The steady-state kinetic mechanism of ATP hydrolysis catalyzed by membrane-bound (Na+ + K+)-ATPase from ox brain. II. Kinetic characterization of phosphointermediates.

(1) The kinetics of the phosphorylated enzymic intermediates of (Na+ + K+)-ATPase from ox brain, which are formed by incubation of the enzyme with 25 microM AT32P, 150 mM Na+ and 1 mM Mg2+, have been studied in dephosphorylation experiments at 1 degree C. The dephosphorylation of the 32P-labelled enzyme was initiated by addition of either 1 mM unlabelled ATP, 2.5 mM ADP or 1 mM unlabelled ATP + ADP in concentrations from 25 to 1000 microM. (2) In the absence of ADP the dephosphorylation curve was linear in a semilogarithmic plot almost from t = 0, whereas by addition of ADP a biphasic behaviour was obtained. The slope of the slow phase of dephosphorylation was virtually independent of the ADP concentration. (3) The results were analysed by the mathematical equation corresponding to the simplest possible model for the interconversion and breakdown of the phosphointermediates: (formula: see text) where alpha, beta, H and G are functions of all the rate constants and H and G furthermore are functions of the initial values for [E1P] and [E2P]. (4) The analysis confirmed the model and enabled the determination of all the rate constants. (5) k-1 was found to be equal to k'-1 + k"-1 . [ADP] indicating an ADP-independent 'spontaneous' dephosphorylation of E1P. The rate constant for this process was close to that for dephosphorylation of E2P, i.e., k'-1 congruent to k3. Also the value of k"-1 was determined. (6) k3 was found to be at least 10 . k-2. The implication of this for the role of the E1P to E2P transition in the Na+ + K+)-stimulated ATP hydrolysis will be discussed in detail in the following paper (Plesner, I.W., Plesner, L., Nørby, J.G. and Klodos, I. (1981) Biochim. Biophys. Acta 643, 483--494). (7) A refinement of the model, accounting for the effect of Na+ on the steady-state ratio between [E1P] and [E2P] is proposed: (formula: see text). At [Na+] = 150 mM as used here, E1P(Na) and E'1P are assumed to be in rapid equilibrium. (8) Comparison of our results with those of others underlines the general validity of the conclusions of the present paper.