HIGH-DENSITY SICKLE-CELL ERYTHROCYTE CORE MEMBRANE SKELETONS DEMONSTRATE SLOW TEMPERATURE-DEPENDENT DISSOCIATION

We have previously demonstrated that slow dissociation of HDSS membran e skeletons in high ionic strength Triton X-100 buffer was related to a posttranslational modification in beta-actin, in which a disulfide b ridge was formed between cysteine 284 and cysteine 373 [Shartava et al : J Cell Biol 128:805, 1995]. These previous dissociation assays were limited to two homozygous (SS) sickle cell patients and a single tempe rature (37 degrees C). In the current work, we have expanded the SS su bjects to 9 and have carried out dissociation assays at 0, 24, 30, 34, and 37 degrees C. At 0 degrees C there was limited dissociation of sp ectrin and actin from normal (AA), low density sickle cell (LDSS), and high density sickle cell (HDSS) core skeleton up to 24 hr, The first order rate constants for dissociation of spectrin, at 0 degrees C, was 0.030-0.035 x 10(-4) sec(-1) for AA, LDSS, and HDSS core skeletons. H owever at 24, 30, 34, and 37 degrees C the rate of dissociation of spe ctrin from HDSS core skeletons was significantly slower than the rate of dissociation from AA core skeletons. Having determined the first or der rate constants for spectrin dissociation at these specified temper atures, we then asked whether dithiothreitol (DTT) would hasten the di ssociation of core skeletons. The presence of DTT caused the rate of d issociation of the HDSS membrane skeleton to become statistically indi stinguishable from the rate of dissociation of AA membrane skeletons. This is consistent with the suggestion that reversible thiol oxidation is responsible for the slow dissociation of the HDSS membrane skeleto n. (C) 1996 Wiley-Liss, Inc.