Reconstitution of glucose-transporting vesicles from erythrocyte membranes disaggregated in detergent.
With the eventual aim of purifying a membrane transport system by using reconstitution of transport activity as an assay, I showed that if, after the erythrocyte membrane is solubilized in deoxycholate, the detergent is removed, membrane vesicles re-form which retain glucose-transport activity. They take up and release D-glucose in preference to L-glucose and the uptake and release are sensitive to Hg2+ and phloretin. Release of tracer D-glucose is competitively inhibited by transported sugars inside the vesicles and increased by unlabelling D-glucose in the outside medium. Uptake of tracer is increased so much by preloading vesicles with unlabelled transported sugars that the tracer is probably concentrated against a gradient. When the membrane is solubilized, two proteins that span the membrane can be separated, suggesting that it will be possible to fractionate the membrane before reconstitution.
D001693:Biological Transport, Active; D003840:Deoxycholic Acid; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D010743:Phospholipids / Q000032:analysis; D012967:Sodium Dodecyl Sulfate; D012995:Solubility
D008565:Membrane Proteins; D010743:Phospholipids; D003840:Deoxycholic Acid; D012967:Sodium Dodecyl Sulfate; D005947:Glucose