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

Abstract

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.

Comparative Properties of Caveolar and Noncaveolar Preparations of Kidney Na+/K+-ATPase

“…Neither in co-immunoprecipitation nor in cross-linking studies, however, do we find evidence of direct contact between caveolin-1 and any specific subunits of the other two oligomers. Our findings, in conjunction with previous extensive evidence of the higher oligomeric states of Na + /K + -ATPase promoters 14,15,29,30,32,39 and caveolin-1, 23,38 clearly indicate that interaction of caveolin-1 with kidney caveolar Na + /K + -ATPase occurs within a large oligomeric complex of caveolin-1, the annexin-2 tetramer, and Na + /K + -ATPase (Figure 9A).…”

“…There are major differences between the lipid compositions of the caveolae–raft microdomains and the remainder of the plasma membrane (Figure 1B and ref (26)), and previous studies have clearly indicated effects of lipid on the catalytic activities and quaternary structure of Na + /K + -ATPase. 28−30 It is also known that quaternary structure has profound effects on the reaction mechanism of the enzyme. 31−33 Hence, we suggest the need for more rigorous investigations, e.g., those using transient kinetics, on the potential differences between the turnover cycles of the caveolar and noncaveolar preparations, and the possible physiological implications of such differences.…”

Structural Analysis of N-glycans attached to Pig Kidney Na+/K+-ATPase

“…Although the α and β subunits are non-covalently linked in a minimal αβ-protomer structural unit [59], solubilized enzyme solutions contain other oligomers, such as (αβ)2, (αβ)3 and (αβ)4 as shown by chemical crosslinking [60]. HPLC analysis of solubilized Na + /K + -ATPase reveals that K + induces the conversion of protomer into diprotomer and/or higher oligomers while Na + has the opposite effect [61,62]. Cation and hence, conformation-dependent alteration in the oligomer equilibrium may occur through changes in exposure of the transmembrane domain of the β subunit as mutational analysis has shown that this portion of the protein mediates β-β homooligomerization, as well as α/β assembly [63].…”

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).…”