Reconstitution of glucose transport activity from erythrocyte membranes without detergent and its use in studying effects of ATP depletion.
The direct reconstitution of unsolubilized membrane proteins by the freeze-thaw procedure avoids possible changes in properties produced by detergent solubilization and fractionation. Glucose transport activity was reconstituted using human erythrocyte membranes, with about 2/3 of the glucose uptake being stereo-specific. The highest specific activity occurred at low ratios of protein to lipid in the reconstitution, where most transport was due to liposomes containing single transporter molecules. Transporters were reconstituted with a scrambling of orientations, indicated by a 50% inactivation by added trypsin. Separation of unreconstituted protein doubled the specific activity. Similar results were obtained using the purified transporter (Wheeler, T.J. and Hinkle, P.C. (1981) J. Biol. Chem. 256, 8907-8914). The same ratio of net uptake to equilibrium exchange was observed for the two preparations. Their relative reconstituted transport activities and cytochalasin B binding activities were equal, indicating that the two were reconstituted with similar efficiencies. The decrease in glucose transport in erythrocytes produced by ATP depletion and the stimulation produced by resealing with ATP (Jacquez, J.A. (1983) Biochim. Biophys. Acta 727, 367-378) were confirmed. However, no difference was observed in reconstituted transport activity using ghosts resealed with or without ATP, indicating that ATP produces indirect effects rather than modifications of the transporter.
D000255:Adenosine Triphosphate / Q000502:physiology*; D001692:Biological Transport; D002021:Buffers; D002498:Centrifugation; D003902:Detergents / Q000494:pharmacology*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008081:Liposomes / Q000032:analysis / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis; D009004:Monosaccharide Transport Proteins / Q000032:analysis; D013501:Surface-Active Agents / Q000494:pharmacology*; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D002021:Buffers; D003902:Detergents; D008081:Liposomes; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D013501:Surface-Active Agents; D000255:Adenosine Triphosphate; D014357:Trypsin; D005947:Glucose