A rapid method of reconstituting human erythrocyte sugar transport proteins.
A rapid reconstitution procedure for human erythrocyte hexose transfer activity is described. The procedure (reverse-phase evaporation) avoids exposure of the isolated proteins to detergent, organic solvent, sonication, or freeze-thaw steps during insertion into synthetic membranes and may be effected within 15 min. The so-formed vesicles are unilamellar structures with a large encapsulated volume, narrow size range, and low passive permeabilities. Contamination by carry-through of endogenous (red cell) lipids is less than 1%. Reconstituted hexose transfer activity was examined by using unfractionated proteins (bands 3, 4.5, and 6) and purified proteins (bands 4.5 and 3). With unfractionated proteins, hexose transport activity is low [0.34 mumol X (mg of protein)-1 X min-1], is inhibited by cytochalasin B, and increases monotonically with protein concentration. Kinetic analysis indicates that Vmax values for both influx and efflux of D-glucose are identical. Reconstitution of the cytochalasin B binding protein (band 4.5) results in hexose transport with high specific activity [5 mumol X (mg of protein)-1 X min-1] and symmetry in transfer kinetics. Band 3 proteins also appear to mediate cytochalasin B sensitive D-glucose transport activity.
D001786:Blood Glucose / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003571:Cytochalasin B / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D009004:Monosaccharide Transport Proteins
D001786:Blood Glucose; D002352:Carrier Proteins; D008081:Liposomes; D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B; D004134:Dimyristoylphosphatidylcholine