The effects of affinity-purified antispectrin γ-globulins on the topographic distribution of anionic residues on human erythrocytes membranes was investigated using collo ida iron hydroxide labeling of mounted, fixed, ghost membranes. Antispectrin γ-globulins were sequestered inside ghosts by hemolysis and the ghosts were incubated for 30 min at 37°C and then fixed with glutaraldehyde. The topographic distribution of colloidal iron hydroxide clusters on ghosts incubated with low (<0.05 mg/ml) or high (>5–10 mg/ml concentrations of sequestered antispectrin was dispersed, but the distribution at intermediate concentrations (0.1–5 mg/ml) was highly aggregated. The aggregation of colloidal iron hydroxide binding sites was time and temperature dependent and required the sequestering of cross-linking antibodies (antispectrin Fab could not substitute for γ-globulin antibodies) inside the ghosts. Prior glutaraldehyde fixation or fixation at the time of hemolysis in antispectrin solutions prevented the antispectrin-induced colloidal iron site aggregation. The antispectrin reacted exclusively at the inner ghost membrane surface and the colloidal iron hydroxide bound to N-acetylneuraminic acid residues on the outer membrane surface which are overwhelming on the sialoglycoprotein glycophorin. These results were interpreted as evidence for a structural transmembrane linkage between the inner surface peripheral protein spectrin and the integral membrane component glycophorin.

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