The stereospecific D-glucose transport activity of cholate extracts from human erythrocyte membranes.
The glucose transport protein of human erythrocyte membranes was solubilized with cholate to facilitate rapid reconstitution and direct glucose transport measurements. This may simplify the isolation of the native glucose transporter. In most experiments the membranes were prepared from fresh blood within 8 h, frozen in liquid nitrogen and stored at -70 degrees C to minimize proteolytic degradation. Solubilization with 25 mM cholate in the presence of 200 mM NaCl at pH 8.4 for 12 min at room temperature gave a high D-glucose transport activity. The solubilized mixture contained 20% of the total membrane protein, only 6% of the polypeptides of molecular weight around 90000, 23% of the polypeptides of molecular weight around 55000, 30% of the phospholipids and at least 6% of the stereospecific D-glucose transport activity. At cholate concentrations up to 22 mM the ratio of solubilized phospholipids to cholate increased steeply, concomitant with an increase in solubilized activity. Above 30 mM cholate the activity diminished. At 4 degrees C the activity of the extract decreased rapidly within the first day and slowly during the next few days. The initial changes seem to have produced a fairly stable, but not native form or fragment of the transporter. When 20 mM EDTA and 5 mM dithioerythritol were included in the solubilization mixture a high activity was preserved for about one day.
D001786:Blood Glucose / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002793:Cholic Acids; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009004:Monosaccharide Transport Proteins; D009005:Monosaccharides / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
D001786:Blood Glucose; D002352:Carrier Proteins; D002793:Cholic Acids; D009004:Monosaccharide Transport Proteins; D009005:Monosaccharides