Characterization and partial isolation of ouabain-insensitive Na+-ATPase in MDCK I cells

We show that MDCK I cells express, besides the classical (Na+ + K+)ATPase, a Na+-stimulated ATPase activity with the following characteristics: (1) K0.5 for Na+ 7.5 ± 1.5 mM and Vmax 23.12 ± 1.1 nmol Pi/mg per min; (2) insensitive to 1 mM ouabain and 30 mM KCl; and (3) inhibited by furosemide and vanadate (IC50 42.1 ± 8.0 and 4.3 ± 0.3 μM, respectively). This enzyme forms a Na+-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate phosphorylated intermediate with molecular weight of 100 kDa. Immunoprecipitation of the (Na+ + K+)ATPase with monoclonal anti-α1 antibody reduced its activity in the supernatant by 90%; the Na+-ATPase activity was completely maintained. In addition, the formation of the Na+-stimulated, furosemide- and hydroxylamine-sensitive ATP-driven acylphosphate intermediate occurred at the same magnitude as that observed before immunoprecipitation. These data suggest that Na+-ATPase and (Na+ + K+)ATPase activities are independent, with Na+-ATPase belonging to a different enzyme entity.