The non-gastric H,K-ATPase is oligomycin-sensitive and can function as an H+,NH4+-ATPase

Abstract

We used the baculovirus/Sf9 expression system to gain new information on the mechanistic properties of the rat non-gastric H,K-ATPase, an enzyme that is implicated in potassium homeostasis. The α2-subunit of this enzyme (HKα2) required a β-subunit for ATPase activity thereby showing a clear preference for NaKβ1 over NaKβ3 and gastric HKβ. NH4+, K +, and Na+ maximally increased the activity of HKα2-NaKβ1 to 24.0, 14.2, and 5.0 μmol Pi·mg-1 protein·h-1, respectively. The enzyme was inhibited by relatively high concentrations of ouabain and SCH 28080, whereas it was potently inhibited by oligomycin. From the phosphorylation level in the presence of oligomycin and the maximal NH 4+-stimulated ATPase activity, a turnover number of 20,000 min-1 was determined. All three cations decreased the steady-state phosphorylation level and enhanced the dephosphorylation rate, disfavoring the hypothesis that Na+ can replace H+ as the activating cation. The potency with which vanadate inhibited the cation-activated enzyme decreased in the order K+ > NH4+ > Na+, indicating that K+ is a stronger E2 promoter than NH4+, whereas in the presence of Na + the enzyme is in the E1 form. For K+ and NH4+, the E2 to E1 conformational equilibrium correlated with their efficacy in the ATPase reaction, indicating that here the transition from E2 to E1 is rate-limiting. Conversely, the low maximal ATPase activity with Na+ is explained by a poor stimulatory effect on the dephosphorylation rate. These data show that NH4+ can replace K+ with similar affinity but higher efficacy as an extracellular activating cation in rat non-gastric H,K-ATPase. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.