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Formation of Ceramide/Sphingomyelin Gel Domains in the Presence of an Unsaturated Phospholipid: A Quantitative Multiprobe Approach

Bruno M. Castro ^*, Rodrigo F. M. de Almeida ^* , Liana C. Silva ^*, Alexander Fedorov ^* and Manuel Prieto ^*

^* Centro de Química-Física Molecular, Instituto Superior Técnico, Lisbon, Portugal; and Departamento de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal

Correspondence: Address reprint requests to Rodrigo F. M. de Almeida, CQFM, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. Fax: 351-218-464-455; E-mail: r.almeida{at}mail.ist.utl.pt.

To better understand how ceramide modulates the biophysical properties of the membrane, the interactions between palmitoyl-ceramide (PCer) and palmitoyl-sphingomyelin (PSM) were studied in the presence of the fluid phospholipid palmitoyl-oleoyl-phosphatidylcholine (POPC) in membrane model systems. The use of two fluorescent membrane probes distinctly sensitive to lipid phases allowed a thorough biophysical characterization of the ternary system. In these mixtures, PCer recruits POPC and PSM in the fluid phase to form extremely ordered and compact gel domains. Gel domain formation by low PCer mol fraction (up to 12 mol %) is enhanced by physiological PSM levels (20–30 mol % total lipid). For higher PSM content, a three-phase situation, consisting of fluid (POPC-rich)/gel (PSM-rich)/gel (PCer-rich) coexistence, is clearly shown. To determine the fraction of each phase a quantitative method was developed. This allowed establishing the complete ternary phase diagram, which helps to predict PCer-rich gel domain formation and explains its enhancement through PSM/PCer interactions.

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