Preparation of a protein-free total brain white matter lipid fraction: characterization of liposomes.
A method of preparing a total lipid extract (TLE), free of protein, by extracting brain white matter with tetrahydrofuran is presented. The optimal conditions of extraction were found to be 50 ml of THF per gram of lyophilized tissue, though fresh tissue can also be used if larger volumes of solvent are employed. The method allowed, in a short time and in a single step, a yield of TLE of 50% on a dry weight basis. Its analytical characterization revealed a qualitative and quantitative composition very similar to the lipid composition of CNS myelin, including all the phospholipid and galactolipid species, cholesterol and gangliosides, but it contained only traces (0.1%) of protein. TLE has been used to prepare liposomes, either multilamellar (MLVs) or unilamellar (LUVs, SUVs), characterized by freeze-fracture electron microscopy. A multilayered, heterogeneous population of liposomes is observed in the MLVs preparation. When these samples were submitted to a freezing and thawing procedure the resulting liposomes were single-walled, and their intravesicular volume was increased. They were quite impermeable to the monovalent cation 86Rb+ and, by contrast, rather permeable to 45Ca+ +. Their complex lipid composition, together with their permeability properties and their response to ionophores, make them very useful to study protein-lipid interactions occurring within the myelin membrane as well as the functional properties of myelin proteins in reconstitution experiments.
Díaz
R S
RS
Cajal Institute, C.S.I.C., Madrid, Spain.
Monreal
J
J
Regueiro
P
P
Lucas
M
M
D000818:Animals; D001923:Brain Chemistry; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002855:Chromatography, Thin Layer; D005612:Freeze Drying; D005614:Freeze Fracturing; D005663:Furans; D005732:Gangliosides / Q000302:isolation & purification; D008055:Lipids / Q000302:isolation & purification*; D008081:Liposomes; D008567:Membranes, Artificial; D004676:Myelin Basic Protein / Q000378:metabolism; D010539:Permeability; D012413:Rubidium / Q000378:metabolism; D012997:Solvents; D014634:Valinomycin
D005663:Furans; D005732:Gangliosides; D008055:Lipids; D008081:Liposomes; D008567:Membranes, Artificial; D004676:Myelin Basic Protein; D012997:Solvents; D014634:Valinomycin; C018674:tetrahydrofuran; D012413:Rubidium; D002118:Calcium