Isolation of stable (alphabeta)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 (alphabeta) 2-diprotomer (D) and alphabeta-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 (alphabeta) 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 degrees 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 (+) congruent with K (+) > Tl (+), 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 degrees C, and the active enzyme chromatography of T showed 34% dissociation into D by exposing it at 25 degrees 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 degrees 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.
Mimura
Kunihiro
K
Department of Environment System Science, Faculty of Risk and Crisis Management, Chiba Institute of Science, Choshi, Chiba 288-0025, Japan.
Tahara
Yoshikazu
Y
Shinji
Nobuko
N
Tokuda
Emi
E
Takenaka
Hitoshi
H
Hayashi
Yutaro
Y
D000818:Animals; D002850:Chromatography, Gel; D004285:Dogs; D005227:Fatty Acids / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010718:Phosphatidylserines / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility; D013552:Swine
D005227:Fatty Acids; D010718:Phosphatidylserines; D021122:Protein Subunits; C042511:1,2-dioleoylphosphatidylserine; D000254:Sodium-Potassium-Exchanging ATPase