EFFECTS OF ALPHA-TOCOPHEROL AND ITS ACETATE ON THE HYDROLYTIC ACTIVITY OF PHOSPHOLIPASE-D IN EGG-YOLK PHOSPHATIDYLCHOLINE BILAYERS

Effects of alpha-tocopherol (Toc) and alpha-tocopherol acetate (TocA) on the hydrolysis activity of phospholipase D (from Streptomyces chromofuscus) were studied in small unilamellar vesicles (SUV) of egg yolk phosphatidylcholine (PC). Choline produced in the reaction was monitored by use of a choline oxidase - oxygen electrode. Addition of 18 mol% Toc into SUV (2 mM PC) brought about a twofold rate of choline production. On the other hand, the effect of 18 mol% TocA in SUV was very small. The apparent maximum velocity, V(max)(app), increased by addition of Toc in SUV. The apparent Michaelis constant, K(m)(app), was unchanged by addition of Toc and TocA in SUV. The Toc and TocA molecules did not have significant effects when PC was solubilized in the micelles of heptaethylene glycol dodecyl ether. The effects of Toc and TocA are, therefore, not due to specific ones on the enzyme itself, but rather upon the bilayer-organization of the substrate. Measurements of spreading pressure showed complete miscibility of PC and Toc, and limited mutual solubility of PC and TocA, suggesting stronger attractive interactions between Toc and PC than those between TocA and PC in the bilayers.