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Journal of Biochemistry Advance Access originally published online on July 25, 2007
Journal of Biochemistry 2007 142(2):157-173; 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; and 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. Tel: +81-422-76-7651, 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 analysed 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. {Delta}Cmax = 26.7%. {Delta}Cmax for the sum of D and H was equivalent to the absolute value of {Delta}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, {Delta}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 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 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

Abbreviations: C12E8, octaethyleneglycol n-dodecylether; CDTA, 1,2-cyclohexylenedinitrilotetraacetic acid;CHES, 2-(cyclohexylamino)ethanesulfonic acid; D, ({alpha}ß)2-diprotomer; {Delta}Cmax, maximum induction in oligomeric protein by changing the K+ or Na+ concentration; E1, enzyme conformation with a higher affinity for Na+ which binds ATP with high affinity and accepts phosphate from ATP; E2, enzyme conformation with a higher affinity for K+, which can accept Pi; E1-P, an ADP-sensitive phosphoenzyme; E2-P, a K+-sensitive phosphoenzyme; E1·Na+, Na+-bound enzyme; E2·K+, K+-bound enzyme; HPGC, high-performance gel chromatography; G, aggregates; H, higher-oligomer; K0.5, concentration of K+ or Na+ to induce a half-maximum change in the amount of oligomer; Kd, dissociation constant for an association-dissociation equilibrium; LALLS, low-angle laser light-scattering photometer; MES, 2-morpholinoethanesulfonic acid, monohydrate; P, {alpha}ß-protomer; Mp, molecular weight of protein moiety for solubilized membrane protein; pNPPase, p-nitrophenyl phosphatase; p-NPP, p-nitrophenyl phosphate; PS, phosphatidylserine; TEA, triethanolamine


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