Granulocyte phospholipase D is activated by a guanine nucleotide dependent protein factor.
When post-nuclear homogenates from HL-60 granulocytes are incubated in the presence of CaCl2, GTP gamma S and ethanol, phospholipase D (PLD) metabolizes both exogenous 2-[14C]arachidonyl-phosphatidylcholine and endogenous phosphatidyl[3H]choline to produce 2-[14C]arachidonyl-phosphatidic acid, 2-[14C]arachidonyl-phosphatidylethanol and [3H]choline. Fractionation of the homogenate by ultracentrifugation into cytosolic and membrane fractions results in the loss of PLD activity. However, when these two fractions are combined in the same proportion as found in the unfractionated homogenate, PLD activity is completely restored. This activity is proportional to the concentration of both the cytosol and the particulate fractions. Release of [14C]arachidonate by PLA2 that occurs under these assay conditions does not require the combined presence of cytosol and membrane fractions. We conclude that, in granulocyte homogenates, PLD activity but not arachidonate release, exhibits an essential requirement for a heat-labile factor whose activity depends on the presence of GTP gamma S.
Anthes
J C
JC
Department of Allergy and Immunology, Schering-Plough Research, Bloomfield, New Jersey 07003.
Wang
P
P
Siegel
M I
MI
Egan
R W
RW
Billah
M M
MM
D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002794:Choline / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D006098:Granulocytes / Q000201:enzymology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D013379:Substrate Specificity
D009363:Neoplasm Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010739:Phospholipase D; D002794:Choline; D002118:Calcium