Incorporation of polypeptide 1-301 of β-spectrin into membrane skeletal junctions in situ and effect of the incorporation on membrane stability. (A) GST-tagged polypeptide 1-301 was introduced into TM-depleted ghosts. Isolated membrane skeletons from the resealed ghosts were analyzed by electrophoresis in 10% SDS-PAGE. (B) Effect of polypeptide 1-301 on membrane stability of TM-depleted ghosts. Membrane stability, expressed as the rate of decline in DI, diminishes (decay curve shifted toward left) with increasing concentration of peptide. (C) Binding of the peptide to ghost membranes, prepared in the absence (•) and presence (○) of magnesium ions. The curves are calculated best-fits for a single set of independent spectrin-4.1R–binding sites, assuming a site concentration of 1.2 μM. The resulting values of K_a are 3.9× 10⁴ M⁻¹ (± 0.6 × 10⁴M⁻¹) (broken line) and 2.0 × 10⁴ M⁻¹ (± 0.2× 10⁴ M⁻¹) (full line). (D) Effect of incorporated peptide on shear resistance of ghosts prepared in the absence (•) or presence (○) of 2 mM MgCl₂, showing reduced shear resistance in the former. Membrane stability was assessed from DI decay curves. The relative stability is defined as the ratio of time under shear for a 50% drop in signal to that for TM-containing ghosts at zero peptide concentration.