Isolation of Stable (αβ)4-Tetraprotomer from Na+/K+-ATPase Solubilized in the Presence of Short-Chain Fatty Acids

Previously, it was demonstrated that acetate anions increase the higher oligomer (H), consuming (αβ) 2 -diprotomer (D) and αβ-protomer (P) of solubilized dog kidney Na + /K + -ATPase [Kobayashi, T. et al. (2007) J. Biochem. 142, 157-173]. Presently, short-chain fatty acids, such as propionate (Prop) and butyrate, have been substituted effectively for acetate. The molecular weight of 6.01 x 10 5 for H and quantitative Na + /K + -dependent interconversion among H, D, and P showed that H was an (αβ) 4 -tetraprotomer (T). T was optimally isolated from the enzyme solubilized in aqueous 40 mM K + Prop at pH 5.6 by gel chromatography performed at 0 °C with elution buffer containing synthetic dioleoyl phosphatidylserine (PS). K 0.5 values of K + -congeners constituting K + Prop for the maximal amount of T were NH 4 + » Rb+ ≅ K + >T1 + , while Na + had no effect. The oligomers of T, D, and P were simultaneously assayed for ATPase upon elution from the gel column, resulting in a specific activity ratio of 1:2:2. The activity of the chromatographically isolated T increased with an increasing dioleoyl PS, giving a saturated activity of 2.38 units/mg at pH 5.6 and 25 °C, and the active enzyme chromatography of T showed 34% dissociation into D by exposing it at 25 °C. On the basis of these data, the specific ATPase activities of T, D, and P were concluded to be 32, 65, and 65 units/mg, respectively, under the conventionally optimal conditions of pH 7.3 and 37 °C, suggesting an equivalence to a fully active enzyme for D and P but half activity for T. The physiological significance of the stable form of T remains to be investigated.