TNP-8N3-ADP photoaffinity labelling of two Na,K-ATPase sequences under separate Na+ plus K+ control

Douglas Ward, M Taylor, KS Lilley, JD Cavieres

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5 Citations (Scopus)


ATP has high- and low-affinity effects on the sodium pump and other P-type ATPases. We have approached this question by using 2‘,3‘-O-(trinitrophenyl)-8-azidoadenosine 5‘-diphosphate (TNP-8N3-ADP) to photoinactivate and label Na,K-ATPase, both in its native state and after covalent FITC block of its high-affinity ATP site. With the native enzyme, the photoinactivation rate constant increases hyperbolically with a KD(TNP-8N3-ADP) of 0.11 μM; TNP-ATP and ATP protect the site with high affinities. The inactivation does not require Na+, but K+ inhibits with a KK‘ of 12 μM; Na+ reverses this effect, with a KNa of 0.17 mM. This pattern suggests that Na+ and K+ are binding at sites in their “intracellular” conformation. It was known that FITC did not abolish the reverse phosphorylation by Pi, or the K+-phosphatase activity, and that TNP-8N3-ADP could subsequently photoinactivate the latter with >100-fold lower affinity; in that case, the cation sites acted as if facing outward [Ward, D. G., and Cavieres, J. D. (1998) J. Biol. Chem. 273, 14277−14284, 33759−33765]. Native and FITC-modified enzymes have now been photolabeled with TNP-8N3-[α-32P]ADP and α-chain soluble tryptic peptides separated by reverse-phase HPLC. With native Na,K-ATPase, three labeled peaks lead to the unique sequence α-470Ile-Val-Glu-Ile-Pro-Phe-Asn-Ser-Thr-Asn-X-Tyr-Gln-Leu-Ser-Ile-His-Lys487, the dropped residue being αLys480. With the FITC enzyme, instead, two independent labeling and purification cycles return the sequence α-721Ala-Asp-Ile-Gly-Val-Ala-Met-Gly-Ile-Ala-Gly-Ser-Asp-Val-Ser-Lys736. These results suggest that Na,K-ATPase also has a low-affinity nucleotide binding region, one that is under distinctive allosteric control by Na+ and K+. Moreover, the cation effects seem compatible with a slow, passive Na+/K+ carrier behavior of the FITC-modified sodium pump.
Original languageEnglish
Pages (from-to)3460-3471
Number of pages12
Issue number10
Publication statusPublished - 14 Mar 2006


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