The interaction of dialkyl ether lecithins with phospholipase A2 (Naja naja naja).
Dialkyl lecithins are nonhydrolyzable substrate analogues for phospholipase A2 (Naja naja naja). Short chain dialkyl lecithins (which form monomers and micelles), and long chain ether-linked lecithins (which form bilayers or can be solubilized in detergent micelles) have been used to study phospholipase A2/phospholipid interactions. The results of gel filtration, 1H and 31P NMR spectroscopy, kinetic assays and UV difference spectroscopy show that binding of the ether-linked lecithins is independent of metal ion in pure lecithin systems (monomer, micelle, or bilayer), but requires Ca2+ for tight binding when Triton X-100 is used to solubilize the lecithin in mixed micelles. The apparent KD for dihexadecyl phosphatidylcholine is 2 mM in sonicated vesicles, but is increased to 6 mM in Triton-mixed micelles in the presence of Ca2+. When phospholipase A2 binds monomeric and micellar dialkyl lecithin, the lecithin phosphate resonance is broadened considerably, indicating that head group motion is restricted. Ca2+ enhances this interaction with the head group in these pure lipid systems. In similar 31P NMR experiments with dihexadecyl phosphatidylcholine solubilized in Triton X-100 mixed micelles, phospholipase A2 does not cause comparable broadening of the phosphate resonance, even in the presence of Ca2+ where gel filtration shows binding occurs. The kinetic effect of the ether-linked lipid inhibitors on phospholipase activity toward ester-linked lecithins is also modulated by the presence of Triton X-100. Ether-linked lecithins are much more potent inhibitors in two-component (dialkyl- diacyl-) lecithin micelles and bilayers than in complex mixed micelles with Triton X-100. Detergent-induced changes in the characteristics of the micellar interface are implicated in this inhibition phenomenon. The interaction of phospholipase A2 with sonicated vesicles of dihexadecyl phosphatidylcholine has also been examined by 1H and 31P NMR spectroscopy. When phospholipase A2 binds to vesicles, it promotes loss of encapsulated lanthanide ions, and appears to accelerate transbilayer phospholipid "flip-flop" rates. These results are discussed in terms of membrane asymmetry studies using phospholipase A2.
D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D004546:Elapid Venoms / Q000032:analysis; D008051:Lipid Bilayers; D008081:Liposomes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D017830:Octoxynol; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010955:Plasmalogens / Q000378:metabolism*; D011092:Polyethylene Glycols; D011663:Pulmonary Surfactants / Q000378:metabolism; D013010:Sonication; D013056:Spectrophotometry, Ultraviolet
D004546:Elapid Venoms; D008051:Lipid Bilayers; D008081:Liposomes; D008823:Micelles; D010955:Plasmalogens; D011663:Pulmonary Surfactants; C032459:choline plasmalogens; D011092:Polyethylene Glycols; D017830:Octoxynol; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium