Frontal affinity chromatographic analysis of membrane protein reconstitution

Abstract

The human red cell glucose transporter Glut1 was solubilized with octaoxyethylene n-dodecyl ether (low critical micelle concentration (CMC)), purified, mixed with egg phospholipids and cholate, and reconstituted by gel filtration on Superdex 75. Free protepliposomes showed relatively high D-glucose transport activity. Frontal affinity chromatographic analysis with the proteoliposomes sterically immobilized in Superdex 200 gel beads revealed that the number of operative cytochalasin B (CB) binding sites increased during the first days of chromatographic runs to become the same as with 1- O- n-octyl β- d-glucopyranoside (high CMC) as solubilizer and Sephadex G-50 as gel filtration medium. The average number of sites per Glut1 monomer was 0.32 ± 0.02. The average K d for CB was 66 ± 3 nM at 150 mM NaCl, similarly as for Glut1 in membrane vesicles, whereas the affinity of d-glucose for reconstituted Glut1 was lower ( K d = 44 ± 3 mM) than for membranous Glut1 ( K d = 15 ± 5 mM). Two theoretical treatments of affinity chromatographic data gave the same values in agreement with competitive and monovalent interactions.