Reactivity of the Na+/K+-ATPase while cycling as Na+-ATPase.

BEAUGÉ, L., BECERRA, A. Y CAMPOS M.

Sapporo, Japón

Conferencia; IXth International Conference of the Na/K-ATPase & related ATPases; 1999

We have continued the studies on the Na+,K' -ATPase reactivity when it acts as Na+-ATPase. Under these conditions the enzyme incorporates inorganic phosphate (Pi) to the E2 form; this incorporation is inhibited by extracellular Na+ and by intracelluIar ATP acting on its regulatory site. At Na+ concentration above 10 mM the reduction in Pi incorporation was associated to an increase in the rate of ATP hydrolysis; this roles out the existence of a reversal of the catalytic cycle. Below 10 mM the external Na+ inhibition with intermediate affinity (K0.5 about 2.5 mM) of the Na+-ATPase activity and the breakdown of E2P coincided with a 50 percent reduction of Pi incorporation. An additional important observation was that, provided the enzyme was cycling, the spontaneous and K+-stimulated dephosphorylation of the E2P complex formed by interaction of the enzyme with [32P]ATP (forward mode) is indistinguishable from that formed by direct [32P]Pi incorporation (reversal mode). These results indicate the following: (i) During the Na+­ATPase activity external Na+ antagonizes the direct [32P]Pi incorporation by two different mechanisms: (a) at [Na+] below 10 mM by the inhibition of E2P breakdown, and (b) at [Na+] above 10 mM by the formation of the E2Na2 complex after the Na+ stimulated dephosphorylation of E2P which is refractory to Pi. (ii) Regarding Vanadate, the effects of ATP and Na+ were qualitatively different from those observed with Pi, suggesting that the Na+­V antagonism requires mechanisms others that the formation of the E2Na2 complex. (iii) The state of the enzyme is crucial for the formation of phosphointermediaries. Thus, while in the absence of cycling the E2P formed by direct Pi incorporation is insensitive to K+, the reactivity towards K+ of the E2P forms attained with ATP and Pi become identical when the enzyme is cycling.