Yoda A, Clark A W, Yoda S
Biochim Biophys Acta. 1984 Dec 5;778(2):332-40. doi: 10.1016/0005-2736(84)90377-8.
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.
用电鳗的膜状（Na⁺ + K⁺）-ATP酶与3-[(3-胆酰胺丙基)-二甲基铵基]-1-丙烷磺酸盐（Chaps）进行增溶处理。通过使用Chaps，50%至70%的增溶酶被重构到含有胆固醇的鸡蛋磷脂脂质体中。所获得的蛋白脂质体主要由直径为134±24 nm的大囊泡组成，其蛋白质/脂质比为1.25±0.07 g蛋白质/摩尔磷脂。在测定过程中，这些蛋白脂质体的囊泡内体积过小，无法持续维持囊泡内配体的浓度，尤其是K⁺的浓度。通过在测定介质中添加20 μM缬氨霉素，K⁺浓度的降低得以消除。蛋白质/脂质比的低值表明这些蛋白脂质体每个囊泡含有一个分子量为280 kDa的Na⁺/K⁺泵颗粒作为主要成分。在这些蛋白脂质体中，（Na⁺ + K⁺）-ATP酶反应的比活性为每分钟10 μmol无机磷酸/毫克蛋白质，ATP水解的周转率为3500 min⁻¹，与相同测定条件下的原始酶相同。转运的Na⁺与水解的ATP的比率为3，与红细胞中的相同。40 - 50 mM的Chaps可使蛋白脂质体解体，且无任何明显的失活现象。与原始蛋白脂质体相比，这种蛋白脂质体的解体使ATP酶活性增加了近两倍，与膜状酶相比，比ATP酶活性增加了一倍，但周转率与原始蛋白脂质体和膜状酶相同。Chaps对蛋白脂质体的这种解体表明（Na⁺ + K⁺）-ATP酶颗粒选择性地掺入脂质体以及该颗粒在囊泡中的不对称取向。
