Department of Pharmacology, the University of Wisconsin, Madison, Wisconsin 53706.
J Gen Physiol. 1969 Jul 1;54(1):327-42. doi: 10.1085/jgp.54.1.327.
The phosphorylated intermediate in the (Na + K)-activated adenosine triphosphatase (Na-K ATPase) has been characterized as an L-glutamyl-gamma-phosphate residue in the enzyme. This has been accomplished by digestion of the phosphorylated and nonphosphorylated forms of the enzyme with pepsin, reaction of the pepsin digests with [2,3-(3)H]N-(n-propyl)hydroxylmine, further digestion of the derivatized peptides with pronase in the presence of carrier L-glutamyl-gamma-N-(n-propyl)hydroxamate and carrier L-aspartyl-N-(n-propyl)hydroxamate, and chromatographic purification. An increment in radioactivity migrated with authentic L-glutamyl-gamma-N-(n-propyl)hydroxamate in a total of seven electrophoretic and chromatographic systems and on gel filtration. No increment in radioactivity was associated with authentic L-aspartyl-beta-N-(n-propyl)hydroxamate in five out of the seven chromatographic and electrophoretic systems. At the last stage of purification the radioactivity from the phosphorylated enzyme which migrated as L-glutamyl-gamma-N-(n-propyl)hydroxamate was 2(1/2) times that from the nonphosphorylated enzyme. On the basis of these results it is concluded that the phosphorylated intermediate in the Na-K ATPase is an L-glutamyl-gamma-phosphate residue. The beef brain Na-K ATPase has been solubilized with the nonionic detergent, Lubrol, and has been purified 10 times over that in the original microsomes. The soluble enzyme remains stable in the presence of ATP and either Na(+) or K(+). If the partially purified enzyme is electrophoresed in 3% polyacrylamide, followed by incubation with ATP, Na(+), K(+), and Mg(++), a single, somewhat diffuse, ATPase band, which is ouabain-sensitive is seen. Protein impurities are also seen on the gel. Gel electrophoresis, after treatment of the partially purified enzyme with phenol-acetic acid-urea, shows about 12 discrete protein bands. Studies on the site-directed alkylation of the (Na + K)-activated adenosine triphosphatase with haloacetate derivatives of cardiotonic steroids are reviewed. Efforts are now underway to specifically alkylate the cardiotonic steroid site of the Na-K ATPase with hellebrigenin 3-[2-(3)H]iodoacetate and to purify the subunit of the enzyme containing the cardiotonic steroid site by following radioactivity. Finally, a working model for the role of the Na-K ATPase in the coupled transport of Na and K is presented.
（Na + K）-激活的三磷酸腺苷酶（Na-K ATPase）的磷酸化中间产物已被鉴定为酶中的 L-谷氨酰-γ-磷酸残基。这是通过用胃蛋白酶消化磷酸化和非磷酸化形式的酶，用 [2,3-（3）H]N-（正丙基）羟胺与胃蛋白酶消化产物反应，在存在载体 L-谷氨酰-γ-N-（正丙基）羟胺和载体 L-天冬酰-N-（正丙基）羟胺的情况下用蛋白酶进一步消化衍生肽，并通过电泳和层析进行色谱纯化。在总共七种电泳和层析系统以及凝胶过滤中，放射性物质的增加与真实的 L-谷氨酰-γ-N-（正丙基）羟胺一起迁移。在七种层析和电泳系统中的五种系统中，没有与真实的 L-天冬酰-β-N-（正丙基）羟胺相关的放射性物质增加。在纯化的最后阶段，从作为 L-谷氨酰-γ-N-（正丙基）羟胺迁移的磷酸化酶中获得的放射性物质是未磷酸化酶的 2（1/2）倍。基于这些结果，可以得出结论，Na-K ATPase 中的磷酸化中间产物是 L-谷氨酰-γ-磷酸残基。牛脑 Na-K ATPase 已用非离子型去污剂 Lubrol 溶解，并比原始微粒体中纯化了 10 倍。在存在 ATP 和 Na(+)或 K(+)的情况下，可溶性酶保持稳定。如果将部分纯化的酶在 3％聚丙烯酰胺中电泳，然后用 ATP、Na(+)、K(+)和 Mg(++)孵育，则可以看到一个单一的，有些弥散的，对哇巴因敏感的 ATP 酶带。在凝胶上还可以看到蛋白质杂质。用苯酚-乙酸-尿素处理部分纯化的酶后，进行凝胶电泳，显示约 12 个离散的蛋白质带。审查了卤代乙酸衍生物对强心甾类药物的（Na + K）-激活的三磷酸腺苷酶的定点烷基化研究。目前正在努力用 hellebrigenin 3-[2-（3）H]碘乙酸特异性烷基化 Na-K ATPase 的强心甾类药物位点，并通过放射性追踪纯化含有强心甾类药物位点的酶亚基。最后，提出了 Na-K ATPase 在 Na 和 K 偶联转运中的作用的工作模型。
