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  • Title: An unexpected effect of ATP on the ratio between activity and phosphoenzyme level of Na+/K(+)-ATPase in steady state.
    Author: Schwarzbaum PJ, Kaufman SB, Rossi RC, Garrahan PJ.
    Journal: Biochim Biophys Acta; 1995 Jan 26; 1233(1):33-40. PubMed ID: 7833347.
    Abstract:
    According to the Albers-Post model the hydrolysis of ATP catalyzed by the Na+/K(+)-ATPase requires the sequential formation of at least two conformers of a phosphoenzyme (E1P and E2P), followed by the K(+)-stimulated hydrolysis of E2P. In this paper we show that this model is a particular case of a more general class of models in all of which the ratio between ATPase activity (v) and total phosphoenzyme level (EP) in steady state is determined solely by the rate constants of interconversion between phosphoconformers and of dephosphorylation. Since these are thought to be unaffected by ATP, the substrate curves for ATPase activity and EP should be identical in shape so that the ratio v/EP ought to be independent of the concentration of ATP. We tested this prediction by parallel measurements of v and EP as a function of [ATP] in the absence or presence of non-limiting concentrations of K+, Rb+ or NH+4. In the absence of K+ or its congeners, both curves followed Michaelis-Menten kinetics, with almost identical Km values (0.16 microM) so that v/EP remained independent of [ATP]. In the presence of either K+, Rb+ or NH+4, v and EP increased with [ATP] along the sum of two Michaelis-Menten equations. The biphasic response of v is well known but, to the best of our knowledge, our results are the first demonstration that the response of EP to [ATP] is also biphasic. Under these conditions, the ratio v/EP increased with [ATP] from 19.8 to 40.1 s-1 along a hyperbola that was half-maximal at 9.5 microM. To preserve the validity of the current model it seems necessary to assume that ATP acts on the E1P <--> E2P transition and/or on the rate of hydrolysis of E2P. The latter possibility was ruled out. We also found that to fit the Albers-Post model to our data, the rate constant for K+ deocclussion from E2 has to be about 10-times higher than that reported from measurements of partial reactions. The results indicate that the Albers-Post model quantitatively predicts the experimental behavior of the Na(+)-ATPase activity but is unable to do this for the Na+/K(+)-ATPase activity, unless additional and yet unproved hypothesis are included.
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