The human red cell glucose transporter in octyl glucoside. High specific activity of monomers in the presence of membrane lipids.
Human red cell membranes were stripped of peripheral proteins and partially solubilized with 50-260 mM octyl glucoside at 2-14 mg protein/ml, to find conditions that afford a high concentration of active glucose transporter after purification on DEAE-cellulose. Transporter-egg yolk phospholipid vesicles were prepared by gel filtration. The specific D-glucose equilibrium exchange activities increased with increasing dilution of the glucose transporter. At 260 mM octyl glucoside the glucose transporter became partially denaturated. At 225 mM detergent the DEAE-cellulose chromatography showed one main and one minor fraction of active glucose transporter. Nucleoside transport activity was enriched in the minor fraction. Solubilization with 75 mM octyl glucoside at 8 mg protein/ml gave a maximal concentration of purified transporter, 0.8 mg/ml, probably corresponding to complete solubilization. The phospholipids were partially retarded on the DEAE-cellulose. The specific D-glucose equilibrium exchange was high, up to 200 nmol glucose/micrograms transporter in two min at 50 mM glucose. High performance gel filtration in octyl glucoside indicated that the transporter formed dimers during the fractionation. These eluted at Mr 125,000, partially separated from the phospholipids, which appeared at Mr 55,000 (cf. Mascher, E. and Lundahl, P. (1987) J. Chromatogr. 397, 175-186). The D-glucose transport activity was low in the main fraction and high in the transporter-phospholipid fraction. Mixing of these fractions did not increase the activity. The glucose transporter is probably dependent on one or more specific membrane lipid(s). Presumably the transporter dimerizes and loses activity upon removal of these lipids.
Mascher
E
E
Department of Biochemistry, Biomedical Center, University of Uppsala, Sweden.
Lundahl
P
P
D001692:Biological Transport; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004910:Erythrocyte Membrane / Q000032:analysis*; D005947:Glucose / Q000378:metabolism; D005960:Glucosides / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008563:Membrane Lipids / Q000502:physiology; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000097:blood* / Q000648:ultrastructure; D012995:Solubility; D013329:Structure-Activity Relationship
D005960:Glucosides; D008563:Membrane Lipids; D009004:Monosaccharide Transport Proteins; C018619:octyl-beta-D-glucoside; D005947:Glucose