Phospholipase D in homogenates from HL-60 granulocytes: Implications of calcium and G protein control

Occupancy of chemotactic peptide receptors leads to rapid initiation of phospholipase D (PLD) activity in intact dimethylsulfoxide-differentiated HL-60 granulocytes (Pai, J.-K, Siegel, M.I., Egan, R.W., and Billah, M.M. (1988) J. Biol. Chem. 263 , 12472). To gain further insight into the activation mechanisms, PLD has been studied in cell lysates from HL-60 granulocytes, using 1-0-alkyl-2-oleoyl-[ 32P]phosphatidylcholine (alkyl-[ 32P]PC), 1-0-[ 3H]alkyl-2-oleoyl-phosphatidylcholine ([ 3H]alkyl-PC) and [ 14C]arachidonyl-phospholipids as substrates. In the presence of Ca 2+ and GTP γS, post-nuclear homogenates degrade alkyl-[ 32P]PC to produce 1-0-alkyl-[ 32P]phosphatidic acid (alkyl-[ 32P]-PA), and in the presence of ethanol, also 1-0-alkyl-[ 32P]phosphatidylethanol (alkyl-[ 32P]PEt). By comparing the 3H/ 32P ratios of PA and PEt to that of PC, it is concluded that PA and PEt are formed exclusively by a PLD that catalyzes both hydrolysis and transphosphatidylation between PC and ethanol. Furthermore, PC containing either ester- or ether-linkage at the sn -1 position is degraded in preference to phosphatidylethanolamine and phosphatidylinositol by PLD in HL-60 cell homogenates. It is concluded that HL-60 granulocytes contain a PC-specific PLD that requires both Ca 2+ and GTP for activation.