J Biol Chem, Vol. 273, Issue 44, 28813-28821, October 30, 1998

Molecular Distance Measurements Reveal an ()₂ Dimeric Structure of Na⁺/K⁺-ATPase
HIGH AFFINITY ATP BINDING SITE AND K⁺-ACTIVATED PHOSPHATASE RESIDE ON DIFFERENT -SUBUNITS

Holger Linnertz, Petra Urbanova^¶, Tomas Obsil^¶, Petr Herman, Evzen Amler^¶, and Wilhelm Schoner

From the  Institute of Biochemistry and Endocrinology, Justus-Liebig-University Giessen, Frankfurter Str. 100, D-35392 Giessen, Germany, ^¶ Institute of Physiology, Czech Academy of Sciences, Videnska 1083, Cz-142 20 Prague 4, Czech Republic, and  Institute of Physics, Charles University, Ke Karlovou 5, Cz-121 16 Prague 2, Czech Republic

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 -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/-subunit of 0.6 and of ErITC/-subunit of 0.48 indicate 2 ATP sites per ()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 -subunit since the diameter of  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 ()₂ diprotomer and works as a functional dimer (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321).