TNP-8N3-ADP photoaffinity labeling of two Na,K-ATPase sequences under separate Na+ plus K+ control.
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-8N(3)-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 K(D(TNP-8N)3(-)(ADP)) of 0.11 microM; TNP-ATP and ATP protect the site with high affinities. The inactivation does not require Na(+), but K(+) inhibits with a K(K)' of 12 microM; Na(+) reverses this effect, with a K(Na) 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 P(i), or the K(+)-phosphatase activity, and that TNP-8N(3)-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-8N(3)-[alpha-(32)P]ADP and alpha-chain soluble tryptic peptides separated by reverse-phase HPLC. With native Na,K-ATPase, three labeled peaks lead to the unique sequence alpha-(470)Ile-Val-Glu-Ile-Pro-Phe-Asn-Ser-Thr-Asn-X-Tyr-Gln-Leu-Ser-Ile-His-Lys(487), the dropped residue being alphaLys480. With the FITC enzyme, instead, two independent labeling and purification cycles return the sequence alpha-(721)Ala-Asp-Ile-Gly-Val-Ala-Met-Gly-Ile-Ala-Gly-Ser-Asp-Val-Ser-Lys(736). 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.
Ward
Douglas G
DG
Transport ATPase Laboratory, Department of Cell Physiology and Pharmacology, Faculty of Medicine and Biological Sciences, University of Leicester, Leicester LE1 9HN, United Kingdom.
Taylor
Mark
M
Lilley
Kathryn S
KS
Cavieres
José D
JD
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry / Q000378:metabolism; D007668:Kidney / Q000201:enzymology / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000378:metabolism; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D019920:Photoaffinity Labels / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D008024:Ligands; D015335:Molecular Probes; D010455:Peptides; D019920:Photoaffinity Labels; C058475:2',3'-O (2,4,6-trinitrophenyl)-8-azidoadenosine diphosphate; D000244:Adenosine Diphosphate; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D008239:Lysine