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J Biol Chem, Vol. 273, Issue 50, 33759-33765, December 11, 1998

Affinity Labeling of Two Nucleotide Sites on Na,K-ATPase Using 2'(3')-O-(2,4,6-Trinitrophenyl)8-azidoadenosine 5'-[-³²P]Diphosphate (TNP-8N₃-[-³²P]ADP) as a Photoactivatable Probe
LABEL INCORPORATION BEFORE AND AFTER BLOCKING THE HIGH AFFINITY ATP SITE WITH FLUORESCEIN ISOTHIOCYANATE

From the Transport ATPase Laboratory, Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, United Kingdom

ATP and its analogues act on the minimal functional unit of Na,K-ATPase, the protomer, with high and low affinity effects. Fluorescein isothiocyanate (FITC) irreversibly blocks the high affinity, or catalytic, ATP site, and yet the surviving K⁺-phosphatase activity of soluble FITC-modified protomers can be photoinactivated by 2'(3')-O-trinitrophenyl (TNP)-8N₃-ADP (Ward, D. G., and Cavieres, J. D. (1998) J. Biol. Chem. 273, 14277-14284). We have now used TNP-8N₃-[-³²P]ADP as a photoaffinity label for Na,K-ATPase. The native enzyme can be photolabeled at 5 µM TNP-8N₃-[-³²P]ADP, and ATP or FITC treatment prevents labeling of the  chain. At 25 µM, however, TNP-8N₃-[-³²P]ADP can be incorporated in the FITC-modified  chain, concurrently with the inactivation of the K⁺-phosphatase activity, to an extrapolated level of 0.5-1.2 mol of ³²P-probe per mol of  chain. Photoinactivation and labeling are prevented by TNP-ADP, vanadate, or strophanthidin and are promoted by Na⁺ or Mg²⁺, but not K⁺. The cation effects suggest that the fluorescein-modified enzyme incorporates the TNP-8N₃-[-³²P]ADP·Mg complex preferentially, and the free probe when in the E1 enzyme form and after occupation of a low-affinity Na⁺ site. Partial trypsinolysis reveals that the point of TNP-8N₃-[-³²P]ADP attachment is on the C-terminal 58-kDa fragment of the FITC-modified  chain. The affinity labeling of the fluorescein enzyme by TNP-8N₃-[-³²P]ADP endorses the view that two nucleotide sites can be occupied simultaneously in each  subunit of Na,K-ATPase.

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