Phospholipase C-delta 3 binds with high specificity to phosphatidylinositol 4,5-bisphosphate and phosphatidic acid in bilayer membranes

In order to acquire an understanding of phospholipase C-delta 3 (PLC-delta 3) action on substrate localized in lipid membrane we have studied the bind ing of human recombinant PLC-delta 3 to large, unilamellar phospholipid ves icles (LUVs). PLC-delta 3 bound weakly to vesicles composed of phosphatidyl choline (PtdCho) or PtdCho plus phosphatidylethanolamine (PtdEtn) or phosph atidylinositol (PtdIns). The enzyme bound strongly to LUVs composed of PtdE tn + PtdCho and phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)). The bin ding affinity (molar partition coefficient) of PLC-delta 3 to PtdEtn + PtdC ho + PtdInsP(2) vesicles was 7.7 X 10(5) M-1. High binding of PLC-delta 3 w as also observed for LUVs composed of phosphatidic acid (PA). Binding of PL C-delta 3 to phosphatidylserine (PtdSer) vesicles was less efficient. Calcu lated molar partition coefficient for binding of PLC-delta 3 to PA and PtdS er vesicles was 1.6 x 10(4) M-1 and 9.4 x 10(2) M-1, respectively. Presence of PA in the LUVs containing PtdInsP(2) considerably enhanced the binding of PLC-delta 3 to the phospholipid membrane. Binding of PLC-delta 3 to phos pholipid vesicles was not dependent on Ca2+ presence. In the liposome assay PA caused a concentration-dependent increase in activity of PLC-delta 3. T he stimulatory effect of PA on PLC-delta 3 was calcium-dependent. At Ca2+ c oncentrations lower than 1 mu M, no effect of PA on the activity of PLC-del ta 3 was observed. PA enhanced PLC-delta 3 activity by increasing the V-max and lowering K-m for PtdInsP(2). As the mol fraction of PA increased from 0-40 mol% the enzyme V-max increased 2.3-fold and K-m decreased threefold. Based on the results presented, we assume that PA supports binding of PLC-d elta 3 to lipid membranes by interaction with the PH domain of the enzyme. The stimulatory effect of PA depends on calcium-dependent interaction with the C2 domain of PLC-delta 3. We propose that binding of PLC-delta 3 to PA may serve as a mechanism for dynamic membrane association and modulation of PLC-delta 3 activity.