Adenosine(5')tetraphospho(5')adenosine-binding protein of calf thymus

An adenosine(5')tetraphospho(5')adenosine (Ap4A) binding protein has been purified from calf thymus. The protein is comprised of a single polypeptide of Mr 54000 and is capable of high-affinity (Kd = 13 microM) binding of Ap4A with great substrate specificity. The Ap4A binding protein has been isolated in two forms: a 'free', or non-polymerase-bound, form which predominates, and a similar form which copurifies with DNA polymerase alpha, but which can be resolved from it. The free form of Ap4A binding protein contains associated adenosine(5')tetraphospho(5')adenosine phosphohydrolase (Ap4Aase) activity, while the form resolved from DNA polymerase alpha contains no such activity. The Ap4Aase activity, which catalyzes the phosphohydrolysis of Ap4A to ATP and AMP, is strongly inhibited by low levels (50-100 microM) of Zn2+ without any effect on the Ap4A binding protein activity. This difference in associated Ap4Aase activity between free and polymerase-bound forms of the protein, plus the copurification mentioned above, indicate a specific association between Ap4A binding protein and DNA polymerase alpha.     

Zinc as a second messenger of mitogenic induction. Effects on diadenosine tetraphosphate (Ap4A) and DNA synthesis

DNA synthesis and adenosine(5')tetraphosphate(5')adenosine (Ap4A) levels decrease in cells treated with EDTA. The inhibitory effect of EDTA can be reversed with micromolar amounts of ZnCl2. ZnCl2 in micromolar concentrations also inhibits Ap4A hydrolase and stimulates amino acid-dependent Ap4A synthesis, suggesting that Zn2+ is modulating intracellular Ap4A pools. Serum addition to G1-arrested cells enhances uptake of Zn, whereas serum depletion leads to a fivefold decrease of the rates of zinc uptake. These results are discussed by regarding Zn2+ as a putative 'second messenger' of mitogenic induction and Ap4A as a possible 'third messenger' and trigger of DNA synthesis.     

Isothermal titration calorimetry reveals a zinc ion as an atomic switch in the diadenosine polyphosphates

Diadenosine polyphosphates (diadenosine 5',5'-P(1),P(n)-polyphosphate (Ap(n)A)) are 5'-5'-phosphate-bridged dinucleosides that have been proposed to act as signaling molecules in a variety of biological systems. Isothermal titration calorimetry was used to measure the affinities of a variety of metal cations for ATP, diadenosine 5',5'-P(1),P(3)-triphosphate (Ap(3)A), diadenosine 5',5'-P(1),P(4)-tetraphosphate (Ap(4)A), and diadenosine 5',5'-P(1),P(5)-pentaphosphate (Ap(5)A). The binding of Mg(2+), Ca(2+), and Mn(2+) to ATP is shown to take place with the beta,gamma-phosphates (primary site) and be endothermic in character. The binding of Ni(2+), Cd(2+), and Zn(2+) to ATP is found to take place at both the primary site and at a secondary site identified as N-7 of the adenine ring. Binding to this second site is exothermic in character. Generally, the binding of metal cations to diadenosine polyphosphates involves a similar primary site to ATP. No exothermic binding events are identified. Critically, the binding of Zn(2+) to diadenosine polyphosphates proves to be exceptional. This appears to involve a very high affinity association involving the N-7 atoms of both adenine rings in each Ap(n)A, as well as the more usual endothermic association with the phosphate chain. The high affinity association is also endothermic in character. A combination of NMR and CD evidence is provided in support of the calorimetry data demonstrating chemical shift changes and base stacking disruptions entirely consistent with N-7 bridging interactions. N-7 bridging interactions are entirely reversible, as demonstrated by EDTA titration. Considering the effects of Zn(2+) on a wide variety of dinucleoside polyphosphate-metabolizing enzymes, we examine the possibility of Zn(2+) acting as an atomic switch to control the biological function of the diadenosine polyphosphates.     

Purification, immunological and biochemical characterization of Ap4A binding protein from Xenopus laevis oocytes.

Diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) binding protein specifically binds Ap4A. The protein has been purified from Xenopus laevis oocytes and presents an estimated molecular weight of 100,000 by gel filtration. In the first stages of the purification, the Ap4A binding activity is found associated to DNA polymerase alpha-DNA primase, forming heterogeneous high molecular weight complexes. A monoclonal antibody has been prepared against the purified Ap4A binding protein. The antibody partially neutralizes the Ap4A binding activity. Using the immunoblot technique, it has been shown that the antibody is able to recognize either native or SDS-denatured Ap4A binding protein. The monoclonal antibody immunoreacted with a polypeptide of 90,000 which coincides with the molecular weight obtained by gel chromatography and indicates that the native Ap4A binding protein from Xenopus oocytes is probably a monomeric protein.     

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.     

Metal requirements of a diadenosine pyrophosphatase from Bartonella bacilliformis: magnetic resonance and kinetic studies of the role of Mn2+

Recombinant IalA protein from Bartonella bacilliformis is a monomeric adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various divalent cations were tested over a 300-fold concentration range, Mg2+, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not. Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound Mn2+ (epsilon b) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/- 1.3 mM) and one on Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex.     

Crystal structures of Salmonella typhimurium propionate kinase and its complex with Ap4A: evidence for a novel Ap4A synthetic activity

Propionate kinase catalyses the last step in the anaerobic breakdown of L-threonine to propionate in which propionyl phosphate and ADP are converted to propionate and ATP. Here we report the structures of propionate kinase (TdcD) in the native form as well as in complex with diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) by X-ray crystallography. Structure of TdcD obtained after cocrystallization with ATP showed Ap4A bound to the active site pocket suggesting the presence of Ap4A synthetic activity in TdcD. Binding of Ap4A to the enzyme was confirmed by the structure determination of a TdcD-Ap4A complex obtained after cocrystallization of TdcD with commercially available Ap4A. Mass spectroscopic studies provided further evidence for the formation of Ap4A by propionate kinase in the presence of ATP. In the TdcD-Ap4A complex structure, Ap4A is present in an extended conformation with one adenosine moiety present in the nucleotide binding site and other in the proposed propionate binding site. These observations tend to support direct in-line transfer of phosphoryl group during the kinase reaction.     

Identification and partial characterization of an adenosine(5'')tetraphospho(5'')adenosine hydrolase on intact bovine aortic endothelial cells.

The biologically active dinucleotides adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')-triphospho(5')adenosine (Ap3A), which are both releasable into the circulation from storage pools in thrombocytes, are catabolized by intact bovine aortic endothelial cells. 1. Compared with extracellular ATP and ADP, which are very rapidly hydrolysed, the degradation of Ap4A and Ap3A by endothelial ectohydrolases is relatively slow, resulting in a much longer half-life on the endothelial surface of the blood vessel. The products of hydrolysis are further degraded and finally taken up as adenosine. 2. Ap4A hydrolase has high affinity for its substrate (Km 10 microM). 3. ATP as well as AMP transiently accumulates in the extracellular fluid, suggesting an asymmetric split of Ap4A by the ectoenzyme. 4. Mg2+ or Mn2+ at millimolar concentration are needed for maximal activity; Zn2+ and Ca2+ are inhibitory. 5. The hydrolysis of Ap4A is retarded by other nucleotides, such as ATP and Ap3A, which are released from platelets simultaneously with Ap4A.     

Diadenosine phosphates and S-adenosylmethionine: novel boron binding biomolecules detected by capillary electrophoresis.

There is evidence that boron has a physiological role in animals and humans, but the search for boron binding biomolecules has been difficult because useful radioactive boron isotopes do not exist. To overcome this limitation we used capillary electrophoresis to identify and quantify boron binding to biomolecules by detecting the negative charge boron imparts to ligands. The effect of molecular structure and proximal electronic charges of adenosine and molecules with adenosine moieties including S-adenosylmethionine (SAM) and diadenosine polyphosphates (Ap(n)A) were compared. The boron affinity of the test species varied with the rank order SAM congruent with Ap(6)A congruent with Ap(5)A>Ap(4)A>Ap(3)A congruent with NAD(+)>Ap(2)A>NADH congruent with 5'ATP>5'ADP>5'AMP>adenosine>3'AMP congruent with 2'AMP congruent with cAMP congruent with adenine. Test species with vicinal cis-diols bound boron; species without those moieties did not. Boron binding affinity increased when proximal cationic moieties were present. Anionic moieties remote from the cis-hydroxyl binding site also positively influenced boron binding affinity. In the Ap(n)A species, cooperative complexing of boron between the terminal ribose moieties apparently occurred. In these species boron affinity greater than expected for two monocomplexes was observed and binding affinities increased as more phosphate groups (beyond three) were present separating the terminal moieties. Our results indicate that Ap(6)A, Ap(5)A, Ap(4)A, Ap(3)A, and SAM have higher affinities for boron than any other currently recognized boron ligand present in animal tissues including NAD(+).     

