Studies on the glucose transfer system in erythrocytes in inhibitor binding, and by kinetic and extraction techniques

Abstract

Glucose transport across the erythrocyte membrane is brought about by facilitated diffusion. Extensive kinetic studies have been made of this system, but as yet relatively little is known of the underlying mechanism. Further characterisation of the component(s) involved was attempted. Various inhibitors and the effects of temperature were studied. Phenolphthalein was shown to be a competitive inhibitor of glucose transfer. The Arrhenius plot for this substance gave a slope of 18,000 cal/mole while those for phloretin and stilboestrol were 20,000 cal/mole and 4,600 cal/mole. N-phenyl malesnide and 1,5-difluoro-2,4-dintrobenzene were not found to be more specific inhibitors than their homologues, but the latter compound was used to follow exchange diffusion of sugars in the erythrocyte. Exchange of glucose appeared to occur more rapidly than net flux. Glucose transfer was also shown to be unaffected by sodium concentration in the medium. Uptake of C-labelled n-ethyl malemide and 1-fluoro-2,4-dinitrobenzene (DNFB) was followed concurrently with the development of inhibition. The two were found to be related, the relationship for DNEB being independent of temperature. A maximum uptake of 400 million molecules per red cell was calculated to give full inhibition. Uptake of 14C-stilboestrol could not be used to give information on the number of sites available for glucose transfer since cell uptake depended solely on the concentration of the inhibitor in the medium. C-labelling from these inhibitors was obtained in butanol extracts, associated with certain lipid fractions, the DNFB being present in a quantity representing 10-20 million molecules per red cell. Several DNP-derivatives were isolated from these fractions. Erythrocytes and erythrocyte ghosts were extracted, after incubation with C-glucose, by various solvents including butanol and isopropanol -chloroform 11:7 to see if evidence of a glucose lipid complex could be demonstrated. Labelling associated with lipid was obtained with several of the solvents in the fractions eluted from silicic acid columns with chloroform methanol 4:1 and 1:4, free glucose being eluted by chloroform methanol 3:2.