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

Abstract

Spermine, at 0.3 mM, inhibits the K +-nitrophenyl phosphatase activity of a dog kidney (Na + + K +)-ATPase preparation, increasing the K 0.5 for K +, reducing the K m 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 K 0.5 for Na + and, to a lesser degree, the K 0.5 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, P i 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 E 2 to E 1 conformational change.