Half-site modification of Lys-480 of the Na+,K+-ATPase alpha-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 approximately 0.5 mol of maximum phosphorylation/mol alpha-chain for ATP or acetyl phosphate (AcP) at steady state conditions. Pyridoxal 5'-diphospho-5'-adenosine (AP2PL)-treated pig kidney enzymes containing approximately 0.5 mol of AP2PL 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 microM ATP to the Mg2+-Na+-bound AP2PL enzyme induced rapid quarter-site phosphorylation (47/s), followed by two different AP2PL 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 AP2PL enzyme induced a slow half-site phosphorylation (3/s), followed by a monophasic AP2PL fluorescence increase (1.2/s). After treatment of the AP2PL enzyme with fluorescein 5'-isothiocyanate to modify Lys-501 fully, the Mg2+-Na+-dependent phosphorylation capacity from ATP of the resulting AP2PL-fluorescein 5'-isothiocyanate enzyme was reduced to approximately 6% without significant changes in half-site phosphorylation capacity with respect to AcP, dynamic AP2PL 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.