AN UNEXPECTED EFFECT OF ATP ON THE RATIO BETWEEN ACTIVITY AND PHOSPHOENZYME LEVEL OF NA+ K+-ATPASE IN STEADY-STATE/

According to the Albers-Post model the hydrolysis of ATP catalyzed by the Na+/K+-ATPase requires the sequential formation of at least two co nformers of a phosphoenzyme (E(1)P and E(2)P), followed by the K+-stim ulated hydrolysis of E(2)P. 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 (upsilon) and total phosphoenzyme level (EP) in steady state is determined solely by the rate constants of in terconversion between phosphoconformers and of dephosphorylation. Sinc e 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 upsilon/EP ought to be independent of the concentration of ATP. We tes ted this prediction by parallel measurements of upsilon and EP as a fu nction of [ATP] in the absence or presence of non-limiting concentrati ons of K+, Rb+ or NH4+. In the absence of K+ or its congeners, both cu rves followed Michaelis-Menten kinetics, with almost identical K-m val ues (0.16 mu M) so that upsilon/EP remained independent of [ATP]. In t he presence of either K+, Rb+ or NH4+, upsilon and EP increased with [ ATP] along the sum of two Michaelis-Menten equations. The biphasic res ponse of upsilon is well known but, to the best of our knowledge, our results are the first demonstration that the response of EP to [ATP] i s also biphasic. Under these conditions, the ratio upsilon/EP increase d with [ATP] from 19.8 to 40.1 s(-1) along a hyperbola that was half-m aximal at 9.5 mu M. To preserve the validity of the current model it s eems necessary to assume that ATP acts on the E(1)P reversible arrow E (2)P transition and/or on the rate of hydrolysis of E(2)P. The latter possibility was ruled out. We also found that to fit the Albers-Post m odel to our data, the rate constant for K+ deocclussion from E(2) has to be about 10-times higher than that reported from measurements of pa rtial reactions. The results indicate that the Albers-Post model quant itatively predicts the experimental behavior of the Na+-ATPase ac tivi ty but is unable to do this for the Na+/K+-ATPase activity, unless add itional and yet unproved hypothesis are included.