The complex ATP–Fe2+ serves as a specific affinity cleavage reagent in ATP-Mg2+ sites of Na,K-ATPase: Altered ligation of Fe2+ (Mg2+) ions accompanies the E1P→E2P conformational change

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The National Academy of Sciences

RESUMO

In the presence of ascorbate/H2O2, ATP–Fe2+ or AMP-PNP–Fe2+ complexes act as affinity cleavage reagents, mediating selective cleavage of the alpha subunit of Na,K-ATPase at high affinity ATP–Mg2+ sites. The cleavages reveal contact points of Fe2+ or Mg2+ ions. In E1 and E1Na conformations, two major cleavages are detected within the conserved 708TGDGVNDSPALKK sequence (at V712 and nearby), and one (E1Na) or two (E1) minor cleavages near V440. In media containing sodium and ATP, Fe2+ substitutes for Mg2+ in activating phosphorylation and ATP hydrolysis. In the E1P conformation, cleavages are the same as in E1. Fe2+ is not bound tightly. By contrast, in the E2P conformation, the pattern is different. A major cleavage occurs near the conserved sequence 212TGES, whereas those in TGDGVNDSPALKK are less prominent. Fe2+ is bound very tightly. On E2P hydrolysis, the Fe2+ dissociates. The results are consistent with E1↔E2 conformation-dependent movements of cytoplasmic domains and sites for Pi and Mg2+ 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 Mg2+ ions in E2P may be crucial in facilitating nucleophilic attack of water on the O—P bond. Mg2+ ions may play a similar role in all P-type pumps. As affinity cleavage reagents, ATP–Fe2+ or other nucleotide–Fe2+ complexes could be widely used to investigate nucleotide binding proteins.

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