title: Phospholipase C-delta3 binds with high specificity to phosphatidylinositol 4,5-bisphosphate and phosphatidic acid in bilayer membranes.
abstract: In order to acquire an understanding of phospholipase C-delta3 (PLC-delta3) action on substrate localized in lipid membrane we have studied the binding of human recombinant PLC-delta3 to large, unilamellar phospholipid vesicles (LUVs). PLC-delta3 bound weakly to vesicles composed of phosphatidylcholine (PtdCho) or PtdCho plus phosphatidylethanolamine (PtdEtn) or phosphatidylinositol (PtdIns). The enzyme bound strongly to LUVs composed of PtdEtn + PtdCho and phosphatidylinositol 4,5-bisphosphate (PtdInsP2). The binding affinity (molar partition coefficient) of PLC-delta3 to PtdEtn + PtdCho + PtdInsP2 vesicles was 7.7 x 105 m-1. High binding of PLC-delta3 was also observed for LUVs composed of phosphatidic acid (PA). Binding of PLC-delta3 to phosphatidylserine (PtdSer) vesicles was less efficient. Calculated molar partition coefficient for binding of PLC-delta3 to PA and PtdSer vesicles was 1.6 x 104 m-1 and 9.4 x 102 m-1, respectively. Presence of PA in the LUVs containing PtdInsP2 considerably enhanced the binding of PLC-delta3 to the phospholipid membrane. Binding of PLC-delta3 to phospholipid vesicles was not dependent on Ca2+ presence. In the liposome assay PA caused a concentration-dependent increase in activity of PLC-delta3. The stimulatory effect of PA on PLC-delta3 was calcium-dependent. At Ca2+ concentrations lower than 1 microm, no effect of PA on the activity of PLC-delta3 was observed. PA enhanced PLC-delta3 activity by increasing the Vmax and lowering Km for PtdInsP2. As the mol fraction of PA increased from 0-40 mol% the enzyme Vmax increased 2.3-fold and Km decreased threefold. Based on the results presented, we assume that PA supports binding of PLC-delta3 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-delta3. We propose that binding of PLC-delta3 to PA may serve as a mechanism for dynamic membrane association and modulation of PLC-delta3 activity.
authors.affiliation: Department of Molecular Medicine, Medical University of Gdansk, Poland. tkpaw@amedec.amg.gda.pl
authors.forename: T
authors.initials: T
authors.lastname: Pawelczyk
authors.forename: A
authors.initials: A
authors.lastname: Matecki
mesh_terms: D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008051:Lipid Bilayers; D010712:Phosphatidic Acids / Q000378:metabolism*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D054803:Phospholipase C delta; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D008051:Lipid Bilayers; D010712:Phosphatidic Acids; D019269:Phosphatidylinositol 4,5-Diphosphate; D011994:Recombinant Proteins; D010738:Type C Phospholipases; C518028:PLCD3 protein, human; D054803:Phospholipase C delta