Detergent‐insoluble glycosphingolipid/cholesterol microdomains of the myelin membrane

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Abstract
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°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°C or (ii) 37°C, or at 4°C after pre‐extraction with (iii) saponin or (iv) methyl‐β‐cyclodextrin (MβCD). 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 MβCD. Furthermore, pre‐extraction with MβCD 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 MβCD. 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.