The isolation and structure of membrane lipid rafts from rat brain

摘要

The action of detergents in the isolation of detergent-resistant membrane fractions from rat brain is reported. Triton X-100 treatment of whole rat brain homogenate at 4℃ produced detergent-resistant membranes with a density of 1.07 g/ml compared with Brij96 where the density of the membrane was only 1.05 g/ml. The DRM fractions isolated using Triton X-100 are considerably heavier than those isolated from homogenates treated with Brij96. The major polar lipid composition of DRMs derived from Brij96 treated homogenates have a higher proportion of amino-phospholipids compared with choline phospholipids than Triton X-100 derived DRMs; this may indicate that DRMs from Brij96 treated homogenates are more closely related to the parent membrane in lipid composition. Solubilization by Triton X-100 at higher temperatures resulted in the appearance of a second detergent-resistant membrane fraction distinctly lighter in density than the membrane recovered at density 1.07 g/ml. Analysis of phospholipid composition of the brain homogenate during detergent treatment for up to 30 min at 37℃ showed a decreasing proportion of sphingomyelin. Treatment of homogenates at 37℃ appears to activate phospholipases/sphingomyelinases that may alter the lipid content of isolated DRMs. The presence of K~+/Mg~(2+) with Brij96 treatment results in DRM fractions with significantly thicker bilayers and of larger vesicle diameter than DRMs isolated from either Triton X-100 or Brij96 treated homogenates in the absence of cations.