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-bisphos phate (PIP2). Sequential chromatography of the brain cytosol yielded f our inhibitor fractions, which exhibited different kinetics to heat tr eatment at 70 degrees C. Purification of the most heat-labile inhibito r to homogeneity yielded two preparations, which displayed apparent mo lecular masses of 150 kDa and 135 kDa, respectively, on SDS-polyacryla mide gels. Tryptic digests of the 150- and 135-kDa proteins yielded si milar elution profiles on a C-18 reverse-phase column, suggesting that the 135-kDa form is a truncated form of the 150-kDa form. Sequences o f 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 depend ence on the 150-kDa inhibitor was not affected by changes in the conce ntrations of Arf, PIP2, or phosphatidylcholine used in the assays, sug gesting that the inhibition is not due to competition with the activat ors or substrate for PLD. The purified inhibitor did not affect the PI P2-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 subs trate vesicles.