Purification and characterization of human erythrocyte glucose transporter in decylmaltoside detergent solution.
The facilitative glucose transporter from human erythrocyte membrane, Glut1, was purified by a novel method. The nonionic detergent decylmaltoside was selected for solubilization on the basis of its efficiency to extract Glut1 from the erythrocyte membrane and its ability to maintain the protein in a monodisperse state. A positive, anion-exchange chromatography protocol produced a Glut1 preparation of 95% purity with little copurified lipid. This protein preparation exhibited cytochalasin B binding in detergent solution, as measured by tryptophan fluorescence quenching. The transporter existed as a monomer in decylmaltoside, with a Stokes radius of 50 A and a molecular mass of 147 kDa for the protein-detergent complex. We screened detergent, pH, additive, and lipid and have found conditions to maintain Glut1 monodispersity for 8 days at 25 degrees C or over 5 weeks at 4 degrees C. This Glut1 preparation represents the best available material for two- and three-dimensional crystallization trials of the human glucose transporter protein.
Boulter
J M
JM
Department of Cell Biology, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, New York 10016, USA.
Wang
D N
DN
D002852:Chromatography, Ion Exchange; D003571:Cytochalasin B / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004355:Drug Stability; D004356:Drug Storage; D004910:Erythrocyte Membrane / Q000737:chemistry*; D051272:Glucose Transporter Type 1; D005960:Glucosides / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D008055:Lipids / Q000302:isolation & purification / Q000494:pharmacology; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D012996:Solutions; D013696:Temperature; D014461:Ultracentrifugation
D003902:Detergents; D051272:Glucose Transporter Type 1; D005960:Glucosides; D008055:Lipids; D009004:Monosaccharide Transport Proteins; C495677:SLC2A1 protein, human; D012996:Solutions; D003571:Cytochalasin B; C040358:dodecyl maltoside