The structure of mixed cholesterol-phospholipid monolayers spread at the air-water interface as probed by interactions with band 3 protein from erythrocyte membranes

Solubilized band 3 protein from human erythrocyte membranes (the anion transport protein) interacts strongly and specifically with monolayers of cholesterol spread at the air-water interface whereas, at pH 7–10, it shows only moderate interactions with phospholipid monolayers (Klappauf, E. and Schubert, D. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1225–1235). When band 3 protein, at pH 7 and an ionic strength of approx. 100 mM, is added to the subphase of mixed cholesterol-glycerophospholipid monolayers, the changes Δπ in monolayer surface pressure induced by the protein depend on the mole fraction X of sterol in the mixture. However, Δπ(X) only increases with increasing X towards the high values of Δπ that are characteristic of cholesterol monolayers if X>0.67±0.04; at lower cholesterol content, Δπ(X) is practically identical to the value obtained with the pure glycerophospholipid. With mixtures of coprostanol and glycerophospholipids, the break in the Δπ(X) curves occurs when X=0.33±0.03. Cholesterol-sphingomyelin and epicoprostanol-phosphatidylethanolamine mixtures show an increase of Δπ(X) when X>0. The data seem to support earlier claims that cholesterol can form stoichiometric complexes with glycerophospholipids, the stoichiometries revealed by the band 3-monolayer interactions being 2:1 and 1:2. They also show that cholesterol-sphingomyelin complexes, if they should exist, must be structurally different from the cholesterol-glycerophospholipid complexes.