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) K(0.5) for Na(+) 7.5+/-1.5 mM and V(max) 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 (IC(50) 42.1+/-8.0 and 4.3+/-0.3 microM, 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-alpha(1) 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.
De Souza
A M
AM
Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
Carvalho
T L G
TL
Sabino
P M
PM
Vives
D
D
Fontes
C F L
CF
Lopes
A G
AG
Caruso-Neves
C
C
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005665:Furosemide / Q000494:pharmacology; D006868:Hydrolysis / Q000187:drug effects; D019811:Hydroxylamine / Q000494:pharmacology; D015151:Immunoblotting; D047468:Immunoprecipitation; D007700:Kinetics; D010042:Ouabain / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D004791:Enzyme Inhibitors; D019811:Hydroxylamine; D014638:Vanadates; D010042:Ouabain; D005665:Furosemide; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase