATP, ADP, AMP, glucose 6-phosphate, p-nitrophenylphosphate, bis-p-nitrophenylphospate, and deoxyribosylthymine-5'-(4-nitrophenylphosphate) were not substrates of the reaction. The enzyme had a molecular mass of 36 kilodaltons (as determined both by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis), an isoelectric point of 4.84 +/- 0.05, and a pH optimum of 8.2 to 8.5. Zn2+, a known potent inhibitor of rat liver bis(5'-adenosyl)-triphosphatase and bis(5'-guanosyl)-tetraphosphatase (EC 3.6 1.17), was without effect. The enzyme differs from the E. coli diadenosine 5',5'-P1, P4-tetraphosphate pyrophosphohydrolase which, in the presence of Mn2+, also hydrolyzes Ap3A.     

Diadenosine 5'',5''"-P1,P4-tetraphosphate in developing embryos of Artemia

Diadenosine 5',5'"-P1,P4-tetraphosphate (Ap4A) has been detected in cysts and developing embryos of the brine shrimp Artemia in amounts 10(4)-10(6) times lower than that of the guanine analogue, Gp4G. The unexpectedly high level of Ap4A in dormant cysts of 2.37 pmol/10(6) cells can be reduced to 0.03 pmol/10(6) cells by decapsulation and storage in saturated NaCl. When development is reinitiated, the Ap4A content of the decapsulated embryos undergoes a rapid 125 -fold increase, reaching a maximum of 3.79 pmol/10(6) cells at the point of emergence when DNA replication begins. If replication is delayed by hypoxia, the Ap4A level is adjusted in order to reach the same maximum value when replication finally begins. As replication proceeds, the level of Ap4A declines again. Unlike mammalian cells, Ap4A in Artemia is less metabolically labile than ATP. These results are consistent with the suggested role of Ap4A in the initiation of DNA synthesis.     

Pig lung 5'-nucleotidase: effect of diadenosine 5',5'-P1, P4-tetraphosphate and its related compounds

1. A 5'-nucleotidase was purified from pig lung to apparent homogeneity. 2. Its kinetic properties were similar to those of the previously reported cytoplasmic 5'-nucleotidase, which preferentially hydrolyses IMP and GMP. 3. It was a tetramer composed of 69 kDa subunit. 4. It was effectively stimulated by diadenosine tetraphosphate and glycerate 2,3-bisphosphate.     

Identification of a unique membrane receptor for adenosine 5',5"'-P1,P4-tetraphosphate

Adenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) has been implicated as a modulator of cell stress. We have performed binding studies which indicate that membranes from all tissues tested bind tritium-labeled Ap4A. The characteristics of Ap4A binding were determined on brain membrane homogenates after development of an optimized in vitro filter-binding assay. Ap4A binding is specific for adenylated dinucleotides and for the length of the phosphate bridge. A Kd of 0.71 microM for Ap4A was determined.     

Yeast diadenosine 5',5'-P1,P4-tetraphosphate alpha,beta-phosphorylase behaves as a dinucleoside tetraphosphate synthetase

The diadenosine 5',5'-P1,P4-tetraphosphate alpha,beta-phosphorylase (Ap4A phosphorylase), recently observed in yeast [Guaranowski, A., & Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547], is shown to be capable of catalyzing the synthesis of Ap4A from ATP + ADP, i.e., the reverse reaction of the phosphorolysis of Ap4A. The synthesis of Ap4A markedly depends on the presence of a divalent cation (Ca2+, Mn2+, or Mg2+). In vitro, the equilibrium constant K = ([Ap4A][Pi])/[(ATP][ADP]) is very sensitive to pH. Ap4A synthesis is favored at low pH, in agreement with the consumption of one to two protons when ATP + ADP are converted into Ap4A and phosphate. Optimal activity is found at pH 5.9. At pH 7.0 and in the presence of Ca2+, the Vm for Ap4A synthesis is 7.4 s-1 (37 degrees C). Ap4A phosphorylase is, therefore, a valuable candidate for the production of Ap4A in vivo. Ap4A phosphorylase is also capable of producing various Np4N' molecules from NTP and N'DP. The NTP site is specific for purine ribonucleotides (N = A, G), whereas the N'DP site has a broader specificity (N' = A, C, G, U, dA). This finding suggests that the Gp4N' nucleotides, as well as the Ap4N' ones, could occur in yeast cells.     

Catabolism of diadenosine 5',5"'-P1,P4-tetraphosphate in procaryotes. Purification and properties of diadenosine 5',5"'-P1,P4-tetraphosphate (symmetrical) pyrophosphohydrolase from Escherichia coli K12

Enzymatic activity which hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) yielding ADP has been identified in extracts of eubacteria, Escherichia coli and Acidaminococcus fermentans, and of a highly thermophilic archaebacterium, Pyrodictum occultum. Specific Ap4A (symmetric) pyrophosphohydrolase from Escherichia coli K12 has been purified almost 400-fold. The preparation was free of phosphatase, ATPase, phosphodiesterase, AMP-nucleosidase, and adenylate kinase. The Ap4A pyrophosphohydrolase molecular weight estimated by gel filtration is 27,000 +/- 1,000. Activity maximum is at pH 8.3. The Km value computed for Ap4A is 25 +/- 3 microM. The sulfhydryl group(s) is essential for enzyme activity. Metal chelators, EDTA, and o-phenanthroline, inhibit Ap4A hydrolysis; I0.5 values are 3 and 50 microM, respectively. Co2+ is a strong stimulator with an almost 100-fold increase in rate of Ap4A hydrolysis and a plateau in the range of 100-500 microM Co2+, when compared with the nonstimulated hydrolysis. Other transition metal ions, Mn2+, Cd2+, and Ni2+, stimulate by factors of 8, 3.5, and 3.5, respectively, with optimal concentrations in the range 200-500, 2-5, and 4-8 microM, respectively. Zn2+, Cu2+, and Fe2+, up to 30 microM, are without effect and they inhibit at higher concentrations. Mg2+ or Ca2+, in the absence of other divalent metal ions, are weak stimulators (1.5-fold stimulation occurs at 1-2 mM concentration), but act synergistically with Co2+ at its suboptimal concentrations. Stimulation in the presence of 10 microM Co2+ and either 1 mM MgCl2 or CaCl2 increases up to 75-fold. The same degree of synergy is found at 10 microM Co2+ and either 2-5 mM spermidine or 0.5-1.5 mM spermine. Besides Ap4A, bacterial Ap4A pyrophosphohydrolase hydrolyzes effectively Ap5A and Gp4G, and, to some extent, p4A, Ap6A, and Ap3A yielding in each case corresponding nucleoside diphosphate as one of the products.