CALPROMOTIN, A CYTOPLASMIC PROTEIN, IS ASSOCIATED WITH THE FORMATION OF DENSE CELLS IN SICKLE-CELL-ANEMIA

We have tested the hypothesis that dense cell formation In sickle cell disease is associated with increased binding of calpromotin to the me mbrane, an event that occurs during the activation of calcium-dependen t potassium transport, By SDS polyacpylamide gel electrophoresis, we f ound that sickle cell membranes contained more calpromotin than did no rmal membranes when stained with Coomassie brilliant blue or when tran sferred to nitrocellulose paper and immunostained with horseradish per oxidase, Also, the membranes from dense sickle cells contained signifi cantly (P=0.00055) higher levels of calpromotin, 2.62 +/- 1.59 mu g/mg membrane protein, compared to light sickle cells, 1.40 +/- 0.70 mu g/ mg membrane protein, when measured by an enzyme-linked immunosorbernt assay. The ratio of calpromotin associated with dense cell membranes t o light cell membranes was significantly greater than 1.0 (P < 0.00005 ). Transmission electron micrographs of immunogold-labelled membranes supported the increase in calpromotin binding in dense sickle cell mem branes. In addition, the immunogold probe demonstrated clustering, whi ch was not observed in light sickle cell membranes nor in normal membr anes, Finally, we incubated HbSS cells in vitro using a repetitive deo xygenation/reoxygenation procedure to produce dense cells and then mea sured the levels of calpromotin associated with their membranes. As ex pected, the levels of calpromotin bound to the membrane doubled during the procedure relative to the basal levels at the beginning of the in cubation, The correlation coefficient, calculated between the increase in dense cell formation and the increase In calpromotin associated wi th the membrane, was statistically significant (P = 0.038), The result s demonstrate that an increase in calpromotin binding to the membrane is associated with dense cell formation presumably through the activat ion of the calcium-dependent potassium channel, (C) 1997 Wiley-Liss, I nc.