Detergent-insoluble glycosphingolipid/cholesterol microdomains of the myelin membrane.
Glycosphingolipids and cholesterol form lateral assemblies, or lipid 'rafts', within biological membranes. Lipid rafts are routinely studied biochemically as low-density, detergent-insoluble complexes (in non-ionic detergents at 4 degrees C; DIGs, detergent-insoluble glycosphingolipid/cholesterol microdomains). Recent discrepancies recommended a re-evaluation of the conditions used for the biochemical analysis of lipid rafts. We have investigated the detergent insolubility of several known proteins present in the glycosphingolipid/cholesterol-rich myelin membrane, using four detergents representing different chemical classes (TX-100, CHAPS, Brij 96 and TX-102), under four conditions: detergent extraction of myelin either at (i) 4 degrees C or (ii) 37 degrees C, or at 4 degrees C after pre-extraction with (iii) saponin or (iv) methyl-beta-cyclodextrin (MbetaCD). Each detergent was different in its ability to solubilize myelin proteins and in the density of the DIGs produced. Brij 96 DIGs floated to a lower density than other detergents tested, possibly representing a subpopulation of DIGs in myelin. DIGs pre-extracted with saponin were denser than DIGs pre-extracted with MbetaCD. Furthermore, pre-extraction with MbetaCD solubilized proteolipid protein (known to associate with cholesterol), whereas pre-extraction with saponin did not, suggesting that saponin is less effective as a cholesterol-perturbing agent than is MbetaCD. These results demonstrate that DIGs isolated by different detergents are not necessarily comparable, and that these detergent-specific DIGs may represent distinct biochemical, and possibly physiological, entities based on the solubilities of specific lipids/proteins in each type of detergent.
Taylor
Christopher M
CM
Department of Neuroscience, University of Connecticut Health Center, Farmington, Connecticut 6030-3401, USA. taylor@neuron.uchc.edu
Coetzee
Timothy
T
Pfeiffer
Steven E
SE
D000818:Animals; D002499:Centrifugation, Density Gradient; D005591:Chemical Fractionation; D002784:Cholesterol / Q000737:chemistry*; D002793:Cholic Acids / Q000737:chemistry; D003902:Detergents / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006028:Glycosphingolipids / Q000737:chemistry*; D046911:Macromolecular Substances; D021962:Membrane Microdomains / Q000737:chemistry*; D051379:Mice; D008810:Mice, Inbred C57BL; D009186:Myelin Sheath / Q000737:chemistry*; D010938:Plant Oils / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D012995:Solubility
D002793:Cholic Acids; D003902:Detergents; D006028:Glycosphingolipids; D046911:Macromolecular Substances; D010938:Plant Oils; D011092:Polyethylene Glycols; C033084:polyethylene glycol oleyl ether; D002784:Cholesterol; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate