Resistance of human erythrocyte membranes to Triton X-100 and C12E8.
Lipid rafts are microdomains enriched in cholesterol and sphingolipids that contain specific membrane proteins. The resistance of domains to extraction by nonionic detergents at 4 degrees C is the commonly used method to characterize these structures that are operationally defined as detergent-resistant membranes (DRMs). Because the selectivity of different detergents in defining membrane rafts has been questioned, we have compared DRMs from human erythrocytes prepared with two detergents: Triton X-100 and C12E8. The DRMs obtained presented a cholesterol/protein mass ratio three times higher than in the whole membrane. Flotillin-2 was revealed in trace amounts in DRMs obtained with C12E8, but it was almost completely confined within the DRM fraction with Triton X-100. Differently, stomatin was found distributed in DRM and non-DRM fractions for both detergents. We have also measured the order parameter (S) of nitroxide spin labels inserted into DRMs by means of electron paramagnetic resonance. The 5- and 16-stearic acid spin label revealed significantly higher S values for DRMs obtained with either Triton X-100 or C12E8 in comparison to intact cells, while the difference in the S values between Triton X-100 and C12E8 DRMs was not statistically significant. Our results suggest that although the acyl chain packing is similar in DRMs prepared with either Triton X-100 or C12E8 detergent, protein content is dissimilar, with flotillin-2 being selectively enriched in Triton X-100 DRMs.
Crepaldi Domingues
Cleyton
C
Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brazil.
Ciana
Annarita
A
Buttafava
Armando
A
Balduini
Cesare
C
de Paula
Eneida
E
Minetti
Giampaolo
G
D015153:Blotting, Western; D002784:Cholesterol / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology*; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000187:drug effects*; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry / Q000187:drug effects*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology*
D003902:Detergents; D008565:Membrane Proteins; C106354:flotillins; D017830:Octoxynol; D002784:Cholesterol