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 microM; the Vmax 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, antiectoATPase 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.
Dombrowski
K E
KE
Department of Veterans Affairs Medical Center, Amarillo, Texas 79106, USA.
Brewer
K A
KA
Maleckar
J R
JR
Kirley
T L
TL
Thomas
J W
JW
Kapp
J A
JA
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001402:B-Lymphocytes / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D002451:Cell Compartmentation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D011233:Precipitin Tests; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D008274:Magnesium; D002118:Calcium