Immobilization of human red cells in gel particles for chromatographic activity studies of the glucose transporter Glut1

Abstract

Chromatography on a novel stationary phase, human red cells immobilized in a gel bed, was introduced for analysis of activities of the glucose transporter Glut1 in the cell membrane. A gel containing positively charged ligands was synthesized from derivatized acrylamide monomers. Red cells were immobilized in gel particles which were packed into a column tube for chromatographic analyses over periods of 10-15 days. D-Glucose was separated from L-glucose on a 1.1-ml bed with a retention volume difference of 0.23 ml, approximately equal to the total inner volume of immobilized intact cells and of ghosts probably formed from lysed cells during the immobilization. The separation was suppressed by the glucose-transport inhibitor cytochalasin B. The interactions between D-glucose, the transport inhibitor forskolin and Glut1 were analyzed by quantitative frontal affinity chromatography. The dissociation constants at room temperature were 6.8 mM for D-glucose binding and 1.8 μM for glucose-displaceable binding of forskolin, in good agreement with published values. The results suggest that chromatography on immobilized cells is a potentially useful tool for studies on cellular membrane functions.