A procedure for membrane-protein reconstitution and the functional reconstitution of the anion transport system of the human-erythrocyte membrane

Abstract

A short procedure for the isolation of band-3 protein, the protein responsible for anion exchange in erythrocytes, in a reasonable degree of purity was developed. Using this protein preparation and a novel procedure for membrane-protein reconstitution, vesicles displaying the basic features of the anion-exchange system of the erythrocyte were obtained. The reconstitution procedure is based on slow direct removal of Triton X-100 from aqueous lipid/detergent solutions. According to the composition of the reconstitution medium, either small single-walled or large multi-walled vesicles are obtained. The procedure conserves protein properties well, as is revealed by the similarity of the rates of SQ42-exchange in erythrocytes and reconstituted vesicles when corrected for the relevant volumes. A number of functional features of the exchange system were studied and compared with those of the native membrane.

The influence of lipid composition on the barrier properties of band 3-containing lipid vesicles

“…It has been reported that by the use of the Triton X-100-Bio-Bead method, band 3 was successfully reconstituted into lipid vesicles [2,[4][5][6]. This suggestion was supported by the findings that band 3-lipid vesicles show an enhanced SO42-permeability compared to protein-free vesicles [4] which could be inhibited by competitive anions [2,8] or high concentrations of specific inhibitors of the native anion transporter, like DIDS [2] and DNDS [4,5].…”

“…The band 3 protein was purified from human erythrocyte membranes, by means of a selective extraction procedure, with increasing concentrations of Triton X-100 as has been described in detail previously [2,5,8]. The incorporation of the band 3 protein was performed as outlined in Ref.…”

“…1) is an attractive candidate for studying protein-lipid interactions in model toluene-phosphatidylcholine extraction [3], adsorption to Bio-Beads [2,[4][5][6] or after replacement of Triton X-100 by N-octylglycoside, by dialysis [7]. It has been reported that by the use of the Triton X-100-Bio-Bead method, band 3 was successfully reconstituted into lipid vesicles [2,[4][5][6].…”

“…1) is an attractive candidate for studying protein-lipid interactions in model toluene-phosphatidylcholine extraction [3], adsorption to Bio-Beads [2,[4][5][6] or after replacement of Triton X-100 by N-octylglycoside, by dialysis [7]. It has been reported that by the use of the Triton X-100-Bio-Bead method, band 3 was successfully reconstituted into lipid vesicles [2,[4][5][6]. This suggestion was supported by the findings that band 3-lipid vesicles show an enhanced SO42-permeability compared to protein-free vesicles [4] which could be inhibited by competitive anions [2,8] or high concentrations of specific inhibitors of the native anion transporter, like DIDS [2] and DNDS [4,5].…”

Functional reconstitution of rat ovarian LH/hCG receptor into proteoliposomes

“…Proteoliposomes were formed as soon as the detergent had been removed by absorption into Bio-Beads SM-2. The procedure of proteoliposome formation in the absence of metal ions and presence of 1 mM EDTA is known to produce unilamellar vesicles [18]. Passing a detergent through a resin column results in almost complete removal of the detergent ( fig.l).…”