A NEW METHOD FOR DETERMINING PHOSPHOLIPASE-D ACTIVITY USING THE MONOMOLECULAR FILM TECHNIQUE

A versatile and continuous assay for phospholipase D (PL D) activity w as developed using the monomolecular film technique. For this purpose, a two-step enzymatic reaction was used. First, PL D hydrolysis of sta ble 1,2-diacyl-sn-glycero-3-phosphocholine (PC) films by PL D generate d a stable 1,2-diacyl-sn-glycero-3-phosphate (PA) film and water-solub le choline. Secondly, the latter acidic phospholipid. in contrast to t he initial PC molecule, was further hydrolysed under the action of por cine pancreatic lipase (PPL) in order to give rise to lysophosphatidi c acid and fatty acid, which were rapidly desorbed from the interface. With this new procedure, it is possible to obtain continuous and accu rate kinetic measurements of the PL D-catalyzed reaction with phosphol ipid monolayers as substrates. The PL D kinetics were linear with lime and the velocities recorded were directly dependent upon the amount o f PL D used. In a preliminary study, we investigated the effects of th e surface pressure on the PL D activity.