An extra phosphorylation of Na+,K(+)-ATPase by paranitrophenylphosphate (pNPP): evidence for the oligomeric nature of the enzyme.
Paranitrophenylphosphate (pNPP) induced fluorescence changes in fluorescence isothiocyanate (FITC)-labeled Na+,K(+)-ATPase preparations. The extents of changes were similar to those induced by acetylphosphate (AcP) accompanying accumulation of a K(+)-sensitive phosphoenzyme (E2P) and an ouabain bound phosphoenzyme in the presence of Mg2+ and 16 mM Na+. Phosphoenzymes formed from [32P]pNPP were shown to turn over. The ratio of the maximum amount of the phosphoenzyme formed from pNPP to that of the phosphoenzyme formed from ATP and that of the ouabain-enzyme complex under steady-state conditions was shown to be close to 1:0.5:1. Such extra phosphorylation has hitherto only been observed in a transient state with the additions of high concentrations of ATP [Peluffo, R.D., Garrahan, P.J., and Rega, A. (1992) J. Biol. Chem. 267, 6596-6601]. Our data are compatible with the simultaneous presence of high and low affinity ATP-binding sites in Na+,K(+)-ATPase [Hamer, E. and Schoner, W. (1993) Eur. J. Biochem. 213, 743-748]. The maximum amount of paranitrophenol-sensitive fraction to synthesize [32P]pNPP in fully accumulated ADP-sensitive phosphoenzyme (E1P) from [32P]ATP was around 1/4 of the amount of ouabain-enzyme complex. These data and others indicate that a much higher degree of oligomerization, rather than (alpha beta)2, may be the functional unit of the enzyme in the membranes.
Yamazaki
A
A
Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo.
Kaya
S
S
Tsuda
T
T
Araki
Y
Y
Hayashi
Y
Y
Taniguchi
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004285:Dogs; D016650:Fluorescein-5-isothiocyanate; D005453:Fluorescence; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism* / Q000494:pharmacology*; D009943:Organophosphorus Compounds / Q000378:metabolism* / Q000494:pharmacology*; D010042:Ouabain / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects* / Q000378:metabolism*; D013552:Swine
D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010761:Phosphorus Radioisotopes; C008644:nitrophenylphosphate; D010042:Ouabain; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate