Characterization and partial purification of liver glucose transporter GLUT2.
GLUT2, the major facilitative glucose transporter isoform expressed in hepatocytes, pancreatic beta-cells, and absorptive epithelial cells, is unique not only with its low affinity and broad substrate specificity as a glucose transporter, but also with its implied function as a glucose-sensor. As a first essential step toward structural and biochemical elucidation of these unique, GLUT2 functions, we describe here the differential solubilization and DEAE-column chromatography of rat hepatocyte GLUT2 protein and its reconstitution into liposomes. The reconstituted GLUT2 bound cytochalasin B in a saturable manner with an apparent dissociation constant (K(d)) of 2.3 x 10(-6) M and a total binding capacity (B(T)) of 8.1 nmol per mg protein. The binding was completely abolished by 2% mercury chloride, but not affected by cytochalasin E. Significantly, the binding was also not affected by 500 mM D-glucose or 3-O-methyl D-glucose (3OMG). The purified GLUT2 catalyzed mercury chloride-sensitive 3OMG uptake, and cytochalasin B inhibited this 3OMG uptake. The inhibition was dose-dependent with respect to cytochalasin B, but was independent of 3OMG concentrations. These findings demonstrate that our solubilized GLUT2 reconstituted in liposomes is at least 60% pure and functional, and that GLUT2 is indeed unique in that its cytochalasin B binding is not affected by its substrate (D-glucose) binding. Our partially purified GLUT2 reconstituted in vesicles will be useful in biochemical and structural elucidation of GLUT2 as a glucose transporter and as a possible glucose sensor.
Lachaal
M
M
The Biophysics Laboratory, Veterans Administration Medical Center, State University of New York, School of Medicine, Medical Research Services, Buffalo 14215, USA.
Rampal
A L
AL
Ryu
J
J
Lee
W
W
Hah
J
J
Jung
C Y
CY
D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D003902:Detergents / Q000737:chemistry; D004492:Edetic Acid; D051273:Glucose Transporter Type 2; D005960:Glucosides / Q000737:chemistry; D008081:Liposomes; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D017830:Octoxynol / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility
D003902:Detergents; D051273:Glucose Transporter Type 2; D005960:Glucosides; D008081:Liposomes; D009004:Monosaccharide Transport Proteins; C017272:dodecyloctaethyleneglycol monoether; D011092:Polyethylene Glycols; C040358:dodecyl maltoside; D017830:Octoxynol; D004492:Edetic Acid