Half-site modification of Lys-480 of the Na+,K+-ATPase α-chain with pyridoxal 5′-diphospho-5′-adenosine reduces ATP-dependent phosphorylation stoichiometry from half to a quarter

Pig and dog kidney Na+,K+-ATPase preparations, irrespective of specific activity, showed similar to 0.5 mol of maximum phosphorylation/mol alpha-chain for ATP or acetyl phosphate (AcP) at steady state conditions. Pyridoxal 5'-diphospho-5'-adenosine (AP(2)PL)-treated pig kidney enzymes containing similar to 0.5 mol of AP(2)PL probe at Lys-480/ mol (Tsuda, T., Kaya, S., Funatsu, H., Hayashi, Y., and Taniguchi, K. (1998) J. Biochem. (Tokyo) 123, 169-174) showed a quarter-site phosphorylation by ATP and half-site phosphorylation from AcP. The addition of 10 mu M ATP to the Mg2+-Na+-bound AP(2)PL enzyme induced rapid quarter-site phosphorylation (47/s), followed by two different AP(2)PL fluorescence changes, a rapid decrease (29/s) and a slow increase (1.1/s). The addition of 1 mM AcP to the Mg2+-Na+-bound AP(2)PL enzyme induced a slow half-site phosphorylation (3/s), followed by a monophasic AP(2)PL fluorescence increase (1.2/s). After treatment of the AP(2)PL enzyme with fluorescein 5'-isothiocyanate to modify Lys-501 fully, the Mg2+-Na+-dependent phosphorylation capacity from ATP of the resulting AP(2)PL-fluorescein 5'-isothiocyanate enzyme was reduced to similar to 6% without significant changes in half-site phosphorylation capacity with respect to AcP, dynamic AP(2)PL fluorescence change by ATP and change by AcP. These data and others support the hypothesis that the functional membrane-bound Na+,K+-ATPase has tetrameric properties.