Adducin-binding proteins in KI-IOVs revealed by label transfer and copelleting experiments. (A) β-Adducin–binding proteins in IOVs revealed by label transfer experiments. Purified β-adducin tail (expressed in E coli) was incubated in the dark with a 2-fold molar excess of sulfo-SBED for 3 hours at 4°C, after which the protein solution was dialyzed overnight against PBS to remove unbound sulfo-SBED. IOVs (200 μg protein) were incubated with the above labeled β-adducin tail for 1 hour at room temperature in the dark to allow membrane association, after which cross-linking to nearest neighbor proteins was activated on ice by exposure for 15 minutes to a 302-nm light source (18.4 W) at a distance of 5 cm. A total of 10 mM dithiothreitol was then added to reduce the disulfide linkage between sulfo-SBED–adducin and its membrane anchor, and the labeled membrane anchor containing the transferred biotin was analyzed by SDS-PAGE, followed by transfer to nitrocellulose and visualization with streptavidin–horseradish peroxidase. Coomassie blue–stained gel lanes A through C contain molecular weight standards (lane A), or IOVs incubated with labeled β-adducin tail either in the absence (lane B) or presence (lane C) of a 20-fold excess of unlabeled β-adducin tail to competitively block all adducin binding sites on the IOVs. Lanes D and E represent streptavidin–horseradish peroxidase blots of lanes B and C. (B) KI-IOVs (100 μg) were incubated with increasing amounts of ¹²⁵I-adducin (purified from mature erythrocytes) or ¹²⁵I-BSA (60 μL total volume) for 2 hours at room temperature in a buffer consisting of phosphate-buffered saline containing 10% sucrose, protease inhibitor mixture, 1 mg/mL BSA, and no Mg²⁺. Bound proteins were separated by centrifugation through a 25% sucrose cushion and quantified by γ counting. The BSA control was subtracted. Data points represent mean ± SD, n = 2. An apparent K_D of approximately 100 nM was calculated assuming a noncooperative, single site–binding equilibrium.