Selective association of spectrin with the cytoplasmic surface of human erythrocyte plasma membranes. Quantitative determination with purified (32P)spectrin.
A specific association between spectrin and the inner surface of the human erythrocyte membrane has been examined by measuring the binding of purified [32P]spectrin to inside out, spectrin-depleted vesicles and to right side out ghost vesicles. Spectrin was labeled by incubating erythrocytes with 32Pi, and eluted from the ghost membranes by extraction in 0.3 mM NaPO4, pH 7.6. [32P]Spectrin was separated from actin and other proteins and isolated in a nonaggregated state as a So20,w = 7 S (in 0.3 mM NaPO4) or So20,w = 8 S (in 20 mM KCl, 0.3 mM NaPO4) protein after sedimentation on linear sucrose gradients. Binding of [32P]spectrin to inverted vesicles devoid of spectrin and actin was at least 10-fold greater than to right side out membranes, and exhibited different properties. Association with inside out vesicles was slow, was decreased to the value for right side out vesicles at high pH, or after heating spectrin above 50 degrees prior to assay, and was saturable with increasing levels of spectrin. Binding to everted vesicles was rapid, unaffected by pH or by heating spectrin, and rose linearly with the concentration of spectrin. Scatchard plots of binding to inverted vesicles were linear at pH 7.6, with a KD of 45 microng/ml, while at pH 6.6, plots were curvilinear and consistent with two types of interactions with a KD of 4 and 19 microng/ml, respectively. The maximal binding capacity at both pH values was about 200 microng of spectrin/mg of membrane protein. Unlabeled spectrin competed for binding with 50% displacement at 27 microng/ml. [32P]Spectrin dissociated and associated with inverted vesicles with an identical dependence on ionic strength as observed for elution of native spectrin from ghosts. MgCl2, CaCl2 (1 to 4 mM) and EDTA (0.5 to 1 mM) had little effect on binding in the presence of 20 mM KCl, while at low ionic strength, MgCl2 (1 mM) increased binding and inhibited dissociation to the same extent as 10 to 20 mM KCl. Binding was abolished by pretreatment of vesicles with 0.1 M acetic acid, or with 0.1 microng/ml of trypsin. The periodic acid-Schiff-staining bands were unaffected by trypsin digestion which destroyed binding; mild digestion, which decreased binding only 50%, converted Band 3 almost completely to a membrane-bound 50,000-dalton fragment resistant to further proteolysis. These experiments suggest that attachment of spectrin to the cytoplasmic surface of the membrane results from a selective protein-protein interaction which is independent of erythrocyte actin. A direct role of the major sialoglycoprotein or Band 3 as a membrane binding site appears unlikely.
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