title: Kinetic study of sunflower phospholipase Dα: interactions with micellar substrate, detergents and metals.
abstract: 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.
authors.affiliation: Organization and Dynamics of Biological Membranes, UMR 5246 ICBMS, CNRS-Université Claude Bernard Lyon 1, Bâtiment Raulin, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne, Cedex, France.
authors.forename: Slim
authors.initials: S
authors.lastname: Abdelkafi
authors.forename: Abdelkarim
authors.initials: A
authors.lastname: Abousalham
mesh_terms: D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D003902:Detergents / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D018525:Germination; D006368:Helianthus / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008823:Micelles; D009391:Nephelometry and Turbidimetry; D010739:Phospholipase D / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012639:Seeds / Q000187:drug effects / Q000201:enzymology*; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D003902:Detergents; D008823:Micelles; D010739:Phospholipase D; D008274:Magnesium; D002118:Calcium