BACKGROUND & AIMS:
:In the presence of ascorbate/H(2)O(2), ATP-Fe(2+) or AMP-PNP-Fe(2+) complexes act as affinity cleavage reagents, mediating selective cleavage of the alpha subunit of Na,K-ATPase at high affinity ATP-Mg(2+) sites. The cleavages reveal contact points of Fe(2+) or Mg(2+) ions. In E(1) and E(1)Na conformations, two major cleavages are detected within the conserved (708)TGDGVNDSPALKK sequence (at V712 and nearby), and one (E(1)Na) or two (E(1)) minor cleavages near V440. In media containing sodium and ATP, Fe(2+) substitutes for Mg(2+) in activating phosphorylation and ATP hydrolysis. In the E(1)P conformation, cleavages are the same as in E(1). Fe(2+) is not bound tightly. By contrast, in the E(2)P conformation, the pattern is different. A major cleavage occurs near the conserved sequence (212)TGES, whereas those in TGDGVNDSPALKK are less prominent. Fe(2+) is bound very tightly. On E(2)P hydrolysis, the Fe(2+) dissociates. The results are consistent with E(1)<-->E(2) conformation-dependent movements of cytoplasmic domains and sites for P(i) and Mg(2+) ions, inferred from previous Fe-cleavage experiments. Furthermore, these concepts fit well with the crystal structure of Ca-ATPase [Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature (London) 405, 647-655]. Altered ligation of Mg(2+) ions in E(2)P may be crucial in facilitating nucleophilic attack of water on the OP bond. Mg(2+) ions may play a similar role in all P-type pumps. As affinity cleavage reagents, ATP-Fe(2+) or other nucleotide-Fe(2+) complexes could be widely used to investigate nucleotide binding proteins.
背景与目标:
: 在抗坏血酸盐/H(2)O(2) 存在下,ATP-Fe(2 +) 或AMP-PNP-Fe(2 +) 配合物作为亲和裂解试剂,介导Na的 α 亚基的选择性裂解,高亲和力ATP-Mg(2 +) 位点的K-ATP酶。裂解揭示了Fe(2) 或Mg(2) 离子的接触点。在E(1) 和E(1)Na构象中,在保守 (708)TGDGVNDSPALKK序列 (在V712和附近) 内检测到两个主要切割,以及在v440附近检测到一个 (E(1)Na) 或两个 (E(1)) 次要切割。在含有钠和ATP的介质中,Fe(2) 代替Mg(2) 激活磷酸化和ATP水解。在E(1)P构象中,裂解与E(1) 相同。Fe(2 +) 结合不紧。相比之下,在E(2)P构象中,模式是不同的。在保守序列 (212)TGES附近发生主要切割,而TGDGVNDSPALKK中的切割较不突出。Fe(2 +) 结合得非常紧密。在E(2)P水解时,Fe(2) 解离。结果与E(1)<->E(2) 胞质结构域和P(i) 和Mg(2) 离子位点的构象依赖性运动一致,这是从先前的Fe裂解实验得出的。此外,这些概念与Ca-ATPase的晶体结构非常吻合 [Toyoshima,C.,Nakasako,M.,Nomura,H.和Ogawa,H. (2000) 自然 (伦敦) 405,647-655]。E(2)P中Mg(2) 离子的连接改变对于促进水对OP键的亲核攻击可能至关重要。Mg(2) 离子可能在所有P型泵中起相似的作用。作为亲和裂解试剂,ATP-Fe(2) 或其他核苷酸-Fe(2) 复合物可广泛用于研究核苷酸结合蛋白。