Biochemical characterization of complex formation by human erythrocyte spectrin, protein 4.1, and actin

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Abstract
Ternary complex formation between the major human erythrocyte membrane skeletal proteins spectrin, protein 4.1, and actin was quantified by measuring cosedimentation of spectrin and band 4.1 with F-actin. Complex formation was dependent upon the concentration of spectrin and band 4.1, each of which promoted the binding of the other to F-actin. Simultaneous measurement of the concentrations of spectrin and band 4.1 in the sedimentable complex showed that a single molecule of band 4.1 was sufficient to promote the binding of a spectrin dimer to F-actin. However, the molar ratio of band 4.1/spectrin in the complex was not fixed, ranging from .apprx. 0.6 to 2.2 as the relative concentration of added spectrin to band 4.1 was decreased. A mol ratio of 0.6 band 4.1/spectrin suggests that a single molecule of band 4.1 can promote the binding of more than 1 spectrin dimer to an actin filament. Saturation binding studies showed that in the presence of band 4.1 every actin monomer in a filament could bind at least 1 molecule of spectrin, yielding ternary complexes with spectrin/actin mol ratios as high as 1.4. Electron microscopy of such complexes showed them to consist of actin filaments heavily decorated with spectrin dimers. Ternary complex formation was not affected by alteration in Mg2+ or Ca2+ concentration but was markedly inhibited by KCl above 100 mM and nearly abolished by 10 mM 2,3-diphosphoglycerate or 10 mM ATP. The data are used to refine the molecular model of the red cell membrane skeleton.

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