BINDING OF ANNEXIN-V TO BILAYERS WITH VARIOUS PHOSPHOLIPID COMPOSITIONS USING GLASS-BEADS IN A FLOW CYTOMETER

Annexin V belongs to the family of calcium-dependent phospholipid bind ing proteins and binds almost solely to phosphatidylserine (PS). When annexin V is used, to detect loss of membrane asymmetry in cellular sy stems, the binding properties under physiological conditions are of im portance, Most biochemical studies use optimized binding conditions, c onditions that are often far from physiological. For the interpretatio n of flow cytometric studies that use fluorescent annexin V to probe P S exposure, it is important to know what mixture of Lipid species expo sed in the outer leaflet of a membrane can evoke a positive annexin V signal,The lipid species is important in this respect as well as the c oncentration that just evokes a positive signal (detection level). Fur thermore, the influence of the composition of the lipid matrix (choles terol content, other phospholipid species) was investigated, as well a s the influence of the calcium concentration on annexin V binding, in this study, we report on the binding of annexin V to phospholipid bila yers (adsorbed to glass beads) as measured by flow cytometry at physio logical conditions. Annexin V binding was found to increase rapidly, w ith increasing PS concentrations up to a certain level (attained at 6 mol% PS). Further increase of the PS concentration resulted only in a slight increase of annexin V binding. Calcium concentrations below 3 m M were found to reduce the sensitivity of the binding assay. Phosphati dylethanolamine incorporated in the phospholipid bilayer resulted in a lower threshold for the binding assay, whereas sphingomyelin had no i nfluence on the binding of annexin V and cholesterol reduces binding o f annexin V to Lipid bilayers. These data may help in the interpretati on of results obtained from binding of annexin V to cell membranes (e. g., involved in apoptosis), (C) 1998 Wiley-Liss,Inc.