Identification and partial characterization of EctoATPase expressed by immortalized B lymphocytes

EctoATPases are extracellular membrane-bound enzymes that catalyze the hydrolysis of the gamma phosphate from ATP, EctoATPase is expressed by activated and immortalized Epstein-Barr virus-transformed human peripheral blood B lymphocytes and murine B cell hybridomas. By contrast, ectoATPase activity is not expressed on nontransformed human peripheral blood B lymphocytes, murine spleen cells, or murine myeloma cells, The K-m for ATP for the B cell ectoATPases ranged from 5 to 77 mu M; the V-max ranged from 48 to 129 pmol/min/10(4) cells, The enzyme required Mg2+ for maximal activity with Little dependence on Ca2+, ADP and purine and pyrimidine nucleoside triphosphates were competitive inhibitors of the catalytic reaction, A putative ectoATPase protein has been identified by Western blot analysis of membrane proteins from the immortalized B cells. Under reducing conditions, anti-ectoATPase antibodies cross-reacted with a 66-kDa protein from murine B cell hybridoma membranes, By contrast a 200-kDa protein from the B cell hybridoma membranes cross-reacted with the antibodies under nonreducing conditions, suggesting a disulfide-linked trimer, The antibodies also cross-reacted with a 66-kDa protein from human B cell membranes under reducing conditions, but did not cross-react with membrane proteins under nonreducing conditions, This suggests that the antibody epitope(s) recognized on the reduced human protein is masked under nonreducing conditions, Thus, this work demonstrates: (1) that ectoATPase may serve as a marker for B cell activation; and (2) mammalian and avian ectoATPases have conserved interspecies immunological epitopes and kinetic properties. (C) 1997 Academic Press.