Functions of the E-ATP and E- P complexes in the membrane ATPase reaction

Abstract

1. 1. In turtle bladder microsomes incubated with 0.01 mM ATP at 0°, the Mg-dependent ATPase is 50 % greater than the Ca-dependent ATPase. The addition of Na + + K + induces a 20 % increase of Mg-ATPase, but has no effect on Ca-ATPase. The addition of Ca 2+ to the (Mg 2+ + Na + + K +)-containing system eliminates the (Na + + K +) increment of its ATPase activity. 2. 2. The E-ATP formed after incubation of microsomes with [U- 14C]ATP (0.01 mM ATP at 0°) amounts to an average value of 13 pmoles/mg in the presence of Ca 2+ alone or of Mg 2+ alone. The addition of either Na + or K + does not affect the underlying E-ATP formation in the presence of Mg 2+ and/or Ca 2+. However, the addition of Na + and K + together induces a 32 % increase in the Ca-dependent formation of E-ATP, but no change in the Mg-dependent formation. The addition of Ca 2+ to the (Mg 2+ + Na + + K +)-containing system causes little or no change in ATP binding. 3. 3. The phospho-protein ( E- P) formed after incubation of microsomes with [γ- 32P]ATP (0.01 mM ATP at 0°) amounts to an average value of 19.2 pmoles/mg in the presence of Ca 2+ alone, and 76 in the presence of Mg 2+ alone. The addition of Na + causes a 3-fold increase in the amount of Ca-dependent formation of E- P, and a 2-fold increase in the Mg-dependent formation. The addition of K + to the (Ca 2+ + Na +)-containing mixture reduces the level of E- P to the underlying Ca-dependent level; while the same K addition to the (Mg 2+ + Na +)-system reduces the level of E- P formation to less than half (31 pmoles) of the underlying Mg-dependent level. The addition of Ca 2+ to either the ((Mg 2+ + Na +) or the (Mg 2+ + Na + + K +) system produces no change in phospho-protein formation. 4. 4. Present data provide support for the hypothesis holding that the presence of Mg 2+, Na + and K + together is required in the first intermediary reaction step of the (Na + + K +)-ATPase sequence. Although Ca can substitute for Mg in the first step of the (Na + + K +)-ATPase sequence, it apparently forms a stable (Ca 2+ + Na + + K +)- E-ATP complex which does not break down into the final hydrolytic products. It is inferred that the Na-induced increment of phosphoprotein formation occurs in a reaction path which is in parallel to that of (Na + + K +)-ATPase. Possible roles of E-ATP and E- P in sodium transport are adduced.