Solubilization and partial characterization of ouabain-insensitive Na(+)-ATPase from basolateral plasma membranes of the intestinal epithelial cells.
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.
Romero
Freddy
F
Laboratorio de Fisiología Gastrointestinal, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela. frromero@ivic.ve
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000201:enzymology; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D005960:Glucosides / Q000494:pharmacology; D006168:Guinea Pigs; D007413:Intestinal Mucosa / Q000201:enzymology*; D007421:Intestine, Small / Q000166:cytology / Q000201:enzymology; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D011136:Polysorbates / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D003902:Detergents; D005960:Glucosides; D008565:Membrane Proteins; D011136:Polysorbates; C018619:octyl-beta-D-glucoside; C017272:dodecyloctaethyleneglycol monoether; C061442:dodecylnonaoxyethylene glycol monoether; D011092:Polyethylene Glycols; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase