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-[C-14]Glucose was eluted later than L-[H-3]glucose from the Glut 1 liposome column (by DELTAV mul), apparently because the D-glucose was transported through the liposomes. The corresponding difference with protein-free liposomes was DELTAV0. The Glut 1 transport retention chromatographic effect, DELTAV(G) = DELTAV - DELTAV0, 40-50 mul at pH 7, was nearly constant at pH 6-10 (400 mM NaCl, 23-degrees-C, internal liposome volume almost-equal-to 240 mul) but decreased steeply below pH 5 to become zero at pH 3.6. The decrease corresponded to a pK(a) of almost-equal-to 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 pK(a) almost-equal-to 4.4-4.8. It seems likely that carboxylate groups form hydrogen bonds to transported D-glucose.