Effects of pH on the activity of the human red cell glucose transporter Glut 1: transport retention chromatography of D-glucose and L-glucose on immobilized Glut 1 liposomes.

The facilitative glucose transporter Glut 1 from human red cells was reconstituted into liposomes that were size-fractionated and immobilized in an octyl sulfide-Sephacryl S-1000 column. D-[14C]Glucose was eluted later than L-[3H]glucose from the Glut 1 liposome column (by delta V microliters), apparently because the D-glucose was transported through the liposomes. The corresponding difference with protein-free liposomes was delta V0. The Glut 1 transport retention chromatographic effect, delta VG = delta V - delta V0, 40-50 microliters at pH 7, was nearly constant at pH 6-10 (400 mM NaCl, 23 degrees C, internal liposome volume approximately 240 microliters) but decreased steeply below pH 5 to become zero at pH 3.6. The decrease corresponded to a pKa of approximately 4.4 and was partly reversible above pH 4.7. Similarly, glucose exchange by non-immobilized freeze-thawed proteoliposomes with Glut 1 slowed down drastically as the pH was lowered from pH 5.5 to 4; and octyl glucoside-solubilized Glut 1 lost half its activity in 15 min at pH 4.5 (low ionic strength, 2 degrees C) as shown by glucose exchange determinations at pH 7.2 The results suggest that Glut 1 is inactivated at low pH upon protonation of carboxylate groups of pKa approximately 4.4-4.8. It seems likely that carboxylate groups form hydrogen bonds to transported D-glucose.