Effect of ATP on the intermediary steps of the reaction of the (Na ++K +)-dependent enzyme system. I. Studied by the use of N-ethylmaleimide inhibition as a tool

Abstract

1. 1. It has been investigated how ATP, Na +, K +, and Mg 2+ influence the inhibition of the (Na ++K +)-activated enzyme system by N-ethylmaleimide. 2. 2. ATP protects against the inhibition by N-ethylmaleimide. With ATP, without Mg 2+ and EDTA, both Na + and K + increase the inhibitory effect of N-ethylmaleimide; K + has the effect in lower concentrations than Na +. The main part of the Na + effect seems to be due to traces of Mg 2+ in the enzyme preparation; these can be removed by EDTA, while EDTA does not influence the effect of K +. 3. 3. With ATP, Mg 2+ increases the inhibitory effect by N-ethlmaleide; Na + potentiates the effect by decreasing the requirement for Mg 2+ for inhibition and it increases the inhibition which can be obtained by Mg 2+. K +, on the other hand, increases the requirement for Mg 2+. 4. 4. Without Mg 2+, with EDTA, K + decreases, while Na + gives a slight increase in the apparent affinity for ATP for the protection against N-ethylmaleimide. 5. 5. With ATP, without Mg 2+, and with EDTA, the increase in inhibition due to K + can be eliminated by titration with Na + and vice versa. The curve for the elimination of the K + effect on the reaction with N-ethylmaleimide is identical with the curve for the activation of the cataltic activity by Na + in the presence of K + and no N-ethylmaleimide. The Na + concentration necessary for half maximum elimination of the K + effect on the inhibition by N-ethylmaleimide decreases with an increase in the ATP concentration. 6. 6. ADP protects against N-ethylmaleimide like ATP; and an increase in the ADP concentration leads to a decrease in the concentration of Na + necessary for the elimination of the K + effect on the reaction with N-ethylmalcimide, but the concentration of ADP necessary to give a certain effect is about two times higher than that of ATP. ITP has practically no protective effect against N-ethylmaleimide. CTP and GTP have a low protective effect and a low effect on the apparent affinity for Na +; 3 mM GTP or CTP has an effect of the same order of magnitudes as 10 μM ATP.