On the oligomeric state of the red blood cell glucose transporter GLUT1.
We stripped human red blood cell membranes of cytoskeleton proteins at pH 12 without reductant, partially solubilized the obtained vesicles by use of octaethylene glycol n-dodecyl ether and purified the glucose transporter GLUT1 by anion-exchange chromatography followed by sulfhydryl-affinity chromatography, which removed most of the nucleoside transporter (NT) and the lipids. Eighty percent of the sulfhydryl-bound GLUT1 could be eluted with sodium dodecyl sulfate (SDS) indicating that the bound protein was multimeric. Matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-ToF-MS) of the trypsinized major SDS-PAGE zone of the purified material identified GLUT1 but no other membrane protein. Transmembrane helices 1 and 8 were among the detected fragments. The reconstituted purified GLUT1 showed glucose transport activity, although only approximately 0.05 high-affinity cytochalasin B (CB) binding sites were present per GLUT1 monomer. The vesicles used as starting material for the purification showed 0.4 CB sites per GLUT1 monomer, similar to vesicles prepared in the presence of dithioerythritol. The data are consistent with the coexistence of monomeric GLUT1 with high-affinity CB-binding activity and preferentially solubilized multimeric GLUT1 with no CB-binding activity in the red blood cell membrane vesicles prepared without reductant.
Zuo
Shusheng
S
Department of Biochemistry, Biomedical Center, Uppsala University, Box 576, S-751 23 Uppsala, Sweden. Shusheng.Zuo@biokem.uu.se
Hellman
Ulf
U
Lundahl
Per
P
D002846:Chromatography, Affinity; D004912:Erythrocytes / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D006801:Humans; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship
D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; C495677:SLC2A1 protein, human; D005947:Glucose