Nucleoside triphosphate diphosphohydrolases 1, 2, 3 and 8 (NTPDases 1, 2, 3 and 8) are the dominant ectonucleotidases and thereby expected to play important roles in nucleotide signaling. Distinct biochemical characteristics of individual NTPDases should allow them to regulate P2 receptor activation differentially. Therefore, the biochemical and kinetic properties of these enzymes were compared. NTPDases 1, 2, 3 and 8 efficiently hydrolyzed ATP and UTP with Km values in the micromolar range, indicating that they should terminate the effects exerted by these nucleotide agonists at P2X1- and P2Y2,4,11 receptors. Since NTPDase1 does not allow accumulation of ADP, it should terminate the activation of P2Y1,12,13 receptors far more efficiently than the other NTPDases. In contrast, NTPDases 2, 3 and 8 are expected to promote the activation of ADP specific receptors, because in the presence of ATP they produce a sustained (NTPDase2) or transient (NTPDases 3 and 8) accumulation of ADP. Interestingly, all plasma membrane NTPDases dephosphorylate UTP with a significant accumulation of UDP, favoring P2Y6 receptor activation. NTPDases differ in divalent cation and pH dependence, although all are active in the pH range of 7.0-.5. Various NTPDases may also distinctly affect formation of extracellular adenosine and therefore adenosine receptor-mediated responses, since they generate different amounts of the substrate (AMP) and inhibitor (ADP) of ecto-5-nucleotidase, the rate limiting enzyme in the production of adenosine. Taken together, these data indicate that plasma membrane NTPDases hydrolyze nucleotides in a distinctive manner and may therefore differentially regulate P2 and adenosine receptor signaling.

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On the basis of sequence homologies observed between members of the E-type ATPases and the phosphate binding motifs of the actin/heat shock protein 70/sugar kinase superfamily, a human ecto-apyrase was analyzed by site-directed mutagenesis of conserved amino acids in apyrase conserved regions (ACR) I and IV. The expressed proteins were analyzed to assess the significance of these amino acids. A conserved aspartic acid residue in ACR IV was mutated to alanine, asparagine, and glutamic acid, and the relative activity and Km for ATP and ADP were determined. Mutation of this Asp 219 to Ala or Asn yielded an enzyme severely reduced in ATP hydrolyzing activity (>90%) and completely devoid of ADPase activity, along with a similar extent of inhibition of hydrolysis of other nucleoside di- and triphosphates. Interestingly, mutation of Asp 219 to Glu completely restored the ability of the enzyme to hydrolyze nucleoside triphosphates at levels above that of the wild-type enzyme, while the ability to hydrolyze nucleoside diphosphates was slightly reduced. Mutation of a second conserved aspartic acid in ACR I (Asp 62) and two invariant glycine residues in both ACR I (Gly 64) and ACR IV (Gly 221) also severely disrupted nucleotidase activity. These results demonstrate that the E-type ATPases contain the nucleoside phosphate binding domains present in the actin/heat shock protein/sugar kinase superfamily. Together with analysis of computer-predicted secondary structures, the results suggest that the ecto-ATPases and ecto-apyrases are part of, or closely related to, the actin superfamily of proteins.

