大鼠脑ATP酶受（Na⁺ + Mg²⁺）和（Na⁺ + Ca²⁺）刺激的磷酸化作用的凝胶电泳鉴定

Gel electrophoretic identity of the (Na+ + Mg-2+)- and (Na+ + Ca-2+)-stimulated phosphorylations of rat brain ATPase.

作者信息

Tobin T, Akera T, Brody T M

出版信息

Biochim Biophys Acta. 1975 Apr 21;389(1):117-25. doi: 10.1016/0005-2736(75)90389-2.

Abstract

The classical E2-P intermediate of (Na+ + K+)-ATPase dephosphorylates readily in the presence of K+ and is not affected by the addition of ADP. To determine the significance in the reaction cycle of (Na+ + K+)-ATPase of kinetically atypical phosphorylations of rat brain (Na+ + K+)-ATPase we compared these phosphorylated components with the classical E2-P intermediate of this enzyme by gel electrophoresis. When rat brain (Na+ + K+)-ATPase was phosphorylated in the presence of high concentrations of Na+ a proportion of the phosphorylated material formed was sensitive to ADP but resistant to K+. Similarly, if phosphorylation was carried out in the presence of Na+ and Ca-2+ up to 300 pmol/mg protein of a K+ -resistant, ADP-sensitive material were formed. If phosphorylation was from [gamma-32-P]CTP up to 800 pmol-32-P/mg protein of an ADP-resistant, K+ -sensitive phosphorylated material were formed. On gel electrophoresis these phosphorylated materials co-migrated with authentic Na+ -stimulated, K+ -sensitive, E2-P-phosphorylated intermediate of (Na+ + K+)-ATPase, supporting suggestions that they represent phosphorylated intermediates in the reaction sequence of this enzyme.

摘要

（Na⁺ + K⁺）-ATP 酶的经典 E2-P 中间体在有 K⁺存在时很容易去磷酸化，并且不受添加 ADP 的影响。为了确定大鼠脑（Na⁺ + K⁺）-ATP 酶动力学非典型磷酸化在其反应循环中的意义，我们通过凝胶电泳将这些磷酸化成分与该酶的经典 E2-P 中间体进行了比较。当大鼠脑（Na⁺ + K⁺）-ATP 酶在高浓度 Na⁺存在下被磷酸化时，形成的一部分磷酸化物质对 ADP 敏感但对 K⁺有抗性。同样，如果在 Na⁺和 Ca²⁺存在下进行磷酸化，会形成高达 300 pmol/mg 蛋白质的对 K⁺有抗性、对 ADP 敏感的物质。如果磷酸化是由[γ-³²P]CTP 进行的，则会形成高达 800 pmol - ³²P/mg 蛋白质的对 ADP 有抗性、对 K⁺敏感的磷酸化物质。在凝胶电泳中，这些磷酸化物质与真正的（Na⁺ + K⁺）-ATP 酶的 Na⁺刺激、K⁺敏感的 E2-P 磷酸化中间体共同迁移，支持了它们代表该酶反应序列中磷酸化中间体的观点。