Hydrolysis of diadenosine polyphosphates by nucleotide pyrophosphatases/phosphodiesterases

Abstract

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 ...

Cellular function and molecular structure of ecto-nucleotidases

“…Mutation of these three glycines into alanine abolished the nucleotide phosphodiesterase activity of NPP1 [366]. Yet, recombinant NPP2 prepared and isolated from a vaccinia virus lysate of BS-C-1 cells was found to hydrolyze both ATP [364] and dinucleoside polyphosphates [351].…”

“…Catalytic activity depends on divalent cations and the enzymes reveal an alkaline pH optimum between pH 9 and 10. Similar to APs, they are also active at pH 7.4 with about 20 % of maximal activity [349][350][351][352]. Enzyme activity is inhibited by EDTA.…”

“…The scheme is based on data presented in the following papers-NPP1: [351,353,412,453,625]; NPP2: [351,353,377,380,403,406]; NPP3: [351]; NPP6: [388]; NPP7: [389,626]. GPC glycerophosphorylcholine, LPC lysophosphatidylcholine, pNPPC p-nitrophenyl phosphorylcholine, SPC sphingosylphosphorylcholine substrate [354][355][356][357].…”

“…K m values for dinucleoside polyphosphates are lower than those for ATP [35]. Heterologously expressed NPP1, NPP2, and NPP3 hydrolyzed dinucleoside polyphosphates with a pH optimum between 8.5 and 9 and K m values for diadenosine 5′,5′″-P 1 ,P 3 -triphosphate (Ap 3 A) were 5.1, 8.0, and 49.5 μM, respectively [351]. K m values in the 3-10-μM range have also been reported for the hydrolysis of diadenosine polyphosphates by a purified ecto-pyrophosphatase (presumably NPP1) from rat brain [352].…”

“…One of the limitations results from the fact that classical inhibitors of P2 receptors can also inhibit E-NPPs [351,369]. In principle, E-NPPs could modulate nucleotide signaling by hydrolyzing purinoceptors agonists and together with APs or ecto-5′-nucleotidase produce adenosine.…”