A H-1 nuclear magnetic resonance method for investigating the phospholipase D-catalyzed hydrolysis of phosphatidylcholine in liposomes

Liposomes with mean diameters between 45 and 73 nm have been prepared from 1-palmitoyl-2 oleoyl-sn-glycero-3-phosphocholine (POPC) at pH 8.0; and a new methodology is described which allows one to quantitatively follow the phospholipase D-catalyzed transformation of POPC to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid and free choline. The method does not require a special sample preparation; it takes advantage of the fact that the chemical shift of the protons of the three methyl groups in free choline differs from the chemical shift of the choline methyl protons in POPC. Measurements have been carried out under different experimental configurations and they have been paralleled by electron and light microscopy studies, partially using a fluorescently labeled phospholipid, It has been found that for a fixed concentration of the Ca2+-independent phospholipase D from Streptomyces sp. AA 586 the initial velocity and the reaction yields depend on the size of the vesicles. The smaller the vesicles, the higher the yields and the lower the initial rates. Furthermore, the size of the liposomes does not change during hydrolysis of the external POPC layer. (C) 1996 Academic Press, Inc.