Preservation of bilayer structure in human erythrocytes and erythrocyte ghosts after phospholipase treatment. A 31P-NMR study

1. 1. Fresh human erythrocytes were treated with lytic and non-lytic combinations of phospholipases A2, C and sphingomyelinase. The 31P-NMR spectra of ghosts derived from such erythrocytes show that, in all cases, the residual phospholipids and lysophospholipids remain organized in a bilayer configuration. 2. 2. A bilayer configuration of the (lyso)phospholipids was also observed after treatment of erythrocyte ghosts with various phospholipases even in the case that 98% of the phospholipid was converted into lysophospholipids (72%) and ceramides (26%). 3. 3. A slightly decreased order of the phosphate group of phospholipid molecules, seen as reduced effective chemical shift anisotropy in the 31P-NMR spectra, was found following the formation of diacylglycerols and ceramides in the membrane of intact erythrocytes. Treatment of ghosts always resulted in an extensive decrease in the order of the phosphate groups. 4. 4. The results allow the following conclusions to be made: 4.1. a. Hydrolysis of phospholipids in intact red cells and ghosts does not result in the formation of non-bilayer configuration of residual phospholipids and lysophospholipids. 4.2. b. Haemolysis, which is obtained by subsequent treatment of intact cells with sphingomyelinase and phospholipase A2, or with phospholipase C, cannot be ascribed to the formation of non-bilayer configuration of phosphate-containing lipids. 4.3. c. Preservation of bilayer structure, even after hydrolysis of all phospholipid, shows that other membrane constituents, e.g. cholesterol and/or membrane proteins play an important role in stabilizing the structure of the erythrocyte membrane. 4.4. d. A major prerequisite for the application of phospholipases in lipid localization studies, the preservation of a bilayer configuration during phospholipid hydrolysis, is met for the erythrocyte membrane.