PHOTOAFFINITY-LABELING OF THE ACTIVE-SITE OF THE NA+ K+-ATPASE WITH 4-AZIDO-2-NITROPHENYL PHOSPHATE/

Na+/K+-ATPase will hydrolyze small acylphosphates such as p-nitropheny l phosphate (pNPP) in addition to ATP and can derive sufficient energy from the hydrolysis of these small molecules to catalyze active ion t ransport. In this report, 4-azido-2-nitrophenyl phosphate (ANPP), a ph otoreactive analog of pNPP, was used as a probe of the substrate bindi ng site of dog renal Na+/K+-ATPase. ANPP was slowly hydrolyzed by Na+/ K+-ATPase with a V-max of 0.19 mu mol mg(-1) min(-1) and with an appar ent K-m of 1.0 mM. The K-m for hydrolysis of pNPP was 1.7 mM. ANPP com petitively inhibited the hydrolysis of pNPP with a K-i of 0.37 mM. Bot h the ATPase and pNPPase activity of the Na+/K+-ATPase were irreversib ly inhibited after photolysis of the enzyme and ANPP with UV light, al though neither activity was completely inhibited by up to 200 mu M ANP P. Inhibition of activity was prevented by including 0.2 mM ATP in the reaction or by excluding Mg2+ from the photolysis buffer. Photolysis with [P-32]ANPP labeled only the a subunit of the Na+/K+-ATPase, and t he amount of labeling was substantially reduced by 0.2 mM ATP or in th e absence of Mg2+. The stoichiometry of labeling extrapolated to a max imum of about 1.2 nmol/mg of protein at 100% inhibition of Mg2+-depend ent activity. Limited proteolytic digestion showed labeling sites on n onoverlapping tryptic peptides derived from the a subunit of Na+/K+-AT Pase, and two radiolabeled peptides were purified from an exhaustive t ryptic digest of [P-32]ANPP-labeled Na+/K+-ATPase. One peptide contain ed amino acids Met-379 to Lys-406, and the second contained amino acid s Ala-655 to Lys-676. Amino acids corresponding to Asn-398 and Pro-668 were missing from the sequences and may represent residues derivatize d by ANPP from within the substrate binding site of Na+/K+-ATPase.