A. Abousalham et al., A NEW METHOD FOR DETERMINING PHOSPHOLIPASE-D ACTIVITY USING THE MONOMOLECULAR FILM TECHNIQUE, Chemistry and physics of lipids, 79(2), 1996, pp. 107-112
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.