Quantification of the Na +/K +-pump in solubilized tissue by the ouabain binding method coupled with high-performance gel chromatography

Abstract

Membrane-bound Na +/K +-ATPase purified from dog kidney outer medulla was solubilized with octaethylene glycol n-dodecyl ether (C 12E 8) and incubated with [ 3H]ouabain in the presence of NaCl, ATP and MgCl 2 for 10 min at 0°C. The resulting enzyme was separated, by high-performance gel chromatography executed at 0.2°C, mainly into its ( αβ) 2-diprotomer and αβ-protomer, which both bound stoichiometrically to [ 3H]ouabain. The amounts of ouabain that bound to the tissue itself and its microsomes could be estimated in the same way, as [ 3H]ouabain was found to bind only to the diprotomer and protomer they possessed. The amounts of ouabain that bound to them in the solubilized state were at least 5-times higher than those that did so when they were non-solubilized, suggesting that the surfactant rendered the enzyme accessible to ouabain. When the solubilized tissue (138 mg ml −1 wet tissue) was reacted with ouabain in the presence of 0.1 M NaCl and 4.8 mM MgCl 2 for 10 min at 0°C, maximal ouabain binding was attained in the presence of 18.3 μM [ 3H]ouabain, 1.2 mM ATP and 3 to 5 mg ml −1 C 12E 8, which was common to the outer medulla and human colon cancer cells. The present method enabled the pump number in protein and tissue samples in the range 7.2 · 10 −9 (purified pump) to 1.5 · 10 −12 (cancer tissue) mol/mg protein to be estimated within 2 h.