MATERIALS AND METHODS

Recently outdated human blood was obtained from the Oxfordshire Blood Transfusion Service, lipids were purchased from Lipid Products, Triton X-100 (reduced form) was obtained from Aldrich Chemical Co., Bio-GelP6-DG desalting gel and the SM2 Bio-Beads were from Bio-Rad,[3H] Triton

X-100 was purchased from New EnglandNuclear Chemicals, Sepharose 4B was obtained from Pharmacia, and [3H] octyl glucoside was obtained from Amersham International. The rest of the materials were of the highest analytical grade. Preparation of Protein Samples.(A) Band 3. Erythrocyte membranes (20 mL; proteincontent, 3 mg/mL), prepared as described in Dodgeet al.(1963), were extracted on ice for 1 h with 2 volumes of extraction buffer (10 mM Tris-HCl, pH 8, 10 mM NaCl, and 0.5 mM EDTA) containig 1% Brij-58. Brij-58 specifically removes glycophorin from the membrane. The extracted ghosts were then centrifuged (30000#, 20 min, 4 C). The supernatant, enriched with glycophorin, was discarded and the pellet was resuspended intwice the original amount of erythrocyte membrane volume of buffer containing 0.5% Triton X-100. This suspension was then extracted on ice for 1 h. The extract was centrifuged (30000#, 20 min, 4 C). The pellet, enriched with peripheralproteins, was discarded and the supernatant was applied to a preequilibrated DEAE-cellulose column. The column was first washed with twice the column volume of buffer containing 0.5% Triton X-100 and 100 mM NaCl, which removed the majority of contaminating proteins. The column was then eluted with twice the column volume of buffer containing 0.5% Triton X-100 and 200 mM NaCl. This eluant, containing band 3 and some peripheral proteins, was then applied directly to a preequilibrated p-cmb affinity gel column. The affinity gel was prepared according to Lukacovic et al.(1981). This column was washed with twice the column volume of buffer containing 0.5% Triton X-100 and 1