Substantial glucose leakage from liposomes on filters and upon molecular-sieve chromatography in determinations of reconstituted glucose-transport activity and liposome volumes.
Transport-protein activities are often determined by procedures that involve isolation of liposomes containing the transported radioactive solute. We determined the activity of the human red cell glucose transporter in liposomes and, by similar procedures, internal volumes of liposomes. For these purposes, we isolated freeze-thawed liposomes loaded with [14C]glucose, either by filtration on cellulose-nitrate and cellulose-acetate filters, or by chromatography on Sephadex. The interaction of liposomes with filters caused substantial leakage of [14C]glucose. About half of the internal [14C]glucose was released on the filters from glucose-transporter liposomes with inhibited transport. Chromatography at high flow rate provided higher and more accurate values than did the filtration procedure. Leakage corrections could be made by use of flow-cell scintillation elution profiles. The ratios between the corrected chromatographic volume values and the filtration values were 1.4-3.0 for liposomes without protein, 2.4-4.0 for glucose-transporter liposomes and 3.6-7.9 for liposomes with several human red cell integral membrane proteins. The D-glucose equilibrium exchange with glucose-transporter liposomes at 50 mM D-glucose was 2.0 nmol D-glucose per microgram transporter per second as determined by use of chromatography at high flow rate. The filtration procedure gave only 0.6 nmol.microgram-1.s-1 due to the [14C]glucose leakage. In our experiments, the chromatographic procedure thus proved superior.
Andersson
L
L
Department of Biochemistry, University of Uppsala, Sweden.
Lundahl
P
P
D001692:Biological Transport; D002250:Carbon Radioisotopes; D002850:Chromatography, Gel; D004058:Diffusion; D004912:Erythrocytes / Q000378:metabolism; D005374:Filtration; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D008081:Liposomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D010743:Phospholipids
D002250:Carbon Radioisotopes; D008081:Liposomes; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D010743:Phospholipids; D005947:Glucose