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The ecto-ATPase from chicken gizzard (smooth muscle) was solubilized, and the 66-kDa cell membrane ectoATPase protein was purified. The protein was then subjected to both enzymatic and chemical cleavage, and the resultant peptides were purified by reverse phase high pressure liquid chromatography and sequenced. Several of these internal peptide sequences were used to design oligonucleotides to screen a chicken muscle library to identify the cDNA encoding the ecto-ATPase. Two overlapping partial clones were sequenced, yielding the complete coding region and a long 3-untrans- Cell membrane ecto-ATPases are millimolar divalent cationdependent, low specificity enzymes that hydrolyze all nucleoside triphosphates (NTPases). They are integral membrane glycoproteins that can be distinguished from ecto-apyrases (ecto-ATP Diphosphohydrolases or ecto-ATPDases) by their inability to hydrolyze ADP and other nucleoside diphosphates at rates that are more than Ϸ1-2% that of their ATP hydrolysis rates. A recent review summarizes the properties and postulated functions of the ecto-ATPases and ecto-apyrases (1). From the results of work on the single-celled parasitic protozoan Toxoplasma gondii, it appears that the ecto-ATPase and ectoapyrase enzymes from that source are closely related as judged by sequence analysis (2, 3). However, significant differences in enzymology and susceptibility to inactivation by detergents exist between ecto-ATPases and ecto-apyrases from a variety of sources (for a review, see Ref. 1), suggesting that they may not be as closely related to each other as is suggested by the sequence homology of the T. gondii enzymes.The physiological functions of the ecto-ATPases and ectoapyrases are not known. However, many functions have been hypothesized, including roles in cellular adhesion, termination of purinergic signaling, and purine recycling (for a review, see Ref. 1), as well as secretion (4) and vesicle trafficking (5). The goal of this study was to clone and sequence the cDNA encoding the chicken muscle ecto-ATPase to gain information about the structure and physiological function of the whole class of ectoATPase enzymes by analysis of sequence homologies with proteins of known function. The sequence reported here represents the first vertebrate ecto-ATPase to be cloned and sequenced. (The rat liver cellular adhesion molecule of 105 kDa "ectoATPase" sequence (6) apparently does not encode the ATPase (7), and the rat liver enzyme is classified as an ecto-apyrase since it hydrolyzes ADP as well as ATP.) The results reported here suggest that at least one physiological function of the ecto-ATPase is involvement in the process of cell adhesion, since it is highly homologous with the lymphoid cell activation antigen (CD39), 1 which is known to be involved in homotypic activated B-cell adhesion (8). This conclusion is consistent with several previous reports obtained from several different species and tissues that suggested by indirect methods that the ectoATPase may be involved in cellular adhesion in rat liv...

The extracellular ATPase (ecto-ATPase) is a divalent cation-dependent nucleoside triphosphatase with an unusually high specific activity. Monoclonal antibodies, described previously [Stout, J. G., Strobel, R. S., & Kirley, T. L. (1995) J. Biol. Chem. 270, 11845-11850], and newly generated polyclonal antibodies, both raised against the chicken gizzard ecto-ATPase, were evaluated for their ability to cross-react with mammalian ecto-ATPases and were used as specific immunochemical probes to identify non-cross-linked and cross-linked ecto-ATPase. Unlike previous results obtained with the rabbit skeletal muscle ecto-ATPase enzyme, cross-linking the chicken gizzard smooth muscle ecto-ATPase with 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP) and dithiobis(succinimidylpropionate) (DSP) increased the activity of the enzyme which corresponded to an increase in a approximately 130 kDa immunoreactive band, proposed to be a ecto-ATPase homodimer, and a concomitant decrease in a approximately 66 kDa immunoreactive band, the ecto-ATPase monomer. Ecto-ATPase was immunochemically identified in chicken, rat, mouse, rabbit, and pig. Interestingly, under nonreducing conditions, the ecto-ATPase activity in rat and pig (unlike chicken and rabbit) was evident on Western blots as an immunoreactive band at approximately 200 kDa, proposed to be an intermolecularly disulfide-linked ecto-ATPase homotrimer. Nonreducing Western blot analysis of various rat tissues with three different monoclonal antibodies that recognize the 66 kDa chicken gizzard ecto-ATPase monomer strengthened the hypothesis that this 200 kDa band indeed represents the trimeric ecto-ATPase. After reduction, ecto-ATPase monomers were found to be approximately 66 kDa in all species examined. The differences in ecto-ATPase quaternary structure stability may account for the observed species differences in ecto-ATPase enzymatic properties. Intermolecular disulfide bonds appear to be one of the species-specific ways to stabilize the native, active ecto-ATPase quaternary structure (the homotrimer). Based on the data obtained, as well as previous data from this and other laboratories, a hypothesis was developed to explain the modulation of ecto-ATPase activity by a variety of agents, including detergents, chemical cross-linkers, lectins, antibodies, and small molecule inhibitors. It is proposed that agents and conditions stabilizing ecto-ATPase oligomers stimulate enzyme activity, whereas agents and conditions destabilizing ecto-ATPase homooligomers would inhibit the ecto-ATPase.