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.     

Isolation and identification of diadenosine 5',5'-P1,P4-tetraphosphate binding proteins using magnetic bio-panning

We report the development of a synthetic, biotin-conjugated diadenosine tetraphosphate (Ap(4)A)-'molecular hook' attached to magnetic beads enabling the isolation of Ap(4)A-binding proteins from bacterial cells or mammalian tissue lysates. Characterisation and identification of isolated binding proteins is performed sequentially by mass spectrometry. The observation of positive controls suggests that these newly observed proteins are putative Ap(4)A-binding partners, and we have expectations that others can be found with further technical improvements in our methods.     

Ap4A induces apoptosis in human cultured cells.

Diadenosine oligophosphates (Ap(n)A) have been proposed as intracellular and extracellular signaling molecules in animal cells. The ratio of diadenosine 5',5'-P1,P3-triphosphate to diadenosine 5',5'-P1,P4-tetraphosphate (Ap3A/Ap4A) is sensitive to the cellular status and alters when cultured cells undergo differentiation or are treated with interferons. In cells undergoing apoptosis induced by DNA topoisomerase II inhibitor VP16, the concentration of Ap3A decreases significantly while that of Ap4A increases. Here, we have examined the effects of exogenously added Ap3A and Ap4A on apoptosis and morphological differentiation. Penetration of Ap(n)A into cells was achieved by cold shock. Ap4A at 10 microM induced programmed cell death in human HL60, U937 and Jurkat cells and mouse VMRO cells and this effect appeared to require Ap4A breakdown as hydrolysis-resistant analogues of Ap4A were inactive. On its own, Ap3A induced neither apoptosis nor cell differentiation but did display strong synergism with the protein kinase C activators 12-deoxyphorbol-13-O-phenylacetate and 12-deoxyphorbol-13-O-phenylacetate-20-acetate in inducing differentiation of HL60 cells. We propose that Ap4A and Ap3A are physiological antagonists in determination of the cellular status: Ap4A induces apoptosis whereas Ap3A is a co-inductor of differentiation. In both cases, the mechanism of signal transduction remains unknown.     

Binding and internalization of p1,p4-diadenosine 5'-tetraphosphate by bovine aortic endothelial cells.

p1,p4-Diadenosine 5'-tetraphosphate (Ap4A) has been implicated as a modulator of blood vessel tone. We have recently demonstrated that the infusion of Ap4A into swine induces vasodilation (Hilderman et al., Am. J. Hypertension 10 (1997) 94A) and that Ap4A induces the release of nitric oxide (NO) from bovine aortic endothelial cells (BAEC) (Hilderman and Christensen, FEBS Lett. 427 (1998) 320-324). However, the interaction of Ap4A with endothelial cells is incompletely understood. Therefore, we determined the characteristics of [3H]-Ap4A binding to BAEC in normal and ATP-depleted cells. These binding studies demonstrate that the interaction of Ap4A with BAEC involves two distinct steps: an ATP independent step and a second ATP dependent step leading to internalization of Ap4A. The initial interaction of Ap4A with BAEC is not affected by either EGTA or iodoacetate; however, both agents block the second step. These data suggest that calcium ions and sulfhydryl groups are required for Ap4A internalization but not for an initial binding event.     

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.     

Diadenosine oligophosphates: peculiarities of synthesis by phenylalanyl-tRNA synthetases from E. coli MRE-600 and Thermus thermophilus HB8.

Temperature and other factors affecting synthesis of bis(5'-adenosyl) tetraphosphate (Ap4A) and bis(5'-adenosyl)triphosphate (Ap3A) catalyzed by phenylalanyl-tRNA synthetases (PheRSs) from Escherichia coli MRE-600 and Thermus thermophilus HB8 have been investigated. Those two synthetases exhibited different temperature-dependent rates of the Ap4A and Ap3A synthesis. However, with respect to the effects of such effectors of the Ap4A synthesis as Zn2+, Mg2+, tRNA and Ap4A phosphonate analogues, as well as some inhibitors of aminoacyl-tRNA synthetase, those two enzymes were apparently undistinguishable.     

