High performance agarose gel chromatography in sodium dodecyl sulfate of integral membrane proteins from human red cells, with special reference to the glucose transporter.
Integral membrane proteins from human red cells were fractionated in sodium dodecyl sulfate solutions by high performance gel filtration on the small-bead cross-linked agarose gel Superose 6. The components were identified by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The combination of Superose chromatography with electrophoresis afforded high resolution. As expected the gel filtration elution volumes depended essentially on the molecular mass, but the elution volumes decreased stepwise as the detergent concentration was increased from 0.6 to 100 mM, with the largest decrease for the glucose transporter. The resolution increased as the flow rate was decreased from 60 to 1 ml X cm-2 X h-1. The Mr values for the anion and glucose transporters as estimated by Superose 6-chromatography at 50 mM detergent were 75-80% of the corresponding Mr values obtained by electrophoresis. At 50 mM dodecyl sulfate the proteins were resolved into four fractions (a-d) which mainly contained: (a) dimer and (b) monomer of the anion transporter, (c) the glucose transporter and (d) components of Mr below 40 000. Monoclonal antibodies that possibly are directed against the glucose transporter (Lundahl, P., Greijer, E., Cardell, S., Mascher, E. and Andersson, L. (1986) Biochim. Biophys. Acta 855, 345-356) interacted only with part of the 4.5-material in fraction c in immunoblotting (Western blotting). Superose 6-chromatography of red cell glucose transporter that had been partially purified on DEAE-cellulose and Mono Q resolved one major and two minor fractions. Electrophoretic analysis showed that components of Mr 90,000, 50,000, and 25,000 had been separated from the major Mr-55,000-4.5-material and revealed size heterogeneity within the major chromatographic fraction. Heating of the glucose transporter in the presence of dodecyl sulfate caused an unexpected retardation of monomeric transporter on Superose 6. The apparent Mr decreased from 44,000 to 29,000.
D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000032:analysis*; D006358:Hot Temperature; D006801:Humans; D008565:Membrane Proteins / Q000097:blood*; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000097:blood*; D012967:Sodium Dodecyl Sulfate
D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D012967:Sodium Dodecyl Sulfate