Cell-surface nucleotidases (NTPDases) contain 10 invariant cysteine residues in their extracellular regions. To investigate disulfide structure in human NTPDase3, we made single and double mutants of these 10 cysteines, and analyzed their enzymatic activity, glycosylation pattern, trafficking to the cell membrane, and sensitivity to reduction. The mutants constituted five distinct phenotypes, thus, strongly suggesting disulfide bonds between C92-C116 (first bond), C261-C308 (second bond), C289-C334 (third bond), C347-C353 (fourth bond), and C399-C422 (fifth bond). Due to conservation of the 10 cysteines, the identified five disulfide bonds are likely to exist in all cell-surface NTPDases. The third and fifth bonds are also present in the soluble NTPDases and are critical for processing, trafficking, and enzymatic activity. The fourth bond has minimal effect on processing and function, while the first and second bonds are of intermediate importance. Most of the N-linked glycosylation sites in the wild-type enzyme are processed to complex oligosaccharides, but at least one site is high-mannose or hybrid in structure. Interestingly, disruption of the first disulfide bond resulted in some enzyme that lost sensitivity to endoglycosidase H, suggesting that the first disulfide bond in the wild-type enzyme shields some high-mannose glycans from terminal glycosylation. Comparative modeling by threading and homology modeling of the NTPDase3 sequence revealed a high degree of structural fold similarity with a bacterial exopolyphosphatase (PDB ). The resultant theoretical 3-D model of the extracellular portion of NTPDase3, based on homology with this exopolyphosphatase, is consistent with the assignment of the disulfide bonds occurring in regions of good fold similarity between NTPDase3 and the exopolyphosphatase. The 3-D model obtained for NTPDase3 also suggests the structural basis for the importance of several apyrase conserved regions for the nucleotidase activities of the NTPDases.

The importance of N-linked glycosylation for the function and oligomerization of an E-type ATPase was examined by using tunicamycin and peptide N-glycosidase F, two agents used to prevent and remove glycosylations, respectively. The cDNA encoding a human ecto-apyrase (HB6), predicted to have seven N-linked glycosylation sites, was transiently expressed in mammalian COS cells and the resulting membrane preparations were treated with peptide N-glycosidase F (PNGase-F). PNGase-F caused a decrease in the apparent molecular weight of the protein (consistent with glycan removal) and a decrease in enzymatic activity over time. The ecto-apyrase was also expressed in the presence of tunicamycin, which completely prevented N-linked glycosylation, resulting in a nonglycosylated core protein devoid of ATP and ADP hydrolyzing activity. However, control and tunicamycin-treated cells expressed the enzyme to similar levels and localization. Interestingly, the quaternary structure of this E-type ATPase appears to be dependent upon the presence of glycan chains. The glycosylated ecto-apyrase exists as a homodimer in situ as assessed by both size-exclusion chromatography of detergent-solubilized ecto-apyrase and cross-linking of membrane-bound ecto-apyrase, in contrast to the enzymatically deglycosylated ecto-apyrase and the tunicamycin-treated ecto-apyrase. These results suggest that glycosylation is necessary for homooligomerization and nucleotide hydrolyzing activity, but not for expression and plasma membrane localization of the E-type ATPase. Similar results were obtained with another human ecto-apyrase, CD39, suggesting that the importance of glycosylation may be generalized to all membrane-bound E-type ATPases.