1-palmitoyl-2-oleoylphosphatidylcholine and 1-2-dimyristoylphosphatidylethanolamine

The structural features of SPM that control the transbilayer distribution of beta-GalCer in POPC vesicles were investigated by (13)C- and (31)P-NMR spectroscopy using lipid analogs that share physical similarities with GalCer or SPM. The SPM analogs included N-palmitoyl-4,5-dihydro-SPM, 3-deoxy-SPM, 1-alkyl-2-amidophosphatidylcholine, and dipalmitoylphosphatidylcholine, a popular model "raft lipid". The transbilayer distributions of the SPM analogs and SPM in POPC vesicles were similar by (31)P-NMR. To observe the dramatic change in GalCer transbilayer distribution that occurs when SPM is included in POPC vesicles, the 3-OH group, 4,5-trans double bond, and amide linkage all were required in SPM. However, inclusion of 2 and 10 mol % dihydroSPM in SPM/POPC (1:1) vesicles mitigated and completely abrogated the effect of SPM on the transbilayer distribution of GalCer. Despite sharing some structural features with GalCer and localizing preferentially to the inner leaflet of POPC vesicles, dimyristoylphosphatidylethanolamine did not undergo a change in transbilayer distribution when SPM was incorporated into the vesicles. The results support the hypothesis that specific interactions may be favored among select sphingolipids in curvature-stressed membranes and emphasize the potential importance of the SPM-dihydroSPM ratio in membrane fission and fusion processes associated with vesicle biogenesis and trafficking.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Biophysics; Chickens; Galactosylceramides; Lipid Bilayers; Lipids; Magnetic Resonance Spectroscopy; Membrane Microdomains; Models, Molecular; Nitrogen; Ovum; Oxygen; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Sphingolipids; Sphingomyelins

We describe the interaction of Crotalus atrox-secreted phospholipase A2 (sPLA2) with giant unilamellar vesicles (GUVs) composed of single and binary phospholipid mixtures visualized through two-photon excitation fluorescent microscopy. The GUV lipid compositions that we examined included 1-palmitoyl-2-oleoyl-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (above their gel-liquid crystal transition temperatures) and two well characterized lipid mixtures, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE):DMPC (7:3) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC) (1:1) equilibrated at their phase-coexistence temperature regime. The membrane fluorescence probes, 6-lauroyl-2-(dimethylamino) napthalene, 6-propionyl-2-(dimethylamino) naphthalene, and rhodamine-phosphatidylethanolamine, were used to assess the state of the membrane and specifically mark the phospholipid domains. Independent of their lipid composition, all GUVs were reduced in size as sPLA2-dependent lipid hydrolysis proceeded. The binding of sPLA2 was monitored using a fluorescein-sPLA2 conjugate. The sPLA2 was observed to associate with the entire surface of the liquid phase in the single phospholipid GUVs. In the mixed-lipid GUV's, at temperatures promoting domain coexistence, a preferential binding of the enzyme to the liquid regions was also found. The lipid phase of the GUV protein binding region was verified by the introduction of 6-propionyl-2-(dimethylamino) naphthalene, which partitions quickly into the lipid fluid phase. Preferential hydrolysis of the liquid domains supported the conclusions based on the binding studies. sPLA2 hydrolyzes the liquid domains in the binary lipid mixtures DLPC:DAPC and DMPC:DMPE, indicating that the solid-phase packing of DAPC and DMPE interferes with sPLA2 binding, irrespective of the phospholipid headgroup. These studies emphasize the importance of lateral packing of the lipids in C. atrox sPLA2 enzymatic hydrolysis of a membrane surface.

Topics: 2-Naphthylamine; Animals; Crotalid Venoms; Crotalus; Dimyristoylphosphatidylcholine; Fluorescent Dyes; Hydrolysis; Kinetics; Laurates; Lipid Metabolism; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipases A; Phospholipases A2; Phospholipids; Photons; Protein Binding; Protein Structure, Tertiary; Time Factors