ARTICLE

ANIONIC SITES OF HUMAN ERYTHROCYTE MEMBRANES : II. Antispectrin-Induced Transmembrane Aggregation of the Binding Sites for Positively Charged Colloidal Particles

Garth L. Nicolson ¹ and Richard G. Painter ¹

¹ From the Cancer Council and Electron Microscopy Laboratories, Armand Hammer Center for Cancer Biology, The Salk Institute for Biological Studies, San Diego, California 92112, and the Department of Biology, University of California, San Diego, La Jolla, California 92037

The effects of affinity-purified antispectrin -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 -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 -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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• Nicolson, G. L., Yanagimachi, R. (1974). Mobility and the Restriction of Mobility of Plasma Membrane Lectin-Binding Components. Science 184: 1294-1296 [Abstract]  

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