GUANINE-NUCLEOTIDE-DEPENDENT AND ADENINE-NUCLEOTIDE-DEPENDENT REGULATION OF PHOSPHOLIPASE-D IN ELECTROPERMEABILIZED HL-60 GRANULOCYTES

We have characterized the regulation of phospholipase D (PLD) in electropermeabilized HL-60 granulocytes in which endogenous phospholipids were pre-labelled with [H-3]oleic acid. Treatment of these permeabilized cells with the nonhydrolysable GTP analogues guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta-gamma-imido]triphosphate induced a sustained (near-linear for up to 60 min) accumulation of phosphatidic acid (PA). In the presence of ethanol a sustained production of phosphatidylethanol (PEt) was also observed. With increasing concentrations of ethanol, PEt formation increased, whereas PA formation declined; this indicated involvement of a PLD-type effector enzyme. The ability of GTP[S] to stimulate this PLD activity was Mg2+-dependent and was inhibited by GDP and its non-hydrolysable beta-thio analogue. Ca2+, at concentrations less-than-or-equal-to nM, had no effect on the GTP[S]-dependent PLD activity. However, higher concentrations of Ca2+ produced a significant potentiation of this activity. Inclusion of MgATP ( greater-than-or-equal-to 0.1 mM), but not other nucleoside triphosphates, also induced a large potentiation of GTP[S]-dependent PLD activation. In the absence of guanine nucleotides, MgATP elicited no significant activation of PLD. Significantly, this effect of ATP was not mimicked by adenosine 5'-[beta-gamma-methylene]triphosphate, a non-hydrolysable ATP analogue. Rather, this analogue inhibited both basal and ATP-potentiated GTP[S]-dependent PLD activity. This suggests that the ability of ATP to potentiate GTP[S]-dependent PLD activity involves phosphotransferase action rather than simple allosteric effects induced by adenine nucleotide binding. The absolute magnitude of the GTP[S]-dependent PLD activity which could be potentiated by MgATP was decreased by 90% when the permeabilized cells were preincubated for various times before addition of these stimulatory agents. This time-dependent loss of MgATP-induced potentiation was prevented when the permeabilized cells were preincubated in the presence of GTP[S]. These results demonstrate that electropermeabilized HL-60 granulocytes can be used to discriminate synergistic roles for a GTP-binding protein(s) and an ATP-dependent process (kinase?) in the regulation of phospholipase D activity.