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.
Tsuda
T
T
Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060, Japan.
Kaya
S
S
Yokoyama
T
T
Hayashi
Y
Y
Taniguchi
K
K
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels / Q000494:pharmacology; D000818:Animals; D004285:Dogs; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D008961:Models, Structural; D010766:Phosphorylation; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D013552:Swine; D013997:Time Factors
D000345:Affinity Labels; D046911:Macromolecular Substances; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; C048236:pyridoxal 5'-diphospho-5'-adenosine; D008239:Lysine