630 UNSICKLING OF “IRREVERSIBLY” SICKLED GHOSTS BY CONDITIONS WHICH INTERFERE WITH SPECTRIN-ACTIN POLYMERIZATION

Red cell (RBC) membrane shape depends, in part, on the RBC membrane skeleton; a protein meshwork of spectrin and actin which laminates the inner membrane surface. We have shown (JCI 58:955, 1976) that irreversibly sickled cells (ISCs) form ISC shaped ghosts and skeletons, which suggests the ISC shape is caused by an acquired skeletal defect. In this study we sought conditions that would alter or reverse this abnormal shape. Ghosts of ISC-rich RBCs were loaded with various agents, incubated at 37°C for 35 min and examined by phase microscopy. In isotonic media ISC ghosts retained an ISC shape. Addition of ATP, ADP, Mg++, Ca++, EDTA (all lmM), DTT (5mM) and various combinations of these agents did not alter this shape. However ISC shaped ghosts became round and indistinguishable from normal in hypertonic NaCl (>400mM), hypotonic NaCl (10-50mM) or isotonic NaCl containing small amounts of Zn++(0.1-0.5mM). ISC shape reversal was time and temperature dependent, and did not correlate with elution or proteolysis of any protein detectable on SDS gels, but did correlate with the formation, in vitro, of a high molecular weight complex of spectrin and actin. Conditions which promoted ISC ghost reversal inhibited formation of this complex.These observations indicate that the ISC shape is not maintained by covalent bonds and suggest that ISCs may be stabilized by abnormal interactions between the spectrin and/or actin components of the membrane skeleton and that disruption of these bonds may allow the skeleton to resume a normal shape.