The yeast gene SNQ2 confers hyper-resistance to the mutagens 4-nitroquinoline-N-oxide (4-NQO) and Triaziquone, as well as to the chemicals sulphomethuron methyl and phenanthroline when present in multiple copies in transformants of Saccharomyces cerevisiae. Subcloning and sequencing of a 5.5 kb yeast DNA fragment revealed that SNQ2 has an open reading frame of 4.5 kb. The putative encoded polypeptide of 1501 amino acids has a predicted molecular weight of 169 kDa and has several hydrophobic regions. Northern analysis showed a transcript of 5.5 kb. Haploid cells with a disrupted SNQ2 reading frame are viable. The SNQ2-encoded protein has domains believed to be involved in ATP binding and is likely to be membrane associated. It most probably serves as an ATP-dependent permease.

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 ...

Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5′-triphosphate agonists at P2Y 1 , P2Y 2 , P2Y 4 , and P2Y 11 receptors, but not P2Y 6 receptors, was established using a ring-constrained methanocarba (a 3.1.0-bicyclohexane) ring as a ribose substitute (Kim et al. J. Med. Chem. 2002, 45, 208-218.). We have now combined the ringconstrained (N)-methanocarba modification of adenine nucleotides with other functionalities known to enhance potency at P2 receptors. The potency of the newly synthesized analogues was determined in the stimulation of phospholipase C through activation of turkey erythrocyte P2Y 1 or human P2Y 1 and P2Y 2 receptors stably expressed in astrocytoma cells. An (N)-methanocarba-2-methylthio-ADP analogue displayed an EC 50 at the hP2Y 1 receptor of 0.40 nM and was 55-fold more potent than the corresponding triphosphate and 16-fold more potent than the riboside 5′-diphosphate. 2-Cl-(N)-methanocarba-ATP and its N 6 -Me analogue were also highly selective, full agonists at P2Y 1 receptors. The (N)-methanocarba-2-methylthio and 2-chloromonophosphate analogues were full agonists exhibiting micromolar potency at P2Y 1 receptors, while the corresponding ribosides were inactive. Although β,γ-methylene-ATP was inactive at P2Y receptors, β,γ-methylene-(N)-methanocarba-ATP was a potent hP2Y 1 receptor agonist with an EC 50 of 160 nM and was selective versus hP2Y 2 and hP2Y 4 receptors. The rates of hydrolysis of Northern (N) and Southern (S) methanocarba analogues of AMP by rat 5′-ectonucleotidase were negligible. The rates of hydrolysis of the corresponding triphosphates by recombinant rat NTPDase1 and 2 were studied. Both isomers were hydrolyzed by NTPDase 1 at about half the rate of ATP hydrolysis. The (N) isomer was hardly hydrolyzed by NTPDase 2, while the (S) isomer was hydrolyzed at one-third of the rate of ATP hydrolysis. This suggests that new, more stable and

We have isolated and characterized the cDNA encoding nucleoside triphosphate diphosphohydrolase 6 (NTPDase6), a novel member of the ecto-nucleoside triphosphate diphosphohydrolase family. The rat-brain-derived cDNA has an open reading frame of 1365 bp encoding a protein of 455 amino acid residues, a calculated molecular mass of 49971 Da and a predicted N-terminal hydrophobic sequence. It shares 86% sequence identity with the human CD39L2 sequence and 48% and 51% identity respectively with sequences of the two related human and murine nucleoside diphosphatases (CD39L4, NTPDase5/ER-UDPase). The mRNA was expressed in all tissues investigated, revealing two major transcripts with differing abundances. PCR analysis suggests a single open reading frame. A Myc-His-tagged NTPDase6 was expressed in Chinese hamster ovary (CHO) and PC12 cells for immunological analysis and protein isolation. The protein was contained in membrane fractions of transfected CHO cells and occurred in a soluble form in the cell culture supernatants. NTPDase6 preferentially hydrolysed nucleoside 5'-diphosphates. With different substrates the order of activity was GDP>IDP>>UDP,CDP>>ADP. Nucleoside 5'-triphosphates were hydrolysed only to a minor extent and no hydrolysis of nucleoside 5'-monophosphates was observed. The enzyme was strongly and equally activated by Ca(2+) and Mg(2+) and had a K(m) for GDP of 211 microM. The immunohistochemical analysis of transfected CHO and PC12 cells suggests that NTPDase6 is associated with the Golgi apparatus and to a small extent also with the plasma membrane. The enzyme might support glycosylation reactions in the Golgi apparatus and, when released from cells, might catalyse the hydrolysis of extracellular nucleotides.