Phospholipase D activity is regulated by product segregation and the structure formation of phosphatidic acid within model membranes. Biophys

ABSTRACT Phospholipase D from Streptomyces chromofuscus (scPLD) hydrolyzes phosphatidylcholines (PC) to produce cho-line and phosphatidic acid (PA), a lipid messenger molecule within biological membranes. To scrutinize the influence of membrane structure on scPLD activity, three different substrate-containing monolayers are used as model systems: pure dipalmitoylphos-phatidylcholine (DPPC) as well as equimolar mixtures of DPPC/n-hexadecanol (C16OH) and DPPC/dipalmitoylglycerol (DPG). The activity of scPLD toward these monolayers is tested by infrared reflection-absorption spectroscopy and exhibits different dependencies on surface pressure. For pure DPPC, the catalytic turnover drastically drops above 20mN/m. On addition of C16OH, this strongdecreasestarts at 5mN/m.For theDPPC/DPGsystem, the reactionyield linearlydecreasesbetween5and25mN/m.The difference in scPLD activity is correlated to the phase state of the monolayers as examined by x-ray diffraction, Brewster angle microscopy, andatomic forcemicroscopy. Because the additivesC16OHandDPGmediate themiscibility ofPCandPA, only a basal activity of scPLD is observed toward themixed systems at higher surface pressures. At pure DPPCmonolayers, scPLD is activated after the segregation of initially formed PA. Furthermore, scPLD is inhibited when the lipids in the PA-rich domains adopt an upright orientation.This phenomenonoffersa self-regulatingmechanism for the concentrationof the secondmessengerPAwithinbiological membranes