Lipid rafts exist as stable cholesterol-independent microdomains in the brush border membrane of enterocytes

Glycosphingolipid/cholesterol-rich membranes ("rafts")can be isolated from many types of cells, but their existence as stable microdomains in the cell membrane has been elusive. Addressing this problem, we studied the distrib ution of galectin-4, a raft marker, and lactase, a protein excluded from ra fts, on microvillar vesicles from the enterocyte brush border membrane. Mag netic beads coated with either anti-galectin-4 or anti-lactase antibodies w ere used for immunoisolation of vesicles followed by double immunogold labe ling of the two proteins. A morphometric analysis revealed subpopulations o f raft-rich and raft-poor vesicles by the following criteria: 1) the lactas e/galectin-4 labeling ratio/vesicle captured by the anti-lactase beads was significantly higher (p less than or equal to 0.01) than that of vesicles c aptured by anti-galectin-4 beads, 2) subpopulations of vesicles labeled by only one of the two antibodies were preferentially captured by beads coated with the respective antibody (p less than or equal to 0.01), 3) the averag e diameter of "galectin-4 positive only" vesicles was smaller than that of vesicles labeled for lactase. Surprisingly, pretreatment with methyl-beta - cyclodextrin, which removed > 70% of microvillar cholesterol, did not affec t the microdomain localization of galectin-4. We conclude that stable, chol esterol-independent raft microdomains exist in the enterocyte brush border.