A SOLUBLE-PROTEIN NEGATIVELY REGULATES PHOSPHOLIPASE-D ACTIVITY - PARTIAL-PURIFICATION AND CHARACTERIZATION

Phosphatidylcholine-specific phospholipase D (PLD) is an important sig nalling phospholipase in mammalian cells. Recently, PLD activity has b een shown to be positively regulated by the GTP-binding protein ARF (A DP-ribosylating factor). In the present work, we document the presence of a factor negatively regulating PLD activity in bovine brain cytoso l. The inhibitory factor is characterized as a large protein or a comp lex of proteins with a molecular mass higher than 300 kDa. Using perme abilized and pre-permeabilized HL-60 cells depleted of their cytosol, we demonstrate that the inhibitor acts on GTP[S]-stimulated PLD activi ty. This effect is immediate, persistent and dose dependent for GTP[S] -stimulated PLD. Different possibilities for a mechanism of action of the inhibitory factor on the regulation of GTP binding to ARF were inv estigated. This inhibitory factor is not the guanine-dissociating inhi bitor (GDI) for the small G-binding proteins Rho (Rho-GDI), reported t o be a PLD inhibitor, since specific antibodies against this protein d id not recognize a protein in the peak containing the inhibitory facto r for PLD activity. Furthermore, the inhibitory factor does not preven t the binding of GTP[S] to ARF in the presence of HL-60 membranes. Thi s excludes its possible role as an inhibitor of an ARF/guanine exchang e factor. The inhibitory factor not only inhibits a pathway of PLD thr ough GTP[S] activation in particular of the small GTP-binding protein, ARE but it also inhibits PLD activated via either protein kinase C (P KC) or tyrosine kinase activation. The inhibitory factor also decrease s PLC activity and this effect seems to be secondary to the inhibition of PLD activity. We discuss a mechanism of action of the inhibitor on PLD and the importance of this enzyme activity for membrane traffic.