Examination of some protein containing fractions from human erythrocyte ghosts for glucose binding as part of the hexose transport system

Abstract

Kinetic studies on the penetration of human red cell membranes by hexoses have shown that transport occurs by a carrier mechanism. Little is known about the biochemical aspects of this carrier mechanism but the carrier is probably lipid, protein or a combination of the two. Studies were carried out to investigate the possibility of membrane proteins from red blood cells being involved in carrier function.Lipid-free membrane proteins were obtained by n-butanol extraction of humanerythrocyte stroma. Electrophoretic techniques showed that these proteins would associate with glucose. However, this was demonstrated by both D- and L-glucose.Specific binding of D-glucose by a lipoprotein material situated at the oil : water interface during butanol extraction was observed. This binding, demonstrated by retention of radioactivity on a millipore filter, could be reduced by about 50% using high concentrations of competitive sugars.The non-ionic detergent, Triton X-100, solubilized both the membrane proteins and lipids. Precipitation of the solubilized material with 50% saturated ammonium sulphate in the presence of radioactive glucose indicated that a component could bind D-glucose in preference to L-glucose. This component was probably protein or lipoprotein as precipitation with 10$ trichloroacetic acid reduced the binding of D-glucose. Assuming all the D-glucose binding, (1.14x 10 g per mg of protein), was involved in carrier function, each red cell would contain 95,000 active sites. Although bovine serum globulins, (and red cell membrane proteins solubilized by butanol extraction), also showed some preferential binding of D-glucose on precipitation by ammonium sulphate, the binding activity was much lower than the Triton X-100 extract. D-glucose binding to the Triton X-100 material could be reduced using the non-competitive inhibitor mercuric chloride and high concentrations of competitive sugars. These studies indicate that protein or lipoprotein might be involved inhexose carrier function, but although the extracts showed specificity towards optical isomers, they failed to mimic a number of other features of the hexose transport system, for example, saturation and the effects of phloretin.