Purification of active Na+-K+-ATPase using a new ouabain-affinity column

Ouabain, aspecific inhibitor ofNa⁺-K⁺-ATPase,was coupled to epoxy agarose via a 13-atom spacer to make an affinitycolumn that specifically bindsNa⁺-K⁺-ATPase.Na⁺-K⁺-ATPasefrom rat and dog kidney was bound to the column and was eluted as afunction of enzyme conformation, altered by adding specificcombinations of ligands.Na⁺-K⁺-ATPasefrom both sources bound to the column in the presence of Na + ATP + Mgand in solutions containing 30 mM K. No binding was observed in thepresence of Na or Na + ATP. These experiments suggest thatNa⁺-K⁺-ATPasebinds to the column under the same conditions that it binds tountethered ouabain.Na⁺-K⁺-ATPasealready bound to the column was competitively eluted with excess freeNa + ouabain or with Na + ATP. The latter eluted active enzyme. Forcomparable amounts of boundNa⁺-K⁺-ATPase,Na + ouabain and Na + ATP eluted more rat than dogNa⁺-K⁺-ATPase,consistent with the lower affinity of the ratNa⁺-K⁺-ATPasefor ouabain. The ouabain-affinity column was used to purify activeNa⁺-K⁺-ATPasefrom rat kidney microsomes and rat adrenal glomerulosa cells. Thespecific activity of the kidney enzyme was increased from ~2 to 15 µmolP_i · mg¹ · min¹.Na⁺-K⁺-ATPasepurified from glomerulosa cells that were prelabeled with ³²P]orthophosphatewas phosphorylated on the -subunit, suggesting that these cellscontain a kinase that phosphorylatesNa⁺-K⁺-ATPase.