Characterization of GLUT3 protein expressed in Chinese hamster ovary cells.
We have expressed GLUT3 protein, an isoform of a facilitative glucose transporter, in Chinese hamster ovary cells by transfection of its cDNA using an expression vector. The expressed GLUT3 protein was detected by Western-blot analysis as a broad band of 45-65 kDa, indicating intensive glycosylation of the protein. The expressed GLUT3 protein was observed, by immunofluorescence staining, to be located mainly at the plasma membrane, and its expression was associated with a marked increase in glucose-transport activity. Kinetic analysis revealed that the Km value of GLUT3 protein for 3-O-methylglucose uptake was approx. 35% of that of GLUT1 protein, whereas the Km value of GLUT3 protein for 2-deoxy-D-glucose uptake was very similar to that of GLUT1 protein. The Vmax. value of GLUT3 protein for 3-O-methylglucose and 2-deoxyglucose uptake was approx. 20-50% of that of GLUT1 protein. GLUT3 protein was well photolabelled with [3H]cytochalasin B or a mannose derivative, 2-N-4-[3H](1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos -4-yloxy)-2- propylamine. Thus GLUT3 protein has very similar characteristics to GLUT1 protein including its subcellular localization, but exhibits lower Km and Vmax. values for 3-O-methylglucose uptake.
Asano
T
T
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.
Katagiri
H
H
Takata
K
K
Tsukuda
K
K
Lin
J L
JL
Ishihara
H
H
Inukai
K
K
Hirano
H
H
Yazaki
Y
Y
Oka
Y
Y
D019325:3-O-Methylglucose; D000345:Affinity Labels; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D006224:Cricetinae; D003571:Cytochalasin B / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics; D003847:Deoxyglucose / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D051274:Glucose Transporter Type 3; D006031:Glycosylation; D007700:Kinetics; D008757:Methylglucosides / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D010777:Photochemistry; D014162:Transfection
D000345:Affinity Labels; D051274:Glucose Transporter Type 3; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D009419:Nerve Tissue Proteins; D019325:3-O-Methylglucose; D003571:Cytochalasin B; D004247:DNA; D003847:Deoxyglucose