Sphingomyelin composition and physical asymmetries in native acetylcholine receptor-rich membranes.
Selective enzymatic hydrolysis, lipid compositional analyses, and fluorescence studies have been carried out on acetylcholine receptor (AChR)-rich membranes from Torpedinidae to investigate the topology of sphingomyelin (SM) in the native membrane and its relationship with the AChR protein. Controlled sphingomyelinase hydrolysis of native membranes showed that SM is predominantly (approximately 60%) localized in the outer half of the lipid bilayer. Differences were also observed in the distribution of SM fatty acid molecular species in the two bilayer leaflets. A fluorescent SM derivative ( N-[10-(1-pyrenyl)decanoyl]sphingomyelin; Py-SM) was used to study protein-lipid interactions in the AChR-rich membrane and in affinity-purified Torpedo AChR reconstituted in liposomes made from Torpedo electrocyte lipid extracts. The efficiency of Förster resonance energy transfer (FRET) from the protein to the pyrenyl-labeled lipid as a function of acceptor surface density was used to estimate distances and topography of the SM derivative relative to the protein. The dynamics of the lipid acyl chains were explored by measuring the thermal dependence of Py-SM excimer formation, sensitive to the fluidity of the membrane. Differences were observed in the concentration dependence of excimer/monomer pyrenyl fluorescence when measured by direct excitation of the probe as against under FRET conditions, indicating differences in the intermolecular collisional frequency of the fluorophores between bulk and protein-vicinal lipid environments, respectively. Py-SM exhibited a moderate selectivity for the protein-vicinal lipid domain, with a calculated relative affinity K(r) approximately 0.55. Upon sphingomyelinase digestion of the membrane, FRET efficiency increased by about 50%, indicating that the resulting pyrenyl-ceramide species have higher affinity for the protein than the parental SM derivative.
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D004735:Energy Transfer; D066298:In Vitro Techniques; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008081:Liposomes / Q000737:chemistry; D046911:Macromolecular Substances; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013109:Sphingomyelins / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature; D014018:Tissue Distribution; D014101:Torpedo / Q000145:classification / Q000378:metabolism*
D008051:Lipid Bilayers; D008081:Liposomes; D046911:Macromolecular Substances; D008563:Membrane Lipids; D008565:Membrane Proteins; D011950:Receptors, Cholinergic; D013109:Sphingomyelins