Interaction of SDS with Na+/K+-ATPase

Abstract

Because nearly all structure/function studies on Na ؉ / K ؉ -ATPase have been done on enzymes prepared in the presence of SDS, we have studied previously unrecognized consequences of SDS interaction with the enzyme. When the purified membrane-bound kidney enzyme was solubilized with SDS or TDS concentrations just sufficient to cause complete solubilization, but not at concentrations severalfold higher, the enzyme retained quaternary structure, exhibiting ␣,␣-, ␣,␤-, ␤,␤-, and ␣,␥-associations as detected by chemical cross-linking. The presence of solubilized oligomers was confirmed by sucrose density gradient centrifugation. This solubilized enzyme had no ATPase activity and was not phosphorylated by ATP, but it retained the ability to occlude Rb ؉ and Na ؉ . This, and comparison of cross-linking patterns obtained with different reagents, suggested that the transmembrane domains of the enzyme are more resistant to SDSinduced unfolding than its other domains. These findings (a) indicate that the partially unfolded oligomer(s) retaining partial function is the intermediate in the SDS-induced denaturation of the native membrane enzyme having the minimum oligomeric structure of (␣,␤,␥) 2 and (b) suggest potential functions for Na ؉ /K ؉ -ATPase with intrinsically unfolded domains. Mixtures of solubilized/ partially unfolded enzyme and membrane-bound enzyme exhibited cross-linking patterns and Na ؉ occlusion capacities different from those of either enzyme species, suggesting that the two interact. Formation of the partially unfolded enzyme during standard purification procedure for the preparation of the membrane-bound enzyme was shown, indicating that it is necessary to ensure the separation of the partially unfolded enzyme from the membrane-bound enzyme to avoid the distortion of the properties of the latter.

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

“…We used one such procedure that we and others had used before, 14,15 but in the final step involving density gradient centrifugation of membrane fragments, we looked not only for fractions enriched with Na + /K + -ATPase but also for those enriched with caveolin-1. The results shown for a typical experiment (Figure 1A,B) indicated clear separation of light membrane fragments containing most of caveolin-1 from heavy fragments containing the major peak of Na + /K + -ATPase activity.…”

“…(16) P i was assayed with Malachite Green as described previously. (7) The maximal level of the phosphoenzyme intermediate of Na + /K + -ATPase was measured using [γ- 32 P]ATP as described previously, 14−16 and the molar activity of the enzyme was calculated as indicated. (17) Cholesterol and protein assays were conducted as described previously.…”

“…Membrane samples were reacted with BS 3 and DDS as described previously(14) and then subjected to SDS–PAGE and immunodetection with the indicated antibodies.…”

“…The customer-designed antibody against the 14 C-terminal residues of the rat γ-subunit of Na + /K + -ATPase(14) was produced by Sigma-Genosys (The Woodlands, TX). BS 3 and DSS were purchased from Thermo Fisher Scientific (Rockford, IL).…”

NMR of membrane proteins in micelles and bilayers: The FXYD family proteins

“…Notable recent examples include a homotetrameric bacterial potassium channel, KcsA [55], and a bacterial mercury transport protein MerF [11]. Furthermore, since SDS purification of the Na,K-ATPase maintains the non-covalent associations of α, β, and FXYD subunits, and yields highly functional enzyme [56][57][58], we reasoned that this detergent would be also be a good choice for FXYD structural studies.…”