Lanthanides as substitutes for calcium ions in the activation of plant α-type phospholipase D

Abstract

Most types of phospholipase D (PLD) from plants contain a C2 domain and are activated by Ca(2+) ions. In this study, other metal ions such as Mg(2+), La(3+), Ce(3+), Tb(3+) and Y(3+) were examined as effectors of recombinantly produced α-type PLD from white cabbage. All the rare earth ions were able to substitute for Ca(2+). The activation curves and displacement experiments reflect a 10- to 50-fold higher affinity of PLD for these ions than for Ca(2+); however, the maximum activity attained only 36% of that in the presence of Ca(2+). Mg(2+) displaced Ca(2+) without being able to activate PLD. All ions were bound to the substrate micelles consisting of phosphatidyl-p-nitrophenol, Triton X-100 and SDS (1:8:1, by mole). The affinity of rare earth ions to the micelles was 100-fold higher than that of Ca(2+) and Mg(2+). A conformational change of the enzyme induced by the low affinity but specific binding of Ca(2+) ions is concluded to be essential for maximal PLD activity. As demonstrated by the measurement of Tb(3+) fluorescence, the substitution of Ca(2+) by rare earth ions provides a new avenue for studying the enigmatic role of Ca(2+) ions in the modulation of PLD activity in plants.