The D-glucose transporter from human erythrocytes has been purified and reconstituted by Kasahara and Hinkle (J Biol Chem 252:7394-7390). Using a similar purification scheme, we have isolated the protein with 65% of the extracted phospholipid at a lipid-protein ratio of 14: 1 by weight. The KD (0.14 pM) and extent (1 1 nmoles/mg protein) for binding of 3H-cytochalasin B was determined by equilibrium dialysis. Glucose was a linear competitive inhibitor of binding of cytochalasin B, with an inhibition constant of 30 mM. To further characterize the protein, samples were filtered in the presence of sodium dodecyl sulfate (SDS) through Sepharose 6B to remove 95% of the lipid followed by filtration of Sephadex G150 to remove the remaining lipid and a contaminating amount of a minor, lower-molecular-weight protein. This preparation contains only 24% acidic and basic amino acids. The protein also contains 5% neutral sugars (of which 3% is galactose), 7% glucosamine, and 5% sialic acid.
Key words: membrane proteins, transport proteins, glucose transport, reconstitution of glucose transport, purification of glucose transporter, cytochalasin B
Recent studies [ l , 21 of isolation and reconstitution of the D-glucose transporter from human erythrocytes have shown that it is a glycoprotein with apparent molecular weight of 55,000. Similar conclusions were reached by others using labeling techniques [3,4]. Now that the protein has been purified, specific questions can be asked about its structure and mechanism using direct chemical measurements. We have begun to answer some of these questions by determining the amino acid and carbohydrate composition of the protein and its ability to bind inhibitors of glucose transport.
METHODS
Materials
Diethylaminoethanol (DEAE) cellulose was purchased from Whatman, phloretin from ICN Pharmaceuticals, cytochalasin B from Aldrich, H-cytochalasin from New