Extraction and fractionation of lipid samples from red cells following a brief incubation with 14C-glucose

Abstract

The facilitated diffusion of glucose across the human erythrocyte membrane has already been established by kinetic studies to be consistent with a carrier mechanism. Little is known biochemically about the carrier or the type of associations formed in its binding with glucose. In the experimental work to be described consideration was given to various possibilities of associations between membrane lipids and glucose molecules. Human erythrocyte stroma were incubated with glucosefor short periods at ice-cold temperatures and were then either extracted with lipid solvents, or freeze-dried prior to such extractions. Separation of the radioactivity and lipids in the extracts was effected by silicic acid/Hyflo column and thin-layer plate chromatography. Where possible a qualitative analysis was made of the lipid fractions associated with the radioactivity. One of the kinetic features of the system in whole cells is the saturation of the carriers with increasing glucose concentration. Indications of this were looked for in experiments with increasing amounts of glucose but recoveries of radioactivity amongst the lipids did not appear to become saturated. The effects of the reversible inhibitors phloretin and stilboestrol and of the irreversible inhibitors dinitrofluorobenzene (DNFB) and mercuric ions on the recovery of labelling and the chromatographic behaviour of the lipids and radioactivity have been studied. Sheep erythrocytes have no detectable sugar transfer system and so a comparison was made between sheep and human erythrocyte lipid extracts following incubation of the cells with C glucose under the same experimental conditions. The main advances resulting from the experiments has been the demonstration of a lipid soluble C glucose-containing material extractable from freeze-dried human ghosts by the methods used by Handa (1963) for glycolipids. The recovery of this material is markedly reduced by treatment of the ghosts with dinitrofluorobenzene or mercuric ions. On thin-layer chromatograms developed in dimethylformamide the complex could be resolved from free glucose.