BINDING OF PHOSPHOLIPASE-C-DELTA(1) TO PHOSPHOLIPID-VESICLES

Binding of phopholipase Cdelta1 (PLCdelta) to phospholipid vesicles was studied using large, unilamellar phospholipid vesicles (LUVs). PLCdelta bound weakly to vesicles composed of phosphatidylserine (PS) or phosphatidylcholine (PC) or phosphatidylethanolamine (PE)+PC, and even more weakly to vesicles composed of phosphatidylinositol. The enzyme bound strongly to LUVs composed of PE+PC and phosphatidylinositol 4,5-bisphosphate (PIP2) or sphingomyelin (SM). Binding of 50% of PLCdelta occurred at 0.25 nmol/ml PIP2 when LUVs composed of PE+PC (molar ratio of 80:20), plus various amounts of PIP2, were used at a constant phospholipid concentration of 300 nmol/ml. When LUVs composed of PE + PC + PIP2 (molar ratio of 79:20:1) were tested as a function of increasing phospholipid concentration, 50% binding of PLCdelta occurred at 1.2 nmol/ml PIP2 and 120 nmol/ml total phospholipid. Similar measurements were conducted with other phospholipids and PIP2 at a molar ratio of 99:1. These showed that 50% binding of PLCdelta occurred at a level of 0.9 nmol/ml PIP2 with 80 nmol/ml PC; at 2.2 nmol/ml PIP2 with 170 nmol/ml PS; at 4.2 nmol/ml PIP2 with 320 nmol/ml PI; and at 0.26 nmol/ml PIP2 with 20 nmol/ml total liver phospholipids. Binding to phosphatidylinositol 4-phosphate was much weaker. When LUVs composed of PE + PC + SM (molar ratio 48:12:40) were tested as a function of increasing phospholipid concentration, 50% binding of PLCdelta occurred at a level of 96 nmol/ml SM. This is well below the concentration of SM that can be calculated to face the cytosol. Binding of PLCdelta to LUVs decreased as the temperature was lowered from 37-degrees-C to 0-degrees-C. Thus PLCdelta shows a high degree of specificity for binding to PIP2 and SM. Under physiological conditions a considerable fraction of PLCdelta may be bound to cellular membranes, either in an inactive form if bound to PIP2 at low resting Ca2+ concentrations, or in the inhibited form if bound to SM.