High-performance agarose gel chromatography in octyl glucoside of integral membrane proteins from human red cells, with special reference to the glucose transporter

Abstract

Integral membrane proteins and lipids from human red cells were fractionated in the presence of octyl glucoside by high-performance gel chromatography on a 22-ml column of the small-bead cross-linked agarose gel Superose 6, at 5°C, pH 7.6 and 30–50 m M detergent. To avoid aggregation a relatively high flow-rate, 9 ml/h, was chosen. At low ionic strength four main fractions were resolved, which contained anion transporter multimers(I), glycophorin oligomers(II), glucose transporter dimers(III) and phospholipids(IV). In 0.5 M sodium chloride the resolution was lower but the yield of the glucose transporter was markedly higher, and chromatography of partially purified glucose transporter gave a protein recovery of about 90%. The apparent M r values for the octyl glucoside complexes of the main components were: anion transporter, 900 000; glycophorin A, 210 000–360 000, dependent on ionic strength; glucose transporter, 110 000–160 000; lipids, 70 000. Some components aggreated with time: at a flow-rate of 1 ml/h mainly glycophorins and the glucose transporter were eluted, but no anion transporter, and fractionation performed 20 h after solubilization showed extensive aggregation of proteins. Superose-6 chromatography of glucose transporter and membrane lipids that had been isolated on DEAE-cellulose partially resolved the transporter and the phospholipid fractions. In this case, the resolution was better with 50 than with 30 m M detergent. The maximum glucose transport activity was approximately one-tenth of that observed before fractionation and appeared in two main fractions, at the main transporter fraction as well as at the overlap between the transporter and the lipids. The activity level was the same in both fractions, although the protein concentration was much lower in the second one. Addition of 2 m M egg-yolk phospholipids to the eluent did not increase the activity. This strongly indicates that the glucose transporter needs some specific membrane lipids to retain high transport activity. At the concentration of ca. 0.3 mg/ml used, the glucose transporter was probably eluted as a dimer in the absence of phospholipids and as a monomer in their presence.