Shono M., Wada M., Fujii T.
Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
Plant Physiol. 1995 Aug;108(4):1615-1621. doi: 10.1104/pp.108.4.1615.
A Na+ -ATPase was partially purified from plasma membranes of the marine alga Heterosigma akashiwo. The plasma membranes of H. akashiwo cells were collected by differential centrifugation with subsequent discontinuous gradient centrifugation. Na+ -ATPase activity was associated with the resultant plasma membrane fraction and was stimulated to the greatest extent in the presence of 100 to 200 mM Na+, 10 mM K+, and 5 mM Mg2+ ions, pH 8.0. The Km value for Na+ ions was 12.2 mM. An apparent Km value for ATP was 880 [mu]M. A 140-kD phosphorylated intermediate was also detected in the same fraction in the presence of both Mg2+ and Na+ ions, and this protein was dephosphorylated upon the addition of K+ ions. We could partially purify the 140-kD protein after solubilization by Suc monolaurate and fractionation by sequential column chromatography on Sephacryl S-300, DEAE-Sepharose CL-6B, and Mono-Q columns. The purified 140-kD polypeptide could also be phosphorylated and be detected after acid sodium dodecyl sulfate-polyacryl-amide gel electrophoresis in the presence of Na+ and Mg2+ ions.
从海洋藻类赤潮异弯藻的质膜中部分纯化出一种钠钾ATP酶。通过差速离心随后进行不连续梯度离心收集赤潮异弯藻细胞的质膜。钠钾ATP酶活性与所得的质膜部分相关，并且在100至200 mM Na⁺、10 mM K⁺和5 mM Mg²⁺离子、pH 8.0存在的情况下受到最大程度的刺激。Na⁺离子的Km值为12.2 mM。ATP的表观Km值为880 μM。在同时存在Mg²⁺和Na⁺离子的同一部分中也检测到一种140-kD的磷酸化中间体，并且在加入K⁺离子后该蛋白质发生去磷酸化。在用月桂酸蔗糖溶解并通过在Sephacryl S-300、DEAE-琼脂糖CL-6B和Mono-Q柱上进行连续柱色谱分离后，我们可以部分纯化140-kD蛋白质。纯化的140-kD多肽在存在Na⁺和Mg²⁺离子的情况下，经酸性十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后也可以被磷酸化并被检测到。
