D-Glucose Uptake by Isolated Human Erythrocyte Membranes versusd-Glucose Transport by Human Erythrocytes

Abstract

Abstract The effects of proteolytic and phospholipase A2 digestion on the transport of d-glucose by intact human erythrocytes and on the uptake of d-glucose by isolated erythrocyte membranes have been studied. This stereospecific uptake of d-glucose was previously shown to be compatible with the over-all monosaccharide transport system in erythrocytes and evidence was presented that it represented the binding of d-glucose to a specific site on the erythrocyte membrane (Kahlenberg, A., Urman, B., and Dolansky, D. (1971) Biochemistry 10, 3154–3162. In the absence of hemolysis, no change is observed in the transport of d-glucose by intact erythrocytes exposed to the above enzymes or in the uptake of d-glucose by membranes isolated from enzyme-treated cells. In contrast, limited proteolytic digestion of isolated erythrocyte membranes, under conditions identical with those for the digestion of intact cells, results in a 3- to 5-fold increase in d-glucose uptake and a 2-fold increase in the apparent dissociation constant for the d-glucose-membrane complex. This increase in the uptake of d-glucose is different from the effects which result from the digestion of isolated membranes with phospholipase A2 (decreased uptake of d-glucose) or neuraminidase (no change in d-glucose uptake). More drastic tryptic digestion leads to a loss of the stimulated d-glucose uptake with no further change in the affinity of the membrane for the sugar. Pronase hydrolysis of 20 to 30% of the proteins of isolated membranes results in the destruction of 50 to 70% of the d-glucose uptake. These results indicate that the transport system in erythrocytes involves membrane components not normally accessible to proteolytic and phospholipase A2 digestion. Isolation of the erythrocyte membrane renders the d-glucose uptake activity of these membrane components susceptible to enzymatic modification. The evidence suggests that the increase in maximum d-glucose uptake resulting from limited proteolytic digestion of isolated membranes represents exposure of latent d-glucose binding sites which are masked by a protease-sensitive covering on the surface of the isolated membrane.