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

摘要

Membrane-bound Na+/K+-ATPase purified from dog kidney outer medulla was solubilized with octaethylene glycol n-dodecyl ether (C12E8) and incubated with [3H]ouabain in the presence of NaCl, ATP and MgCl2 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 MgCl2 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 C12E8, 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.