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Journal of Biochemistry Advance Access published online on July 25, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm150
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© 2007 The Japanese Biochemical Society

Na+- and K+-Dependent Oligomeric Interconversion among {alpha}ß-Protomers, Diprotomers and Higher Oligomers in Solubilized Na+/K+-ATPase

Takayuki Kobayashi1, Yoshikazu Tahara1, Hitoshi Takenaka1, Kunihiro Mimura2 and Yutaro Hayashi1,*

1Department of Biochemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611;
2Department of Environmental Security System, Faculty of Risk and Crisis Management, Chiba Institute of Science, Choshi, Chiba 288-0025, Japan

*To whom correspondence should be addressed: Yutaro Hayashi, Tel. & Fax: +81-422-76-7651; E-mail : yutahaya{at}kyorin-u.ac.jp

Received May 4, 2007; Accepted May 28, 2007


  Abstract

Protein fractions of a higher-oligomer (H), ({alpha}ß)2-diprotomer (D) and {alpha}ß-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 analyzed using HPLC at 0° C. With increasing KCl concentrations, the content or amount of D increased from 27.6 to 54.3% of total protein, i.e., {triangleup}Cmax = 26.7%. {triangleup}Cmax for the sum of D and H was equivalent to the absolute value of {triangleup}Cmax 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, {triangleup}Cmax increased linearly with the remaining ATPase activity. The magnitude of the interconversion could be explained based on an equilibrium of D {rightleftharpoons}2P, assuming 50-fold difference in the Kd between KCl and NaCl, and coexistence of un-convertible oligomers, which comprised as much as 39% of the eluted protein. Oligomeric interconversion, determined as a function of the KCl or NaCl concentration, showed K0.5s of 64.8 µM and 6.50 mM for KCl and NaCl, respectively, implying that oligomeric interconversion was coupled with Na+/K+-binding to their active transport sites.

Key Words: Dog kidney, Oligomeric interconversion, Oligomeric structure, Na+/K+-ATPase, Solubilized membrane protein


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