New approaches for the reconstitution and functional assay of membrane transport proteins. Application to the anion transporter of human erythrocytes

The human red blood cell anion transport protein, band 3, was isolated and reconstituted into lipid vesicles. The main feature of the new reconstitution is the replacement of native lipids and of solubilizing detergent by externally added lipids, while band 3 protein is immobilized on a gel matrix. The vesicles formed upon detergent removal and sonication are unilamellar and sealed, and band 3 protein is the major polypeptide detectable in them. The method consists of: (a) solubilization of alkali-treated red blood cell membranes by Triton X-100; (b) binding of glycophorin and band 3 protein to diethylaminoethyl (DEAE)-cellulose in Triton X-100 solution, followed by high ionic strength elution; (c) band 3 protein complexation to organomercurial Sepharose; (d) exchange of the Triton X-100 with the dialyzable detergent octylglucopyranoside, while band 3 protein is complexed to the column; (e) elution of band 3 by cysteine (5 mM) in the presence of octylglucopyranoside; (f) addition of lipids (asolectin or egg phosphatidylcholine) to the protein-detergent suspension; and (g) dialysis of the mixture against 1% bovine serum albumin to remove the detergent completely. The vesicles were assayed for anion transport capacity by a novel procedure which is based on the fluorescent substrate N-(2-aminoethylsulfonate)7-nitrobenz-2-oxa-1,3-diazole (NBD-taurine) and on anti-NBD-antibodies as quenchers of extravesicular NBD-taurine fluorescence. Efflux of NBD-taurine from vesicles was monitored in a continuous mode as a decrease in intravesicular fluorescence. The band 3-mediated flux was approx. 50% inhibitable by externally added disulfonic stilbenes, indicating the random distribution of band 3 protein in reconstituted vesicles. Both the specific transfer rate (i.e., nmol substrate/mg protein per min) of band 3 and its energy of activation (E_a) in the artificial lipid milieu were similar to those obtained with the native system. Glycophorin incorporation into this milieu had no significant effect on the associated anion transport properties.