In vitro competition between adenosine(5')tetraphospho(5')adenosine and deoxyribonucleic acid in the reaction with diamminedichloroplatinum(II)

Competition between adenosine(5')tetraphospho(5')adenosine (Ap4A) and DNA for the synthesis of adducts with the cis or trans isomer of diamminedichloroplatinum(II) was measured in the presence and absence of magnesium and spermidinium ions. Reaction products were analysed by circular dichroism, poly(ethyleneimine) thin-layer chromatography and reversed-phase chromatography. Competition was affected by the oligovalent cations that bound specifically to the dinucleotide. Platination of DNA was favoured under all conditions. Chromatin was less competitive. The mechanism was kinetic competition, DNA reacting considerably faster than Ap4A. Platinum(II) did not exchange between adducts and free DNA and Ap4A, respectively. On that basis only low amounts of Ap4A adducts were estimated to be formed under conditions of clinical chemotherapy. The cis and trans isomers of diamminedichloroplatinum(II) were equally effective. Platinum(II) adducts of Ap4A were neither degraded by Ap4A-specific pyrophosphohydrolases nor by phosphodiesterase nor in the presence of unfractionated extract of calf thymus. Unphysiologically high concentrations of Crotalus durissus phosphodiesterase I were required for hydrolytic splitting, the amount of which was similar for both platinum(II) isomer adducts. The results suggest that Ap4A platinum(II) adducts might accumulate during chemotherapy of cancer treatment.     

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.     

Influence of the aminoacyl-tRNA synthetase inhibitors and the diadenosine-5'-tetraphosphate phosphonate analogues on the catalysis of diadenosyl oligophosphates formation

Well-known aminoacyl-tRNA synthetase (ARSase) inhibitors, namely the analogues of amino acids and aminoacyl adenylates (aminoalkyl- and aminophosphonyl adenylates with Ki congruent to 0.1 microM) as well as the diadenosine 5',5'-p1,p4-tetraphosphate (Ap4A) phosphonoanalogues, were for the first time used for the Ap4A biosynthesis regulation. Effects of a set of such compounds on lysyl-, phenylalanyl- and alanyl-tRNA synthetases from E. coli, capable of synthesizing Ap4A in the presence of Zn2+ ions and pyrophosphatase, have been studied. The adenylate analogues were found to inhibit the Ap4A and Ap3A formation (I50 congruent to 6 mM). Aminophosphonic and aminophosphonous acids are not involved in Ap3A and Ap4A biosynthesis and inhibited it at high concentrations. The Ap4A phosphoanalogues slightly inhibited the major reactions of ARSases, as well as the biosynthesis of Ap3A and Ap4A, at a concentration of 5 mM.     

Investigation into the interactions between diadenosine 5',5'-P1,P4-tetraphosphate and two proteins: molecular chaperone GroEL and cAMP receptor protein

Diadenosine 5',5'-P(1),P(4)-tetraphosphate (Ap(4)A) is a dinucleoside polyphosphate found ubiquitously in eukaryotic and prokaryotic cells. Despite Ap(4)A being universal, its functions have proved to be difficult to define, although they appear to have a strong presence during cellular stress. Here we report on our investigations into the nature and properties of putative Ap(4)A interactions with Escherichia coli molecular chaperone GroEL and cAMP receptor protein (CRP). We confirm previous literature observations that GroEL is an Ap(4)A binding protein and go on to prove that binding of Ap(4)A to GroEL involves a set of binding sites (one per monomer) distinct from the well-known GroEL ATP/ADP sites. Binding of Ap(4)A to GroEL appears to enhance ATPase rates at higher temperatures, encourages the release of bound ADP, and may promote substrate protein release through differential destabilization of the substrate protein-GroEL complex. We suggest that such effects should result in enhanced GroEL/GroES chaperoning activities that could be a primary reason for the improved yields of the refolded substrate protein observed during GroEL/GroES-assisted folding and refolding at >or=30 degrees C in the presence of Ap(4)A. In contrast, we were unable to obtain any data to support a direct role for Ap(4)A interactions with CRP.     

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.