REGULATION OF PHOSPHOLIPASE-D ACTIVITY BY NEUTRAL LIPIDS IN EGG-YOLK PHOSPHATIDYLCHOLINE SMALL UNILAMELLAR VESICLES AND BY CALCIUM-ION IN AQUEOUS-MEDIUM

Hydrolysis activity of phospholipase D from Streptomyces chromofuscus (PLD) was studied in small unilamellar vesicles (SUV) of egg yolk phosphatidylcholine (PC). The enzyme was associated with PC-SUV in a Ca2+-dependent manner. Both apparent maximum velocity, V-max(app), and reciprocal of apparent Michaelis constant, i.e., apparent binding constant, 1/K-m(app), increased with Ca2+ concentration, and the maximum values of these kinetic parameters were obtained at about 20 mu M Ca2+. Incorporation of 1,2-diacyl-glycerol (DAG), cholesterol (Cho1) or alpha-tocopherol (Toc) into PC-SUV induced shift of the antisymmetric PO2- stretching band of PC to lower frequency. The neutral lipids in SUV brought about increase of the V-max(app) value (Yamamoto et al. (1993) Biochim. Biophys. Acta 1145, 293-297). On the basis of these findings we discussed the regulation of PLD activity in terms of the Ca2+-dependent complex formation of PLD with SUV, and the enhancement of susceptibility of the P-O bond in PC molecule by neutral lipids.