Molecular Distance Measurements Reveal an (αβ)2Dimeric Structure of Na+/K+-ATPase

Abstract

ATP hydrolysis by Na+/K+-ATPase proceeds via the interaction of simultaneously existing and cooperating high (E1ATP) and low (E2ATP) substrate binding sites. It is unclear whether both ATP sites reside on the same or on different catalytic α-subunits. To answer this question, we looked for a fluorescent label for the E2ATP site that would be suitable for distance measurements by Förster energy transfer after affinity labeling of the E1ATP site by fluorescein 5′-isothiocyanate (FITC). Erythrosin 5′-isothiocyanate (ErITC) inactivated, in an E1ATP site-blocked enzyme (by FITC), the residual activity of the E2ATP site, namely K+-activated p-nitrophenylphosphatase in a concentration-dependent way that was ATP-protectable. The molar ratios of FITC/α-subunit of 0.6 and of ErITC/α-subunit of 0.48 indicate 2 ATP sites per (αβ)2 diprotomer. Measurements of Förster energy transfer between the FITC-labeled E1ATP and the ErITC-labeled or Co(NH3)4ATP-inactivated E2ATP sites gave a distance of 6.45 ± 0.64 nm. This distance excludes 2 ATP sites per α-subunit since the diameter of α is 4–5 nm. Förster energy transfer between cardiac glycoside binding sites labeled with anthroylouabain and fluoresceinylethylenediamino ouabain gave a distance of 4.9 ± 0.5 nm. Hence all data are consistent with the hypothesis that Na+/K+-ATPase in cellular membranes is an (αβ)2 diprotomer and works as a functional dimer (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem.272, 16315–16321).