Anionic Sites of Human Erythrocyte Membranes

The effects of affinity-purified antispectrin gamma-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 gamma-globulins were sequestered inside ghosts by hemolysis and the ghosts were incubated for 30 min at 37 degrees C and then fixed with glutaraldehyde. The topographic distribution of colloidal iron hydroxide clusters on ghosts incubated with low (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 gamma-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.