IDENTIFICATION AND PARTIAL CHARACTERIZATION OF AN ECTOATPASE EXPRESSED BY HUMAN NATURAL-KILLER-CELLS

An extracellular membrane-associated ectoATPase has been identified on the human natural killer cell line NK3.3. The enzyme is distinct from other classes of ATPases, kinases, and phosphatases. NK3.3 ectoATPase demonstrated a K(m) for ATP of 41 muM and a V(max) of 0.2 mumol/min a nd required both Ca2+ and Mg2+ for maximal activity. Purine and pyrimi dine nucleotides were competitive inhibitors of the catalytic reaction . Inhibition increased with the addition of increasing negative charge of the phosphate side chain and was also dependent on contributions f rom the nucleoside. NK3.3 ectoATPase activity was inhibited by reactio n with the affinity label [p-(fluorosulfonyl)benzoyl]-5'-adenosine (5' -FSBA), which is shown to modify the enzyme at or near the ATP-binding domain. Photoaffinity labeling of intact NK3.3 cells with [alpha-P-32 ]-8-azidoATP demonstrated an ATP-binding protein of 68-80 kDa unique t o NK3.3 cells. A positive correlation was observed between the ability of the various nucleotides to block photoincorporation into the 68-80 -kDa protein and their ability to inhibit ectoATPase activity. NK3.3 c ells which were made ectoATPase-deficient by reaction with 5'-FSBA dem onstrated that this enzyme does not have a major role in the protectio n of this cytolytic effector cell from the possible lytic effects of e xtracellular ATP.