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 Ca2+ and GTP gamma S, 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 Ca2+ and GTP for activation.
Anthes
J C
JC
Department of Allergy and Inflammation Schering Plough Corp. Bloomfield, NJ 07003.
Eckel
S
S
Siegel
M I
MI
Egan
R W
RW
Billah
M M
MM
D002118:Calcium / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000502:physiology*; D020404:Glycerophospholipids; D006098:Granulocytes / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D013379:Substrate Specificity; D013873:Thionucleotides / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D020404:Glycerophospholipids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D013873:Thionucleotides; C051521:phosphatidylethanol; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D010740:Phospholipases; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D002118:Calcium