Human erythrocyte hexose transporter activity is governed by bilayer lipid composition in reconstituted vesicles.

Purified membrane protein band 4.5 (a sugar transport protein) from human erythrocytes was reconstituted by reverse-phase evaporation into a variety of bilayers formed from the synthetic lecithins. The number of proteins reconstituted was estimated by determination of D-glucose-sensitive cytochalasin B binding sites. D-Glucose transport activity in reconstituted vesicles was assessed by monitoring cytochalasin B sensitive D-glucose fluxes using microturbidimetric analysis. The major points are as follows: The sugar transport activity of the reconstituted system is directly proportional to the number of cytochalasin B binding sites reconstituted. The ratio of cytochalasin B binding sites per band 4.5 protein is 0.8. These data suggest that the functional protein unit is a monomer. Inhibitor studies of reconstituted hexose transfer support the notion that the kinetics of reconstituted transport are intrinsically symmetric. The turnover number for transport is not consistent with transport proceeding via water-filled pores. The absolute activity of the reconstituted system (Vmax per reconstituted cytochalasin B binding protein) is governed by the bulk lipid composition of the synthetic membrane. At temperatures where bilayers formed from dimyristoyl- or dipalmitoyllecithin (DML and DPL, respectively) are "crystalline", hexose transport activity is not observed. Over the same temperature range, however, crystalline bilayers formed from the longer chain lecithins, distearoyl-, diarachidonoyl-, and dielaidoyllecithin (DSL, DAL, and DEL, respectively), support significant protein-mediated transport activity. In a given synthetic membrane, the bilayer transition from the liquid-crystalline to the fluid state results in increased protein-mediated sugar transport activity. In the one synthetic membrane (DEL) in which the activation energy (Ea) for transport could be measured both above and below the bilayer phase transition, Ea was unaffected by the phase change. Ea and the Arrhenius constant (A) for transport are dependent on lecithin acyl chain length and saturation. For both parameters, the order of increase is DML less than DPL = dipalmitoleoyllecithin (DPOL) less than DSL less than DAL less than DEL = dioleoyllecithin (DOL). This means that at 59-60 degrees C, the order of catalytic activity follows the lipid sequence DML less than DPOL less than DAL less than DOL much less than DPL = DEL less than DSL. Cholesterol (48 mol%) restores protein-mediated transport activity to crystalline DPL bilayers and reduces the activity supported by fluid DPL bilayers. This effect is not simply related to the effects of cholesterol on the bilayer partial specific volume. T