Diadenosine polyphosphates (Ap n As) act as extracellular signaling molecules in a broad variety of tissues. They were shown to be hydrolyzed by surface-located enzymes in an asymmetric manner, generating AMP and Ap n-1 from Ap n A. The molecular identity of the enzymes responsible remains unclear. We analyzed the potential of NPP1, NPP2, and NPP3, the three members of the ecto-nucleotide pyrophosphatase/phosphodiesterase family, to hydrolyze the diadenosine polyphosphates 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), and the diguanosine polyphosphate, diguanosine 5¢,5¢¢¢-P 1 ,P 4 -tetraphosphate (Gp 4 G). Each of the three enzymes hydrolyzed Ap 3 A, Ap 4 A, and Ap 5 A at comparable rates. Gp 4 G was hydrolyzed by NPP1 and NPP2 at rates similar to Ap 4 A, but only at half this rate by NPP3. Hydrolysis was asymmetric, involving the a,b-pyrophosphate bond. Ap n A hydrolysis had a very alkaline pH optimum and was inhibited by EDTA. Michaelis constant (K m ) values for Ap 3 A were 5.1 lM, 8.0 lM, and 49.5 lM for NPP1, NPP2, and NPP3, respectively. Our results suggest that NPP1, NPP2, and NPP3 are major enzyme candidates for the hydrolysis of extracellular diadenosine polyphosphates in vertebrate tissues.Keywords: diadenosine polyphosphate; diguanosine polyphosphate; ectonucleotidase; nucleotide pyrophosphatase; nucleotide phosphodiesterase.Diadenosine polyphosphates [adenosine-(5¢)-oligophospho-(5¢)-adenosines, Ap n As] comprise two adenosine residues linked together by a polyphosphate chain through phosphoester bonds at their ribose 5¢ carbons. Ap n As are present intracellularly in prokaryotic and eukaryotic cells [1]. Recently, this group of nucleotides has attracted considerable interest because its members act as extracellular signaling molecules in a broad variety of tissues [2,3]. They are involved, for example, in the modulation of synaptic transmission and sensory nerve function [2-4], inhibition of platelet aggregation [5], or in the control of vascular tone [6][7][8][9]. Vasoactive effects were also observed with adenosine polyphosphoguanosines (Ap n Gs) and diguanosine polyphosphates (Gp n Gs) [10].The diadenosine polyphosphates 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) are stored in chromaffin granules at millimolar concentrations together with noradrenaline and other nucleotides such as ATP and ADP [11,12]. In cholinergic synaptic vesicles, Ap 4 A and Ap 5 A were found to be co-stored with acetylcholine [13]. They can be released from secretory cells in a stimulus-dependent manner [2]. Besides the adrenal medulla, platelets are thought to represent the main source of Ap n As in blood. Stimulated platelets release, from their storage granules, a mixture of Ap n As (up to Ap 7 A), as well as Ap n Gs and Gp n Gs, together with ATP, ADP and serotonin [14,15]. Ap n ...

A capillary electrophoresis (CE) method for the characterization of recombinant NTPDase1, 2, and 3, and for assaying NTPDase inhibitors has been developed performing the enzymatic reaction within the capillary. After hydrodynamic injection of plugs of substrate solution with or without inhibitor in reaction buffer, followed by a suspension of an enzyme-containing membrane preparation, and subsequent injection of another plug of substrate solution with or without inhibitor, the reaction took place close to the capillary inlet. After 5 min, the electrophoretic separation of the reaction products was initiated by applying a constant current of j 60 mA. The method employing a polyacrylamide-coated capillary and reverse polarity mode provided baseline resolution of substrates and products within a short separation time of less than 7 min. A 50 mM phosphate buffer (pH 6.5) was used for the separations and the products were detected by their UV absorbance at 210 nm. The MichaelisYMenten constants (K m ) for the recombinant rat NTPDases 1, 2, and 3 obtained with this method were consistent with previously reported data. The inhibition studies revealed pronounced differences in the potency of reactive blue 2, pyridoxalphosphate-6-azophenyl-2 0 ,4 0 -disulfonic acid (PPADS), suramin, and N 6 -diethyl-b,g-dibromomethylene-ATP (ARL67156) towards the NTPDase isoforms. Notably, ARL67156 does not inhibit all NTPDases, having only a minor inhibitory effect on NTPDase2. Dipyridamole is not an inhibitor of the NTPDase isoforms investigated. The new method is fast and accurate, it requires only tiny amounts of material (nanoliter scale), no sample pretreatment and can be fully automated; thus it is clearly superior to the current standard methods.Abbreviations: ARL67156 -N 6 -diethyl-b,g-dibromomethylene-ATP; CE -capillary electrophoresis; CHO -Chinese hamster ovary; EMMA -electrophoretically mediated microanalysis; (E)-NTPDase -(ecto)-nucleoside triphosphate diphosphohydrolase; I.S. -internal standard; PPADS -pyridoxalphosphate-6-azophenyl-2 0 ,4 0 -disulfonic acid; RB2 -reactive blue 2

The physiological action of extracellular ATP and other nucleotides in the nervous system is controlled by surfacelocated enzymes (ecto-nucleotidases) of which several families with partially overlapping substrate speci®cities exist. In order to identify ecto-nucleotidases potentially associated with neural cells, we chose PC12 cells for analysis. PC12 cells revealed surface-located ATPase and ADPase activity with apparent K m -values of 283 mM and 243 mM, respectively. Using PCR we identi®ed the mRNA of all members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated (NTPDase1 to NTPDase3, NTPDase5/6), of ecto-nucleotide pyrophosphatase/phosphodiesterase3 (NPP3), tissue-non-speci®c alkaline phosphatase and ecto-5 H -nucleotidase. The surface-located catalytic activity differed greatly between the various enzyme species. Our data suggest that hydrolysis of ATP and ADP is mainly due to members of the ecto-nucleoside triphosphate diphosphohydrolase family. Activity of ecto-5 H -nucleotidase and alkaline phosphatase was very low and activity of NPP3 was absent.For a detailed analysis of the cellular distribution of ectonucleotidases single and double transfections of PC12 cells were performed, followed by¯uorescence analysis. Ectonucleotidases were distributed over the entire cell surface and accumulated intracellularly in varicosities and neurite tips. PC12 cell ecto-nucleotidases are likely to play an important role in terminating autocrine functions of released nucleotides and in producing extracellular nucleosides supporting the survival and neuritic differentiation of PC12 cells.