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Am J Physiol Cell Physiol 275: C1167-C1177, 1998;
0363-6143/98 $5.00
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Vol. 275, Issue 4, C1167-C1177, October 1998

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

Douglas R. Yingst1, Shang-You Yang2, and Rick Schiebinger2

Departments of 1 Physiology and 2 Internal Medicine, Division of Endocrinology, Wayne State University School of Medicine, Detroit, Michigan 48201

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 ~2 to 15 µmol 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.

phosphorylation; kinase; phosphatase; sodium; potassium; rat adrenal glomerulosa; rat kidney; dog kidney; affinity chromatography


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