Effect of ATP on the intermediary steps of the reaction of the (Na ++K +)-dependent enzyme system. II. Effect of a variation in the ATP/Mg 2+ ratio

Abstract

1. 1. It has been investigated how ATP in the presence of varying concentrations of Mg 2+ (a) influence the apparent affinity for Na + relative to K + for the site on the (Na ++K +)-dependent enzyme system where Na + activates — the “Na +-site”; (b) influences the catalytic activity in the presence of optimum concentrations of Na ++K +. 2. 2. The “Na +-site” has an apparent affinity for K + which is at least 2.5 times higher than for Na + without ATP in the medium. ATP increases the apparent affinity of this site for Na + relative to K + and with saturating concentrations of ATP the apparent affinity for Na + is about 3 times higher than for K +, i.e. ATP increases the apparent affinity for Na + relative to K + at least 7–8 times. 3. 3. It is the concentration of total ATP in the solution which determines the effect on the Na +:K + affinity ratio. At a given concentration of added ATP a wide variation in the concentration of Mg 2+ has no effect on the apparent Na +:K + affinity ratio. It means (a) that the effect is independent of the catalytic activity, (b) that free ATP as well as MgATP must have the effect, and (c) that the system must have about the same affinity for free ATP and for MgATP at the site where they exert their effect. 4. 4. With K ++Na +, the activity as a function of MgATP gives a bell-shaped curve when the Mg 2+ concentration is varied at a given ATP concentration. The activity at the maximum increases with the ATP concentration up to 3 mM and the maximum moves towards higher MgATP concentrations. The slope of the ascending part of the curve decreases with an increase in the concentration of added ATP, i.e. the activity at a given MgATP concentration decreases with an increase in the free ATP: free Mg 2+ (ATP f: Mg f 2+) ratio. For the descending part of the curve the activity decreases with a decrease in the ATP f: Mg f 2+ ratio, showing that maximum activity at a given MgATP concentration is obtained with an intermediary ATP f:Mg f 2+ ratio. The relationship between the effects of MgATP, free ATP and free Mg 2+ on the system is discussed.