Dipalmitoyl-phosphatidylcholine/phospholipase D interactions investigated with polarization-modulated infrared reflection absorption spectroscopy

The hydrolysis of 1,2-dipalmitoylphosphatidylcholine (DPPC) catalyzed by St reptomyces chromofuscus phospholipase D (PLD) has been investigated using m onolayer techniques and polarization-modulated infrared absorption reflecti on spectroscopy. The spectroscopic analysis of the phosphate groups provide s a quantitative estimation of the hydrolysis yield. The hydrolysis kinetic s was investigated in dependence on the phase state of the lipid monolayer. It was found that PLD exhibits maximum activity in the liquid-expanded pha se, whereas PLA, has its activity maximum in the two-phase region, A lag ph ase was observed in all experiments indicating that small amounts of the hy drolysis product 1,2-dipalmitoylphosphatidic acid (DPPA) are needed for ini tiating the fast hydrolysis reaction. Higher concentrations of DPPA inhibit the hydrolysis. The critical inhibition concentration of DPPA is a functio n of the monolayer pressure.