Dephosphorylation kinetics of pig kidney Na+,K+-ATPase.
The kinetics of K+-stimulated dephosphorylation of the Na+,K+-ATPase were investigated at pH 7.4, 24 degrees C, and an ATP concentration of 1.0 mM via the stopped-flow technique using the fluorescent label RH421. Two different mixing procedures were used: (a) premixing with ATP to allow phosphorylation to go to completion, followed by mixing with KCl; and (b) simultaneous mixing with ATP and KCl. Using mixing procedure (a), the dephosphorylation rate constant of enzyme complexed with K+ ions could be determined directly to be </=366 s-1 and the rate constant for spontaneous dephosphorylation (without K+) </=60 s-1. The K+ concentration dependence of the observed reciprocal time constant showed half-saturation at a K+ concentration of 2.4-2.6 mM with positive cooperativity involved in the occupation of the K+ binding sites on the E2P conformation of the enzyme. Using mixing procedure (b), it was found that at saturating K+ concentrations the dephosphorylation of the enzyme is rate-limited by its phosphorylation, which occurs with a rate constant of approximately 190 s-1 (1). These results show that all reactions occurring after phosphorylation and prior to dephosphorylation, i.e., the E1P to E2P conformational transition as well as Na+ release and K+ binding steps, must be fast (>190 s-1).
Kane
D J
DJ
Department of Biophysical Chemistry, Max-Planck-Institut für Biophysik, Kennedyallee 70, D-60596 Frankfurt am Main, Germany.
Grell
E
E
Bamberg
E
E
Clarke
R J
RJ
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine
D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium