Human erythrocyte band 3. Solubilization and reconstitution into two-dimensional crystals.
Various polyoxyethylene alkylethers were used to extract integral proteins from human erythrocyte membranes. The solubilization power of these detergents and the oligomerization of solubilized band 3 were studied. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that short-chain detergents induced oligomers larger than the band 3 dimer. In contrast, after solubilization with long-chain detergents, the predominant band on SDS-containing gels was the monomeric band 3. Oligomerization in short-chain detergents occurred preferentially at room temperature whereas monomeric band 3 prevailed at 4 degrees C. Consistent with these results, negative stain electron microscopy of solubilized isolated band 3 showed larger complexes with short-chain detergents than with long-chain detergents. Cu2+/o-phenanthroline-induced crosslinking had no effect on size or shape of band 3 particles. Despite their rather heterogeneous dimensions, octylpolyoxyethylene-solubilized band 3 complexes assembled into two-dimensional trigonal lattices (a = b = 11 (+/- 0.5) nm) in the presence of dimyristoyl phosphatidylcholine. The unit cell exhibited a pronounced stain-filled region surrounded by three elongated morphological subunits. Each subunit most likely represents a band 3 dimer. Freeze-drying/metal-shadowing of reconstituted lattices revealed one large elevation per unit cell protruding from an otherwise smooth surface.
Dolder
M
M
Maurice E. Müller-Institute for High-resolution Electron Microscopy, Biocenter, Basel, Switzerland.
Walz
T
T
Hefti
A
A
Engel
A
A
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D003902:Detergents; D004134:Dimyristoylphosphatidylcholine; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D013816:Thermodynamics; D014961:X-Ray Diffraction
D001457:Anion Exchange Protein 1, Erythrocyte; D003902:Detergents; D046911:Macromolecular Substances; D004134:Dimyristoylphosphatidylcholine