An immunochemical approach for the analysis of membrane protein alterations in Ca2+-loaded human erythrocytes.

An increase in the intracellular concentration of Ca2+ in human erythrocytes results in the formation of gamma-glutamyl-epsilon-lysine cross-linked membrane protein polymers. Following solubilization of the membranes with SDS, these polymers can be isolated on a Lubrol-containing sucrose gradient. Immunoelectrophoresis of the polymeric material with a polyspecific rabbit antibody against human ghosts gave rise to a single, but heterogeneous, precipitate. The polymer was amphiphilic and, on addition to Triton-solubilized erythrocyte membrane proteins, it coprecipitated with spectrin. When the antihost antibody was absorbed with the polymer prior to cross immunoelectrophoresis of normal erythrocyte membrane proteins, the precipitates of glycophorin, acetylcholinesterase, and hemoglobin were normal, whereas the antibody titers against band 3 protein, spectrin, and ankyrin became reduced. Furthermore, a rabbit antibody raised against the isolated human polymer reacted selectively with the same three membrane proteins. No reactions occurred with lysate proteins.