Cholate-solubilized erythrocyte glucose transporters exist as a mixture of homodimers and homotetramers

Biochemistry. 1991 May 14;30(19):4654-8. doi: 10.1021/bi00233a003.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Catalysis
  • Cholic Acids / chemistry*
  • Chromatography, Gel
  • Cytochalasin B / chemistry
  • Dithiothreitol
  • Enzyme-Linked Immunosorbent Assay
  • Erythrocyte Membrane / chemistry
  • Erythrocytes / chemistry*
  • Humans
  • Membrane Proteins / chemistry
  • Monosaccharide Transport Proteins / chemistry*
  • Ultracentrifugation

Substances

  • Cholic Acids
  • Membrane Proteins
  • Monosaccharide Transport Proteins
  • Cytochalasin B
  • Dithiothreitol