Multiple forms of phospholipase D inhibitor from rat brain cytosol: Purification and characterization of heat-labile form

Rat brain cytosol contains proteins that markedly inhibit the activity of partially purified brain membrane phospholipase D (PLD) stimulated by ADP-ribosylation factor (Arf) and phosphatidylinositol 4,5-bisphosphate (PIP₂). Sequential chromatography of the brain cytosol yielded four inhibitor fractions, which exhibited different kinetics to heat treatment at 70°C. Purification of the most heat-labile inhibitor to homogeneity yielded two preparations, which displayed apparent molecular masses of 150 kDa and 135 kDa, respectively, on SDS-polyacrylamide gels. Tryptic digests of the 150- and 135-kDa proteins yielded similar elution profiles on a C₁₈ reverse-phase column, suggesting that the 135-kDa form is a truncated form of the 150-kDa form. Sequences of two tryptic peptides were determined. A data base search revealed no proteins with these sequences. The purified 150-kDa inhibitor negated the PLD activity stimulated by Arf, RhoA, or Cdc42. The concentration required for half-maximal inhibition was 0.4 nM. Concentration dependence on the 150-kDa inhibitor was not affected by changes in the concentrations of Arf, PIP₂, or phosphatidylcholine used in the assays, suggesting that the inhibition is not due to competition with the activators or substrate for PLD. The purified inhibitor did not affect the PIP₂-hydrolyzing activity of a phospholipase C isozyme that was measured with substrate vesicles of lipid composition identical with that used for the PLD assay. Thus, the mechanism of inhibition appears to be a specific allosteric modification of PLD rather than disruption of substrate vesicles.