Comparison of the cytoskeleton fractions of rat red blood cells prepared with non-ionic detergents.
The characteristics of cytoskeleton fractions prepared from rat red cell ghosts with four non-ionic detergents were studied. One percent (w/v) solutions of Triton X-100, Emulgen 911, MEGA-9 (nonanoyl-N-methylglucamide), and octylglucoside solubilized 78, 68, 80, and 92% of the ghost phospholipid, while they solubilized 82, 78, 72, and 62% of the ghost band 3, a transmembrane protein, respectively. There was no correlation between the solubilization percentages of phospholipid and band 3. Phospholipids retained in cytoskeleton fractions were shown to exist as blebs on the surface by electron microscopic observation. The cytoskeleton fraction prepared with octylglucoside retained about two-fold more band 3 than that with Triton X-100 (Triton shells). However, cytoskeleton fractions prepared from p-chloromercuribenzoate-treated ghosts with the two detergents retained almost equal amounts of band 3, less than 5% of that in the ghosts. Under this condition, most of band 2.1, a protein linking band 3 to the spectrin-actin network, was released from the cytoskeleton fractions. The band 3 solubilized with octylglucoside sedimented faster in a linear sucrose gradient and had a larger Stokes' radius than that with Triton X-100, which is known to exist as dimer. These results strongly suggest that octylglucoside does not disturb the association of tetrameric band 3 with the spectrin-actin network, while Triton X-100 dissociates tetrameric band 3 to the dimer, resulting in the difference in the amount of band 3 retained in cytoskeleton fractions. In conclusion, octylglucoside can produce a more native cytoskeleton fraction of red cell membranes than Triton shells.
Kunimoto
M
M
Basic Medical Sciences Division, National Institute for Environmental Studies, Ibaraki.
Shibata
K
K
Miura
T
T
D000818:Animals; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003599:Cytoskeleton / Q000032:analysis*; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis; D004912:Erythrocytes / Q000032:analysis*; D005960:Glucosides; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D017830:Octoxynol; D010743:Phospholipids / Q000032:analysis; D011092:Polyethylene Glycols; D011189:Potassium Chloride; D011487:Protein Conformation; D051381:Rats
D003902:Detergents; D005960:Glucosides; D010743:Phospholipids; C018619:octyl-beta-D-glucoside; D011092:Polyethylene Glycols; D011189:Potassium Chloride; D017830:Octoxynol