Affinity labelling with MgATP analogues reveals coexisting Na⁺ and K⁺ forms of the α‐subunits of Na⁺/K⁺‐ATPase

<jats:p>To test the hypothesis that Na<jats:sup>+</jats:sup>/K<jats:sup>+</jats:sup>‐ATPase works as an (αβ)<jats:sub>2</jats:sub>‐diprotomer with interacting catalytic α‐subunits, tryptic digestion of pig kidney enzyme, that had been inactivated with substitution‐inert MgATP complex analogues, was performed. This led to the demonstration of coexisting C‐terminal Na<jats:sup>+</jats:sup>‐like 80‐kDa as well as K<jats:sup>+</jats:sup>‐like 60‐kDa peptides and N‐terminal 40‐kDa peptides of the α‐subunit. To localize the ATP binding sites on tryptic peptides, studies with radioactive MgATP complex analogues were performed: Co(NH<jats:sub>3</jats:sub>)<jats:sub>4</jats:sub>‐8‐N<jats:sub>3</jats:sub>‐ATP specifically modified the E<jats:sub>2</jats:sub>ATP (low affinity) binding site of Na<jats:sup>+</jats:sup>/K<jats:sup>+</jats:sup>‐ATPase with an inactivation rate constant (<jats:italic>k</jats:italic><jats:sub>2</jats:sub>) of 12 × 10<jats:sup>−3</jats:sup>·min<jats:sup>−1</jats:sup> at 37 °C and a dissociation constant (<jats:italic>K</jats:italic><jats:sub>d</jats:sub>) of 207 ± 28 µ<jats:sc>m</jats:sc>. Tryptic digestion of the [γ<jats:sup>32</jats:sup>P]Co(NH<jats:sub>3</jats:sub>)<jats:sub>4</jats:sub>‐8‐N<jats:sub>3</jats:sub>‐ATP‐inactivated and photolabelled α‐subunit (<jats:italic>M</jats:italic><jats:sub>r</jats:sub> = 100 kDa) led, in the absence of univalent cations, to a K<jats:sup>+</jats:sup>‐like C‐terminal 60‐kDa fragment which was labelled in addition to an unlabelled Na<jats:sup>+</jats:sup>‐like C‐terminal 80‐kDa fragment. Tryptic digestion of [α<jats:sup>32</jats:sup>P]‐or [γ<jats:sup>32</jats:sup>P]Cr(H<jats:sub>2</jats:sub>O)<jats:sub>4</jats:sub>ATP – bound to the E<jats:sub>1</jats:sub>ATP (high affinity) site – led to the labelling of a Na<jats:sup>+</jats:sup>‐like 80‐kDa fragment besides the immediate formation of an unlabelled K<jats:sup>+</jats:sup>‐like N‐terminal 40‐kDa fragment and a C‐terminal 60‐kDa fragment. Because a labelled Na<jats:sup>+</jats:sup>‐like 80‐kDa fragment cannot result from an unlabelled K<jats:sup>+</jats:sup>‐like 60‐kDa fragment, and because unlabelled α‐subunits did not show any catalytic activity, the findings are consistent with a situation in which Na<jats:sup>+</jats:sup>‐ and K<jats:sup>+</jats:sup>‐like conformations are stabilized by tight binding of substitution‐inert MgATP complex analogues to the E<jats:sub>1</jats:sub>ATP and E<jats:sub>2</jats:sub>ATP sites. Hence, all data are consistent with the hypothesis that ATP binding induces coexisting Na<jats:sup>+</jats:sup> and K<jats:sup>+</jats:sup> conformations within an (αβ)<jats:sub>2</jats:sub>‐diprotomeric Na<jats:sup>+</jats:sup>/K<jats:sup>+</jats:sup>‐ATPase.</jats:p>