Na+- and K+-dependent oligomeric interconversion among alphabeta-protomers, diprotomers and higher oligomers in solubilized Na+/K+-ATPase.
Protein fractions of a higher-oligomer (H), (alphabeta)(2)-diprotomer (D) and alphabeta-protomer (P) were separated from dog kidney Na(+)/K(+)-ATPase solubilized in the presence of NaCl and KCl. Na(+)/K(+)-dependent interconversion of the oligomers was analysed using HPLC at 0 degrees C. With increasing KCl concentrations, the content or amount of D increased from 27.6 to 54.3% of total protein, i.e. DeltaC(max) = 26.7%. DeltaC(max) for the sum of D and H was equivalent to the absolute value of DeltaC(max) for P, regardless of the anion present, indicating that K(+) induced the conversion of P into D and/or H, and Na(+) had the opposite effect. When enzymes that had been denatured to varying degrees by aging were solubilized, DeltaC(max) increased linearly with the remaining ATPase activity. The magnitude of the interconversion could be explained based on an equilibrium of D <==> 2P, assuming 50-fold difference in the K(d) between KCl and NaCl, and coexistence of unconvertible oligomers, which comprised as much as 39% of the eluted protein. Oligomeric interconversion, determined as a function of the KCl or NaCl concentration, showed K(0.5)s of 64.8 microM and 6.50 mM for KCl and NaCl, respectively, implying that oligomeric interconversion was coupled with Na(+)/K(+)-binding to their active transport sites.
Kobayashi
Takayuki
T
Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan.
Tahara
Yoshikazu
Y
Takenaka
Hitoshi
H
Mimura
Kunihiro
K
Hayashi
Yutaro
Y
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D011189:Potassium Chloride / Q000494:pharmacology*; D021122:Protein Subunits / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D012995:Solubility
D021122:Protein Subunits; D012965:Sodium Chloride; D011189:Potassium Chloride; D000254:Sodium-Potassium-Exchanging ATPase