Kinetic study of sunflower phospholipase Dα: interactions with micellar substrate, detergents and metals.

Phospholipase Dα (PLDα) purified from six-day post-germinated sunflower seeds was inactive in vitro on bilamellar substrates. It was fully active on mixed micelles made with phospholipids and a mixture of Triton-X100 and SDS at equal concentrations. It had an absolute need for divalent ions and calcium ions at millimolar concentration were the most efficient. Calcium had two effects. Firstly, using the fluorescent probe 2-p-toluidinylnaphtalene-6-sulfonate, we showed that the enzyme was able to bind calcium with a dissociation constant of 40-50 mM. This high value is probably due to the modification of the C2 domain which lacks some coordination residues allowing the binding of the metal. Secondly, using turbidity measurements, we showed that the metal ions interact with the SDS contained in the mixed micelles thus leading to an aggregated form of the substrate which is more easily hydrolyzed by PLDα.