Geisler Markus
Institut für Biochemie der Pflanzen. Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf. Germany.
Biol Proced Online. 1998 May 14;1:70-80. doi: 10.1251/bpo9.
In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993) et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20-25% of the membrane protein. The purification of the Synecho-cystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC(50)= 119 &mgr;M) and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.
为了表征集胞藻6803的P型ATP酶[盖斯勒（1993年）等人，《分子生物学杂志》234卷，第1284页]并便于其纯化，我们在一株缺乏ATP酶的大肠杆菌菌株中表达了带有N端6xHis标签的ATP酶版本。表达的ATP酶经免疫检测为一条约97 kDa的主带，定位于大肠杆菌质膜，约占膜蛋白的20 - 25%。本文描述了在天然和变性条件下通过单步镍亲和层析纯化集胞藻6xHis - ATP酶的方法。ATP酶活性和磷酸中间体的形成证实了该酶的完整功能：该ATP酶受钒酸盐抑制（IC(50)= 119 μM），酸性PAGE显示的磷酸化酶中间体的形成依赖于钙，表明集胞藻P - ATP酶作为一种钙泵发挥作用。
