Does diamide treatment of intact human erythrocytes cause a loss of phospholipid asymmetry?

Diamide-treated human erythrocytes have been compared with native red cells as to the accessibility of their amino phospholipids to both phospholipase A2 hydrolysis and fluorescamine labeling. In agreement with observations by others (Haest, C.W.M., Plasa, G., Kamp, D. and Deuticke, B. (1978) Biochim. Biophys. Acta 509, 21-32), treatment of intact human erythrocytes with diamide resulted in considerably enhanced degradation of amino phospholipids upon subsequent incubation of the cells with bee venom phospholipase A2. The hydrolysis of phosphatidylethanolamine (PE) in control cells reached a plateau value at 5% after 10 min. In diamide-treated cells, on the other hand, PE hydrolysis did not level off. Contrastingly, dose-response curves recorded for the labeling of PE with the very fast reacting NH2-group-specific reagent, fluorescamine, showed identical results for both native and diamide-treated erythrocytes. In each of these two cases, a plateau was reached after approx. 15% of the PE had been labeled. These results strongly suggest that the enhanced phospholipase-A2-induced hydrolysis of amino phospholipids in diamide-treated erythrocytes may reflect a destabilization of the lipid bilayer, rather than an in situ loss of phospholipid asymmetry.