Reconstitution of (Na+ + K+)-ATPase proteoliposomes having the same turnover rate as the membranous enzyme.
Membranous (Na+ + K+)-ATPase from the electric eel was solubilized with 3-[3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate (Chaps). 50 to 70% of the solubilized enzyme was reconstituted in egg phospholipid liposomes containing cholesterol by using Chaps. The obtained proteoliposomes consisted of large vesicles with a diameter of 134 +/- 24 nm as the major component, and their protein/lipid ratio was 1.25 +/- 0.07 g protein/mol phospholipid. The intravesicular volume of these proteoliposomes is too small to consistently sustain the intravesicular concentrations of ligands, especially K+, during the assay. The decrease in K+ concentration was cancelled by the addition of 20 microM valinomycin in the assay medium. The low value of the protein/lipid ratio suggests that these proteoliposomes contain one Na+/K+-pump particle with a molecular mass of 280 kDa per one vesicle as the major component. In these proteoliposomes, the specific activity of the (Na+ + K+)-ATPase reaction was 10 mumol Pi/mg protein per min, and the turnover rate of the ATP-hydrolysis was 3500 min-1, the same as the original enzyme under the same assay condition. The ratio of transported Na+ to hydrolyzed ATP was 3, the same as that in the red cell. The proteoliposomes could be disintegrated by 40-50 mM Chaps without any significant inactivation. This disintegration of proteoliposomes nearly tripled the ATPase activity compared to the original ones and doubled the specific ATPase activity compared to the membranous enzyme, but the turnover rate was the same as the original proteoliposomes and the membranous enzyme. This disintegration of proteoliposomes by Chaps suggests the selective incorporation of the (Na+ + K+)-ATPase particle into the liposomes and the asymmetric orientation of the (Na+ + K+)-ATPase particle in the vesicle.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002784:Cholesterol / Q000494:pharmacology; D002793:Cholic Acids; D004593:Electrophorus; D006868:Hydrolysis; D008854:Microscopy, Electron; D011510:Proteolipids / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013997:Time Factors
D002793:Cholic Acids; D011510:Proteolipids; C020485:proteoliposomes; D000255:Adenosine Triphosphate; D002784:Cholesterol; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate