Purification of active Na+-K+-ATPase using a new ouabain-affinity column.
Ouabain, a specific inhibitor of Na+-K+-ATPase, was coupled to epoxy agarose via a 13-atom spacer to make an affinity column that specifically binds Na+-K+-ATPase. Na+-K+-ATPase from rat and dog kidney was bound to the column and was eluted as a function of enzyme conformation, altered by adding specific combinations of ligands. Na+-K+-ATPase from both sources bound to the column in the presence of Na + ATP + Mg and in solutions containing 30 mM K. No binding was observed in the presence of Na or Na + ATP. These experiments suggest that Na+-K+-ATPase binds to the column under the same conditions that it binds to untethered ouabain. Na+-K+-ATPase already bound to the column was competitively eluted with excess free Na + ouabain or with Na + ATP. The latter eluted active enzyme. For comparable amounts of bound Na+-K+-ATPase, Na + ouabain and Na + ATP eluted more rat than dog Na+-K+-ATPase, consistent with the lower affinity of the rat Na+-K+-ATPase for ouabain. The ouabain-affinity column was used to purify active Na+-K+-ATPase from rat kidney microsomes and rat adrenal glomerulosa cells. The specific activity of the kidney enzyme was increased from approximately 2 to 15 micromol Pi . mg-1 . min-1. Na+-K+-ATPase purified from glomerulosa cells that were prelabeled with [32P]orthophosphate was phosphorylated on the alpha-subunit, suggesting that these cells contain a kinase that phosphorylates Na+-K+-ATPase.
Yingst
D R
DR
Department of Physiology, Division of Endocrinology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Yang
S Y
SY
Schiebinger
R
R
D000255:Adenosine Triphosphate; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D007668:Kidney / Q000201:enzymology*; D008274:Magnesium; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D010042:Ouabain; D051381:Rats; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification* / Q000378:metabolism
D007202:Indicators and Reagents; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium