Lipid translocation across the human erythrocyte membrane. Regulatory factors

A simple method based on the differential extraction of lysophosphatidylcholine (LPC) by saline and albumin solutions has been developed to study the factors that influence lipid translocation across intact human erythrocyte membrane. With this assay, the rate of LPC translocation across the bilayer at 37 degrees C was found to be 1.87% h (0.0187 h-1). Identical translocation rates were derived for normal cells and cells in which the ATP was totally depleted, implying that the metabolic state of the cell had no influence. In contrast, the translocation rate was strongly influenced by temperature. Above 21 degrees C, the rate doubled for every 51 degrees C increase in temperature, suggesting an important role for diffusion through the lipid phase. Denaturation of a single major skeletal protein, spectrin, by heating cells to 5 degrees C did not alter the translocation rate. However, oxidative cross-linking of a complex of membrane proteins by treatment with diamide significantly increased the rate of translocation at 37 degrees C. Cholesterol enrichment of the cells decreased the apparent rate of translocation but not the total quantity of LPC translocated. Taken together, these data suggest that lipid translocation across the intact human erythrocyte membrane is not energy dependent, and that it is influenced by the organizational state of both the lipid and protein moieties of the membrane.