Chignell C F, Titus E
Proc Natl Acad Sci U S A. 1969 Sep;64(1):324-9. doi: 10.1073/pnas.64.1.324.
A microsomal adenosine triphosphatase (ATPase) that requires both sodium and potassium ions is thought to be identical with, or an integral part of, the active cation transport system located in cell membranes. Attempts to isolate and purify (Na(+) + K(+))-ATPase have met with limited success because solubilization of microsomal protein causes partial, if not complete, loss of enzymatic activity. We now report the isolation from rat kidney microsomes of proteins which, though enzymatically inactive, could still be identified as components of the (Na(+) + K(+))-ATPase system. Phosphoproteins known to be intermediates in the hydrolysis of ATP by (Na(+) + K(+))-ATPase were prepared by incubating rat kidney microsomes with gamma-labeled ATP(33) in the presence of sodium or with P(32)-orthophosphate in the presence of ouabain. After the P(32)- and P(33)-labeled microsomes had been dissolved in phenol-acetic acid-urea, the resultant solutions were mixed and subjected to polyacrylamide gel electrophoresis. The radioactivity from both phosphorus isotopes was found almost exclusively in one of the resultant 21 protein bands. In contrast, the radioactive protein from DFP(32)-labeled microsomes moved slightly faster than the radioactive protein from microsomes labeled with P(33)-orthophosphate in the presence of ouabain. DFP inhibits (Na(+) + K(+))-ATPase by reacting with a nucleophilic site at or near the active site. These results suggest that while a single protein component of (Na(+) + K(+))-ATPase accepts the terminal phosphate from ATP, the final splitting of this phosphoprotein intermediate may be catalyzed by nucleophilic sites on a second protein.
一种需要钠离子和钾离子的微粒体腺苷三磷酸酶（ATP酶）被认为与位于细胞膜中的活性阳离子转运系统相同，或是其不可或缺的一部分。分离和纯化（Na⁺ + K⁺）-ATP酶的尝试取得的成功有限，因为微粒体蛋白的溶解会导致酶活性部分丧失，即便不是完全丧失。我们现在报告从大鼠肾脏微粒体中分离出的蛋白质，这些蛋白质虽然没有酶活性，但仍可被鉴定为（Na⁺ + K⁺）-ATP酶系统的组成部分。已知（Na⁺ + K⁺）-ATP酶水解ATP过程中的中间产物磷蛋白，是通过在有钠存在的情况下用γ标记的ATP³³孵育大鼠肾脏微粒体，或在有哇巴因存在的情况下用³²P-正磷酸盐孵育制备的。在³²P和³³P标记的微粒体溶解于苯酚-乙酸-尿素后，将所得溶液混合并进行聚丙烯酰胺凝胶电泳。两种磷同位素的放射性几乎完全集中在所得的21条蛋白带中的一条上。相比之下，在有哇巴因存在的情况下，来自DFP³²标记微粒体的放射性蛋白比来自用³³P-正磷酸盐标记的微粒体的放射性蛋白迁移得稍快一些。DFP通过与活性位点或其附近的亲核位点反应来抑制（Na⁺ + K⁺）-ATP酶。这些结果表明，虽然（Na⁺ + K⁺）-ATP酶的单一蛋白质成分从ATP接受末端磷酸基团，但这种磷蛋白中间体的最终裂解可能由第二种蛋白质上的亲核位点催化。
