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

Abstract

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.

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

“…Natural phosphatidylserine (PS) purified from ox brain and four kinds of synthetic PS with various lengths of acyl chains, such as dioleoyl (C18:1), dipalmytoyl (C16:0), dimyristoyl (C14:0), and dioctanoyl (C8:0), were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Other chemicals used have been described previously (10). Special care was taken to minimize contamination by Na + and K + (10).…”

“…Other chemicals used have been described previously (10). Special care was taken to minimize contamination by Na + and K + (10).…”

“…These findings strongly indicate that the Na + /K + -dependent interconversion among the oligomers and the exhibited enzymatic action are closely related to each other. Furthermore, it has also been demonstrated that the solubilized enzyme is mainly composed of D and P, regardless of the other anions, other than the chloride anion, present in the solubilizing solution (10). The exception was acetate, which was observed to behave in a characteristic manner, producing more H (with the addition of K + , without fixing the oligomeric structure of D) than the other anions ( Figure 4B in ref 10).…”

Active Detergent-solubilized H+,K+-ATPase Is a Monomer

“…In this way, indeed, it was established that sarcoplasmic reticulum Ca 2ϩ -ATPase, solubilized as a monomer by C 12 E 8 , retains all of the basic features required to function as a Ca 2ϩ -transporting unit, regardless of the tendency of this Ca 2ϩ pump to undergo reversible and irreversible self-association (20). For Na ϩ ,K ϩ -ATPase, this also appears to be the case (28 -30), although dimerization of the ␣,␤-protomer is a more pronounced feature of this ATPase than it is for Ca 2ϩ -ATPase (27,(31)(32)(33). Application of the same surfactant strategy to H ϩ ,K ϩ -ATPase is fraught with the problem that it is difficult to avoid either irreversible or reversible inhibition of enzyme activity in the detergent-solubilized state.…”

“…Despite the probable presence of protein-protein interactions in the native membrane, which may affect enzyme activity (25,26), they all indicate monomeric Ca 2ϩ -ATPase as the fundamental Ca 2ϩ -transporting unit. For Na ϩ ,K ϩ -ATPase, the evidence is more equivocal in that both monomeric and dimeric forms with retention of enzyme activity have been detected by analytical ultracentrifugation (27)(28)(29)(30) and by HPLC (31)(32)(33). The requirement of an oligomeric structure as a sine qua non to preserve transport and enzyme activity, as claimed for H ϩ ,K ϩ -ATPase and reported in a recent review (34), is not indicated by either the detergent data or by the available threedimensional x-ray diffraction structures of Ca 2ϩ -ATPase (35,36), Na ϩ ,K ϩ -ATPase (37)(38)(39), and H ϩ -ATPase (40,41).…”