Maintenance of lipid asymmetry in red blood cells and ghosts: effect of divalent cations and serum albumin on the transbilayer distribution of phosphatidylserine

The maintenance of lipid asymmetry in the plasma membrane of human red blood cells (RBC) was investigated by assessing the equilibrium distribution of exogenously inserted NBD-labeled phosphatidylserine (PS) and endogenous PS in RBC and hypotonically lysed ghosts. PS distribution was determined by the ability to 'back-exchange' NBD-lipids into acceptor membranes and bovine serum albumin, and by prothrombinase complex assay for endogenous PS. To maintain the normal asymmetric distribution of PS in RBC, ghosts required Mg²⁺ in the lysis buffer. The inclusion of Ca²⁺, even in the presence of Mg²⁺ resulted in complete randomization of endogenous and exogenously inserted PS. These results indicate that NBD-labeled PS analogs faithfully monitor the distribution of endogenous PS during ghost preparation. In contrast, treatment of RBC with bovine serum albumin had no effect on the distribution of endogenous PS, although it resulted in a time-dependent movement of NBD-labeled PS from the inner to the outer leaflet (flop). This phenomenon was dependent on continuous incubation in the presence of albumin and could not be duplicated when pure acceptor membranes were used.