REQUIREMENT OF ADENOSINE 5'-TRIPHOSPHATE AND CA2+ FOR GUANOSINE 5'-TRIPHOSPHATE-BINDING PROTEIN-MEDIATED PHOSPHOLIPASE-D ACTIVATION IN RAT PHEOCHROMOCYTOMA PC12 CELLS

In digitonin-permeabilized PC12 cells labeled with [H-3]palmitic acid, formation of [H-3]phosphatidylethanol ([H-3]PEt), a marker of phospholipase D (PLD) activity, was increased with increasing concentrations of Ca2+ in the presence of ATP, Mg2+ and ethanol. Guanosine-5'-O-(3-thio-triphosphate) (GTPgammaS) significantly enhanced Ca2+-stimulated [H-3]PEt formation. The effect of GTPgammaS was abolished when any one of Ca2+, ATP or Mg2+ was excluded. ATP could not be replaced by other nucleotides except for ADP. Thus, the GTP-binding protein-mediated PLD activation absolutely required both Ca2+ and Mg-ATP.