The isoelectric point of the human red cell glucose transporter.
The isoelectric point (pI) of the human red cell glucose transporter (Glut 1) was determined. Inconsistent values of 6.0, 6.4-6.5 and 8 have been reported earlier. Integral membrane proteins from human red cells were analyzed by two-dimensional electrophoresis with isoelectric focusing and sodium dodecyl sulfate gel electrophoresis (2D-PAGE). A zone of monomeric Glut 1 was found at pH 8.7, but most of the Glut 1 focused at pH 6-7 together with the anion transporter and other components. Purified Glut 1 focused only at pH 8.5 +/- 0.2 (S.D., n = 12) and deglycosylated purified Glut 1 only at pH 8.4 +/- 0.1 (n = 5), as shown by 2D-PAGE. The absence of Glut 1 below pH 8 in the latter cases was confirmed by immunoblotting with a monoclonal antibody. Furthermore, Glut 1 was photoaffinity-labelled with [3H]cytochalasin B and subjected to isoelectric focusing in one dimension. The pI of the labelled Glut 1 was 8.6 +/- 0.3 (n = 11). A pI of 9.1 was calculated for the Glut 1 polypeptide on the basis of amino acid composition and pKa values for amino acid side groups. The sialic acid content of the glycosylated transporter from fresh red cells was determined at approximately 2.1 sialic acid residues per transporter, which corresponds to a calculated pI of 8.8. The pI values of other human glucose transporter polypeptides of the facilitative diffusion type (Glut 2, 3, 4 and 5) were calculated at 8.4, 7.4, 7.1 and 6.2, respectively.
Englund
A K
AK
Department of Biochemistry, Biomedical Center, Uppsala University, Sweden.
Lundahl
P
P
D000345:Affinity Labels; D003571:Cytochalasin B; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007526:Isoelectric Point; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000097:blood / Q000737:chemistry*; D019158:N-Acetylneuraminic Acid; D010777:Photochemistry; D012794:Sialic Acids / Q000032:analysis
D000345:Affinity Labels; D009004:Monosaccharide Transport Proteins; D012794:Sialic Acids; D003571:Cytochalasin B; D019158:N-Acetylneuraminic Acid