Cholate-solubilized erythrocyte glucose transporters exist as a mixture of homodimers and homotetramers.
The molecular size of purified, human erythrocyte glucose transport protein (GLUT1) solubilized in cholic acid was determined by size-exclusion chromatography (SEC) and sucrose gradient ultracentrifugation. GLUT1 purified in the presence of dithiothreitol (GLUT1 + DTT) is resolved as a complex of average Stokes' radius 5.74 nm by SEC. This complex displays D-glucose-inhibitable cytochalasin B binding and, upon reconstitution into proteoliposomes, catalyzes cytochalasin B inhibitable D-glucose transport. GLUT1 purified in the absence of dithiothreitol (GLUT1-DTT) is resolved by SEC as at least two particles of average Stokes' radii 5.74 (minor component) and 7.48 nm (major component). Solubilization of GLUT1-DTT in the presence of dithiothreitol reduces the amount of 7.48-nm complex and increases the amount of 5.74-nm complex resolved by SEC. GLUT1-DTT displays D-glucose-inhibitable cytochalasin B binding and, upon reconstitution into proteoliposomes, catalyzes cytochalasin B inhibitable D-glucose transport. Sucrose gradient ultracentrifugation of GLUT1 + DTT in cholate resolves GLUT1 into two components of 4.8 and 7.6 S. The 4.8S complex is the major component of GLUT1 + DTT. The reverse profile is observed upon sucrose gradient ultracentrifugation of GLUT1-DTT. SEC of human erythrocyte membrane proteins resolves GLUT1 as a major broad peak of average Stokes' radius 7.48 nm and a minor component of 5.74 nm. Both components are characterized by D-glucose-inhibitable cytochalasin B binding. Purified GLUT1 is associated with approximately 26 tightly bound lipid molecules per monomer of transport protein. These data suggest that purified GLUT1 exists as a mixture of homodimers and homotetramers in cholate-lipid micelles and that the presence of reductant during solubilization favors dimer formation.
Hebert
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DN
Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester 01605.
Carruthers
A
A
D002384:Catalysis; D002793:Cholic Acids / Q000737:chemistry*; D002850:Chromatography, Gel; D003571:Cytochalasin B / Q000737:chemistry; D004229:Dithiothreitol; D004797:Enzyme-Linked Immunosorbent Assay; D004910:Erythrocyte Membrane / Q000737:chemistry; D004912:Erythrocytes / Q000737:chemistry*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry; D009004:Monosaccharide Transport Proteins / Q000737:chemistry*; D014461:Ultracentrifugation
D002793:Cholic Acids; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D003571:Cytochalasin B; D004229:Dithiothreitol