Detergent-free domain isolated from Xenopus egg plasma membrane with properties similar to those of detergent-resistant membranes.
Microdomains known as "rafts" have been isolated from many cell types as detergent-resistant membranes (DRMs) and are enriched in sphingolipids and cholesterol. However, there has been considerable controversy over whether such domains are found in native membranes or are artificially generated by the purification procedure. This controversy is based at least in part on the fact that raft membranes were first detected following detergent extraction in the cold. We isolated two plasma membrane fractions, without detergent treatment, using a discontinuous sucrose density gradient. One fraction was designated "light" and the other "heavy." These fractions were compared with DRMs, which were isolated in the presence of 1% Triton X-100. We found that Xenopus DRMs are enriched with sphingomyelin and cholesterol and exhibit a phase state similar to the liquid-ordered phase. Comparison of DRM complexes with the light and heavy plasma membrane fractions revealed some physical and biochemical similarities between the light fraction of the plasma membrane and the DRM complexes, based on (1) the phosphatidylcholine/sphingomyelin ratio and (2) the protein composition visualized on a two-dimensional gel. These two fractions are also quite similar in their thermotropic phase behavior, and their high levels of ganglioside GM1. We conclude that the light membrane fraction isolated in a detergent-free environment has many of the characteristics normally associated with DRMs.
Luria
Ayala
A
Section of Molecular and Cellular Biology, University of California, Davis, California 95616, USA. arluria@ucdavis.edu
Vegelyte-Avery
Vaida
V
Stith
Brad
B
Tsvetkova
Nelly M
NM
Wolkers
Willem F
WF
Crowe
John H
JH
Tablin
Fern
F
Nuccitelli
Richard
R
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003902:Detergents / Q000737:chemistry*; D004351:Drug Resistance; D004527:Egg Proteins / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional; D005677:G(M1) Ganglioside / Q000378:metabolism; D008563:Membrane Lipids / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification; D008970:Molecular Weight; D010063:Ovum / Q000737:chemistry* / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D014982:Xenopus laevis
D003902:Detergents; D004527:Egg Proteins; D008563:Membrane Lipids; D008565:Membrane Proteins; D010743:Phospholipids; D005677:G(M1) Ganglioside