Immobilization of active facilitated glucose transporters (GLUT-1) in supported biological membranes

Membrane fragments or membrane proteins within a lipid mixture were immobilized over metal electrodes. This procedure has been developed to study biological membranes without interference from cell machinery. To obtain a smooth hydrophilic biomembrane support and a mode of binding of the membrane, either a crosslinked gel or an aromatic polyamine-polymer doped with avidin was deposited at the metal electrode by electropolymerization. This layer (less than 10 nm thick) also served as a submembrane compartment. The facilitated glucose transporter (GLUT-1) purified from human erythrocytes was integrated into a lipid membrane containing artificial biotinylated lipids and reacted with the activated surface of the glucose sensitive electrode. It was demonstrated that the lipid layer was attached to the polymer-containing avidin and could only be removed by detergent extraction. The presence of an active membrane transporter was demonstrated by electrochemical detection of glucose in the submembrane compartment, and by inhibition of glucose transport with the specific inhibitor Cytochalasin-B.