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Mammalian ectoapyrase (CD39) is an integral membrane protein with two transmembrane domains and a large extracellular region. The enzymatic activity of ectoapyrase is inhibited by most detergents used for membrane protein solubilization. In contrast, the enzymatic activities of soluble E-type ATPases, including potato tuber (Solanum tuberosum) apyrase and parasite ectoATPase, are not affected by detergents. Here we show that ectoapyrase is a tetramer and that detergents that reduce the activity of the enzyme promote dissociation of the tetramer to monomers. We expressed a secreted form of the ectoapyrase in COS-7 cells by fusing the signal peptide of murine CD4 with the extracellular domain of the ectoapyrase. The soluble ectoapyrase is catalytically active and its activity is not affected by detergents. Mutants of the ectoapyrase with only the NH 2 -or the COOH-terminal transmembrane domain are membrane-bound, and their activity is no longer affected by detergents. The enzymatic activity of all of the mutant proteins is less than that of the native enzyme. These results suggest that the proper contacts between the transmembrane domains of the monomers in the tetramer are necessary for full enzymatic activity.Ectoapyrases, or ATP diphosphohydrolases, hydrolyze extracellular nucleotide tri-and diphosphates in the presence of Ca 2ϩ or Mg 2ϩ (1). The rate of hydrolysis of nucleotide diphosphates is ϳ50% of that of the triphosphates. Mammalian ectoapyrases play important roles in many biological processes, including the modulation of neural cell activities (for a review, see Ref.2), prevention of intravascular thrombosis (3, 4), and regulation of immune responses (5). The enzymatic activity of ectoapyrases suggests that their functions might be to regulate purinergic signaling systems (6).The amino acid sequence of potato apyrase (7) revealed regions of similarity between the potato apyrase and several other proteins: garden pea nucleotide triphosphatase (8), Toxoplasma gondii nucleotide triphosphatase (9), yeast guanosine diphosphatase (10), and the lymphocyte cell activating antigen CD39 (11). We showed that CD39 has ectoapyrase activity (5) and that it is likely that the gene for CD39 is the only ectoapyrase gene in the mammalian genome (12). Subsequently, it was shown that CD39 also has ecto-ADPase activity in endothelial cells and is responsible for inhibition of ADP-induced platelet aggregation (3, 4). The ecto-ATPases in chicken gizzard (13), humans (CD39L1 (14)), and rats (15) are also encoded by genes similar to that for CD39. These latter enzymes hydrolyze nucleoside diphosphates at less than 10% of the rate of ATP hydrolysis (15).Mammalian ectoapyrases (CD39) are integral membrane proteins with two transmembrane domains (one at each end of the protein), small cytoplasmic NH 2 -and COOH-terminal segments, and a large extracellular domain (11) with enzymatic activity (5). This arrangement is unusual for ecto-enzymes, since these are usually attached to the membrane by a single protein or lipid link (16, 17)...

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CD39, a 70-to 100-kDa molecule expressed primarily on activated lymphoid cells, was previously identified as a surface marker of Epstein Barr virus (EBV)-transformed B cells. In this report, we show that an ecto-(Ca 2؉ ,Mg 2؉)-apyrase activity is present on EBV-transformed B cells, but not on B or T lymphomas. The coincidence between CD39 expression and ecto-apyrase activity on immune cells suggests that CD39 may be an ecto-apyrase. This supposition is supported by the observation that the amino acid sequence of CD39 is significantly homologous to those of several newly identified nucleotide triphosphatases. Finally, we show that CD39 indeed has ecto-apyrase activity by expression in COS-7 cells.

The multidrug resistance (mdrl) gene product, P-glycoprotein, is responsible for the ATP-dependent extrusion of a variety of compounds, including chemotherapeutic drugs, from cells. The data presented here show that cells with increased levels of the P-glycoprotein release ATP to the medium in proportion to the concentration of the protein in their plasma membrane. Furthermore, measurements of whole-cell and single-channel currents with patch-clamp electrodes indicate that the P-glycoprotein serves as an ATPconducting channel in the plasma membrane. These findings suggest an unusual role for the P-glycoprotein.Multidrug resistance in tumor cells is mediated by increased expression of the product of the mdrl gene (MDRI, now PGYJ, in human gene nomenclature), the P-glycoprotein (1-5). Its primary structure (6, 7) is similar to that of various transporters of ions, amino acids, peptides, or proteins in bacterial, yeast, and animal cells, which share ATP-binding motifs (8). The P-glycoprotein also resembles structurally the mitochondrial ATP/ADP exchanger (9, 10).The hydrolysis of ATP apparently provides the energy for drug extrusion from the cell (11-19). However, the mechanism of the transport process is not known for any of the transporters of the ATP-binding cassette (ABC) superfamily, including one reconstituted in artificial vesicles (20).In this paper we-show that P-glycoprotein in plasma membranes serves as a channel for steady ATP release from the cell. The possible physiological significance ofthis export will be briefly discussed. MATERIALS AND METHODSCell Lines. Chinese hamster ovary (CHO) cells, designated AUX B1, and P-glycoprotein-overexpressing drug-resistant cells, CHRC5 and CHRB30, were provided by Victor Ling (Ontario Cancer Institute, Toronto) (21). The human lung tumor cell line SW-1573 mutants that express the P-glycoprotein in different amounts were obtained by culturing the cells with adriamycin at 5 or 7 ,ug/ml, yielding SW10 or SWadria,.Drug-sensitive CHO cells (LR73) (25) transfected with multiple copies of murine mdrl cDNA (EX4N), or transfected with the murine mdrl cDNA in which the codons for lysines 432 and 1074 had been replaced by those for arginine residues to modify the two nucleotide-binding sites (88-8), were obtained from Philippe Gros (McGill University, Montreal).Immunofluorescence. P-glycoprotein concentration on cell surfaces was measured by staining with fluorescent mouse monoclonal antibodies directed to surface epitopes of the protein, followed by analysis on a FACScan (Becton Dickinson) using LYSYS software. The hamster cell lines were stained with antibody 4A6B3/10 to the hamster P-glycoprotein (26), and the human cell lines were stained with antibody 4E3 to the human P-glycoprotein (R.J.A., unpublished results).ATP measurements. (i) HPLC. Nucleotides, in the Hanks buffer bathing the cells, were separated by HPLC (Waters) and quantitated as described by Fellenz and Gerweck (27).(ii) Luminometry. After stable baseline measurements were obtained from 1 ml of Hanks...