The isolation and structure of membrane lipid rafts from rat brain.
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 degrees C produced detergent-resistant membranes with a density of 1.07g/ml compared with Brij96 where the density of the membrane was only 1.05g/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 aminophospholipids 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 1.07g/ml. Analysis of phospholipid composition of the brain homogenate during detergent treatment for up to 30min at 37 degrees C showed a decreasing proportion of sphingomyelin. Treatment of homogenates at 37 degrees C appears to activate phospholipases/sphingomyelinases that may alter the lipid content of isolated DRMs. The presence of K+/Mg2+ 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.
Chen
X
X
Department of Biochemistry, King's College London, 150 Stamford Street, London SE1 9NH, UK.
Morris
R
R
Lawrence
M J
MJ
Quinn
P J
PJ
D000818:Animals; D001923:Brain Chemistry; D002784:Cholesterol / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D002855:Chromatography, Thin Layer; D005260:Female; D008297:Male; D008563:Membrane Lipids / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D021962:Membrane Microdomains / Q000737:chemistry*; D017830:Octoxynol / Q000737:chemistry; D010743:Phospholipids / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats; D013109:Sphingomyelins / Q000032:analysis / Q000302:isolation & purification
D008563:Membrane Lipids; D010743:Phospholipids; D013109:Sphingomyelins; D017830:Octoxynol; D002784:Cholesterol