The modulatory effect of membrane viscosity on structural and functional properties of the anion exchange protein of human erythrocytes.

The sterol content of human erythrocyte membranes was modified by polyvinylpyrrolidone (PVP)-mediated enrichment or depletion of cholesterol (CHL) or incorporation of cholesteryl hemisuccinate (CHS). The effects of these modifications on osmotic fragility and anion exchange protein (AEP) disposition and function were evaluated. CHS enrichment was fast (1 hr, 37 degrees C) and led to a concentration-dependent crenation as well as a decrease in osmotic cell fragility, in parallel with increased membrane microviscosity. CHL caused similar but considerably less marked effects due to slower incorporation rates into membranes. CHS enrichment of cells induced susceptibility of AEP to trypsin, a protease which otherwise does not affect AEP in intact cells. Although transport rates of monosaccharides, nucleosides, and anions were markedly slowed down by CHS enrichment of cells in parallel with increased membrane viscosity, anion transport was the most affected. The temperature profile of anion transport in CHS-enriched cells revealed a 10-kcal/mol increase in the enthalpy of activation relative to normal cells. Anion transport measured in heteroexchange conditions (Cl in--pyruvate out) and (Cl in-sulfate out) was relatively more susceptible to CHS modification than when it was measured in homoexchange conditions (Cl in-Cl out). The results of these measurements indicate that CHS-mediated increase in membrane viscosity affects AEP translocation capacity and transmembrane disposition via changes in lipid compressibility. Specific effects of CHS on AEP function, however, could not be ruled out.

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 (Ea) 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.

The anion-transfer system of erythrocyte membranes. N-(7-Nitrobenzofurazan-4-yl)taurine, a fluorescent substrate-analogue of the system.

The fluorescent probe Nbd-Tau [N-(7-nitrobenzofurazan-4-yl)taurine] was synthesized and evaluated as a potential substrate of the anion-transport system of human erythrocyte membrane. The probe inhibited Cl- exchange in a competitive manner from either surface of the membrane, displaying Ki values in the mM range at the inner surface and in the microM range at the outer surface. Inhibition from within cells was via interaction with Cl--transport sites, whereas from it was via interaction with sites of unidentified nature. Nbd-Tau efflux from cells was monitored fluorimetrically in a continuous mode by a novel method that circumvents separation of the cells from the medium. Using this method, it is shown that Nbd-Tau efflux fulfils the following criteria of a substrate of the anion transport system: (a) susceptibility to classical and specific inhibitors of the system; (b) competitive inhibition with Cl- for anion-transport sites; and (c) temperature coefficient comparable with that of Cl- exchange. The fluorometric method is highly sensitive, versatile, and kinetically informative. With minor modifications it can be used for measuring anion transport across "ghost" and isolated membrane vesicles.