Quantification of the Na+/K(+)-pump in solubilized tissue by the ouabain binding method coupled with high-performance gel chromatography.
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 degrees C. The resulting enzyme was separated, by high-performance gel chromatography executed at 0.2 degrees C. Mainly into its (alpha beta)2-diprotomer and alpha beta-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 degrees C, maximal ouabain binding was attained in the presence of 18.3 microM [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 x 10(-9) (purified pump) to 1.5 x 10(-12) (cancer tissue) mol/mg protein to be estimated within 2 h.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003110:Colonic Neoplasms / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004285:Dogs; D006801:Humans; D007679:Kidney Medulla / Q000201:enzymology / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008861:Microsomes / Q000201:enzymology; D010042:Ouabain / Q000032:analysis / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011485:Protein Binding; D012492:Salts / Q000494:pharmacology; D012542:Scattering, Radiation; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D014407:Tumor Cells, Cultured
D003902:Detergents; D008565:Membrane Proteins; D012492:Salts; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase