Freezing Injury and Phospholipid Degradation in Vivo in Woody Plant Cells: II. Regulatory Effects of Divalent Cations on Activity of Membrane-bound Phospholipase D.

Activity of membrane-bound phospholipase D in microsomes from bark tissues of black locust tree (Robina pseudoacacia L.) was demonstrated to be regulated by a competitive binding of divalent cations. Binding of Ca(2+) at high concentrations (1 to 50 millimolar) modified the pH activity profile, shifting the optimum pH by 0.5 unit toward neutral and increasing the activity in the neutral pH. Mg(2+), on the other hand, inhibited the reaction of membrane-bound phospholipase D without added Ca(2+), and competitively inhibited the Ca(2+) stimulation. The regulatory effects of those ions were dependent on pH. Reduction in pH resulted in a decrease in the apparent dissociation constant for Ca(2+) and an increase in that for Mg(2+). From Lineweaver-Burk double reciprocal plots of Ca(2+) and the initial velocity, it was suggested that the binding of Ca(2+) in the higher concentration resulted in nearly the same conformational change of enzyme as reduction in pH. Mg(2+), on the other hand, counteracted those effects of Ca(2+) and lower pH on the enzyme conformation in such a manner as to inactivate. The membrane-bound phospholipase D because more sensitive to Ca(2+) and less sensitive to Mg(2+) as the hardiness of the tissues decreased. This fact may indicate that some qualitative changes in membranes are involved in the hardiness changes and also in the susceptibility of phospholipid to degradation by phospholipase D in plant cells.