Role of Magnesium in the Plasma Membrane ATPase of Red Beet

The phosphorylation technique was used to assess the role of Mg in the red beet (Beta vulgaris L.) plasma membrane ATPase. When an excess of ethylenediaminetetraacetate (Tris salt, pH 6.5) was added to phosphorylation reactions at steady-state, the phosphorylation level declined exponentially and the rate constant for dephosphorylation was similar to that observed when phosphorylation reactions were chased with unlabeled ATP. When KCl was included with the EDTA chase, a 2.4-fold increase in the turnover of the phosphoenzyme was observed. Thus, the formation of the phosphorylated intermediate but not its breakdown requires free Mg to be present. When an excess of unlabeled ATP containing MgSO(4) was added to plasma membranes incubated for 20 seconds with [gamma-(32)P]ATP in the absence of MgSO(4), a burst of phosphorylation was observed that declined exponentially. The rate constant for this decline was similar to that observed for phosphoenzyme turnover after initial labeling in the presence of MgSO(4). Extrapolation of this kinetic plot to zero time indicated that ATP binding can occur when MgSO(4) is absent. It is proposed that Mg has a specific role in the transphosphorylation reaction of the terminal phosphate group of ATP to the enzyme.