THE ROLE OF MG2- STUDY BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY( IN THE HYDROLYTIC ACTIVITY OF THE ISOLATED CHLOROPLAST ATPASE )

The influences of total magnesium ion concentration at different total ATP concentrations, and of total ATP concentration, for different tot al magnesium ion concentrations, on the enzymatic rate of the isolated chloroplast F-1 ATPase, have been followed by a chromatographic metho d consisting in the separation and determination of ADP. From the vari ous series of curves, it is concluded that the experimental results (p osition of the maxima, K-m values) are better fitted by a mechanism in volving the activation of the enzyme by magnesium ion and hydrolysis o f free ATP, rather than by the classical mechanism, for which the enzy me hydrolyzes the MgATP complex and is inhibited by Mg2+. Although the equations giving the reaction rate are similar in the two cases, the calculated values of K-m are widely different. The value obtained from the classical mechanism does not agree with K-D, the dissociation con stant of the enzyme-substrate complex, measured by the Hummel and Drey er method. Moreover, when the total ATP concentration tends toward the total magnesium ion concentration, the nucleotide binding to the enzy me tends toward zero, although it should be maximum if MgATP were the true substrate. Finally, the inhibitory effect of Na+ is more easily e xplained as a competition between this ion and the activating Mg2+, th an by the classical mechanism.