A POSTTRANSLATIONAL MODIFICATION OF BETA-ACTIN CONTRIBUTES TO THE SLOW DISSOCIATION OF THE SPECTRIN-PROTEIN 4.1-ACTIN COMPLEX OF IRREVERSIBLY SICKLED CELLS

Irreversibly sickled cells (ISCs) remain sickled even under conditions where they are well oxygenated and hemoglobin is depolymerized. In ou r studies we demonstrate that triton extracted ISC core skeletons cont aining only spectrin, protein 4.1, and actin also retain their sickled shape; while reversibly sickled cell (RSC) skeletons remodel to a rou nd or biconcave shape. We also demonstrate that these triton extracted ISC core skeletons dissociate more slowly upon incubation at 37 degre es C than do RSC or control (AA) core skeletons. This observation may supply the basis for the inability of the ISC core skeleton to remodel its shape. Using an in vitro ternary complex dissociation assay, we d emonstrate that a modification in beta-actin is the major determinant of the slow dissociation of the spectrin-protein 4.1-actin complex iso lated from the ISC core skeleton. We demonstrate that the difference b etween ISC and control beta-actin is the inaccessibility of two cystei ne residues in ISC beta-actin to labeling by thiol reactive reagents; due to the formation of a disulfide bridge between cysteine(284) and c ysteine(373) in ISC beta-actin, or alternatively another modification of cysteine(284) and cysteine(373) which is reversible with DTT and ad ds less than 100 D to the molecular weight of beta-actin.