Purification of two spectrin-binding proteins: biochemical and electron microscopic evidence for site-specific reassociation between spectrin and bands 2.1 and 4.1.

Abstract

Two peripheral proteins of the human eryth- The human erythrocyte membrane provides a model system for investigating protein-membrane associations at the molecular level. Of particular interest is the existence of an extensive cytoskeletal network that may control cell shape and deformability (1-3) and the distribution of intramembrane particles (4) and surface markers (5). Interactions between spectrin, the major cytoskeletal protein, and the cytoplasmic surface of the erythrocyte membrane have been studied in detail and partially characterized (6-10). Bennett and Branton (6) have demonstrated the existence of a class of sites to which spectrin binds with high affinity, and recent evidence (8,11,12) indicates that band 2

Spectrin's E2/E3 ubiquitin conjugating/ligating activity is diminished in sickle cells

“…RBC membrane preparation and isolation of purified spectrin was performed as described [33]. Human red blood cell inside-out vesicles (IOV), depleted of spectrin and actin, were prepared as described [49]. The isolated spectrin was homogenous as indicated by SDS-PAGE [50].…”

Functional diversity among spectrin isoforms

“…These heterodimers further self-associate to form tetramers and larger oligomers. Both morphological studies directly visualizing spectrin tetramers and oligomers [Tyler et al, 1979Morrow and Marchesi, 198 11 and biochemical studies isolating and mapping the self-association domain [Morrow et al, 1980Morrow and Marchesi, 19811 indicate that spectrin tetramers and oligomers are composed of heterodimers arranged in a head-to-head fashion, in which the amino terminus of a-spectrin interacts with the carboxyl terminus of P-spectrin.…”

Protein 4.1 deficiency associated with an altered binding to the spectrin-actin complex of the red cell membrane skeleton.

“…Under these conditions, the spectrin extract was devoid of protein 4.1 as assessed by Western blot. Protein 4.1 was extracted from spectrin-depleted vesicles with 1 M KCl (20) and then purified by selective interaction with inositol hexaphosphate (21). Spectrin and protein 4.1 were dialyzed overnight against buffer A (130 mM KCl, 20 mM NaCl, 0.1 mM EGTA, 10 mM Tris, pH 7.4).…”