Cation sites, spermine, and the reaction sequence of the (Na+ + K+)-dependent ATPase

Spermine, at 0.3 mM, inhibits the K+-nitrophenyl phosphatase activity of a dog kidney (Na+ + K+)-ATPase preparation, increasing the K0.5 for K+, reducing the Km for substrate, and affecting little the inhibition by Na+. These actions can be attributed, in a model of the phosphatase reaction, to parallel decreases in affinity for K+ and Na+ at their cytoplasmically accessible sites. In the (Na+ + K+)-ATPase reaction, spermine increases the K0.5 for Na+ and, to a lesser degree, the K0.5 for K+ as activators. With spermine, the double-reciprocal plots of velocity vs. ATP concentration (in the range 0.3-3 mM), at fixed levels of K+ (from 1 to 10 mM), remain parallel but are rotated clockwise and spread somewhat, reflecting stimulation at low ATP concentrations and inhibition at high ATP but low KCl concentrations. These actions can be attributed, in a steady-state ping-pong model of the ATPase reaction, solely to decreased rates of binding of Na+ and K+ to their sites, with major effects at the cytoplasmically accessible sites for Na+ (acceptance) and K+ (discharge), and with a lesser effect at the extracellularly accessible sites for K+ (acceptance). On these grounds, spermine is a highly specific and potentially valuable reagent for studying the reaction. Furthermore, the model for K+-ATP interactions not only supports a specific reaction sequence (K+ addition, Pi release, ATP addition, K+ release) but also argues against the availability of low-affinity substrate sites except during sharply restricted segments of the reaction sequence, thereby favoring proposals that the low-affinity substrate sites are transformed into high-affinity substrate sites with the E2 to E1 conformational change.