Investigacion clinica 2009-09-01

Solubilization and partial characterization of ouabain-insensitive Na(+)-ATPase from basolateral plasma membranes of the intestinal epithelial cells.

Freddy Romero

Index: Invest. Clin. 50 , 303-14, (2009)

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Abstract

It has been proposed that intestinal sodium transport is mediated by two different active mechanisms: the ouabain-sensitive Na+/K(+)-ATPase and ouabain-insensitive Na(+)-ATPase. In order to determine the optimum conditions to solubilize the membrane-bound Na(+)-ATPase of enterocyte, basolateral plasma membranes were solubilized using different amounts of octyl glucoside (O.G), Tween 20, octaethylene glycol monododecyl ether (C12E8), and polyoxyethylene 9-lauryl ether (C12E9). Solubilized fractions were assayed for protein concentration and ATPase activity and characterized by electrophoresis analysis. Optimal solubilization of Na(+)-ATPase was obtained after mixing of 1 mg of basolateral plasma membrane with 1.5 mg of C12E9. Under these conditions, C12E9 solubilized over 60% membrane protein and Na(+)- and Na+/K(+)-ATPases activities were recovered over 80% in the soluble fraction without inactivation. In addition, when 25% glycerol and 2 mM ATP were added, the solubilized Na(+)-ATPase was stable after 3 days at 4 degrees C. The C12E9-solubilized Na(+)-ATPase presented the following kinetic characteristics: 1) is only stimulated by the Na+ salt, 2) K0.5 for Na+ = 4.62 +/- 0.06 mM, 3) is similarly stimulated by the Na+ salt of different anions, 4) optimal pH = 7.0, 5) inhibited by furosemide (IC50 = 0.52 +/- 0.10 nm). These kinetic properties of the solubilized Na(+)-ATPase were similar to those described to the native membrane-bound enzyme. This work reports for the first time, solubilization and characterization of a fully active and stable Na(+)-ATPase from basolateral plasma membranes of enterocyte using C12E9.