INHIBITION OF PHOSPHOLIPASE-D BY A PROTEIN FACTOR FROM BOVINE BRAIN CYTOSOL - PARTIAL, PURIFICATION AND CHARACTERIZATION OF THE INHIBITION-MECHANISM

A specific protein inhibitor of partially purified bovine brain phosph olipase D (PLD) was identified from bovine brain cytosol. The PLD inhi bitor has been enriched through several chromatographic steps and char acterized with respect to size and mechanism of inhibition, The inhibi tor showed an apparent molecular mass of 30 kDa by Superose 12 gel exc lusion chromatography and inhibited PLD activity with an IC50 of 7 nM. The inhibitor had neither proteolytic activity nor phospholipid-hydro lyzing activity. Because phosphatidylinositol 4,5-bisphosphate (PIP2), which is included in substrate vesicles, is an essential cofactor for PLD, we examined whether the inhibition might be mediated by sequestr ation of PIP2. PIP2 hydrolysis by phospholipase C (PLC)-beta 1 was not affected by the inhibitor and the inhibitor did not bind to substrate vesicles containing PIP2. In contrast, a PH domain derived from PLC-d elta 1, which could bind to PIP2, showed a nearly identical inhibition of both PLC-beta 1 and PLD activities, Thus, the PLD inhibition by th e inhibitor is due to the specific interaction with not PIP2 but PLD. The suppression of PLD activity by the inhibitor was largely eliminate d by the addition of ADP-ribosylation factor (ARF) and GTP gamma S. We propose that the inhibitor plays a negative role in regulation of PLD activity by PIP2 and ARF.