PRELIMINARY CHARACTERIZATION OF A STRUCTURAL DEFECT IN HOMOZYGOUS SICKLED CELL ALPHA-SPECTRIN DEMONSTRATED BY A RABBIT AUTOANTIBODY

We have identified a rabbit autoantibody that strongly reacts with the core membrane skeleton of control red blood cells, and does not react with low- or high-density sickle cell core skeletons upon indirect im munofluorescence. Western blot analysis of red blood cell membrane pro teins, utilizing this autoantibody, indicated no reactivity to any pro tein when SDS-PAGE was conducted in the presence of the reducing agent , dithiothreitol. However when SDS-PAGE was performed on control red b lood cell membrane proteins separated in the absence of dithiothreitol , the autoantibody specifically reacted with a high molecular weight p olypeptide (apparent M-r congruent to 310 kD) representing a DTT sensi tive form of control alpha spectrin, which we refer to as alpha' spect rin, There was no staining of high density or low density sickle cell alpha or alpha' spectrin, This autoantibody should be an excellent too l for the fine mapping of structural change(s) in control vs, sickle c ell alpha spectrin, and determination of whether the structural altera tion effects spectrin dimer-tetramer interconversion and/or the spectr in-actin interaction. The modification in alpha spectrin, detected by this antibody, is very specific for homozygous SS a spectrin because s ickle cell beta(+) thalassemic alpha spectrin and sickle cell trait al pha spectrin react intensely with the autoantibody. Am. J. Hematol, 58 :200-205, 1998, (C) 1998 Wiley-Liss, Inc.