A new method for the reconstitution of the anion transport system of the human erythrocyte membrane

Abstract

The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. The sample was dialyzed overnight against detergent-free buffer. Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30 degrees C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100 mM, inhibition of the flux by H2DIDS and flufenamate (approx. KI-values at 30 degrees C: 0.1 and 0.7 microM, respectively), and "right-side-out" orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H2DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.

Monomeric erythrocyte band 3 protein transports anions.

“…Band 3 dimers covalently crosslinked via S-S bridges were obtained by treating the isolated protein with CuS04/ophenanthroline (13) and subsequent purification by gel filtration on a Sephacryl S-300 column (120 x 2.6 cm). Reconstitution ofthe anion-transport system in liposomes made ofegg yolk phosphatidylcholine (Lipid Products, Nutley, U.K.) was performed as described by Scheuring et al (12,14). However, Triton X-100 was substituted by nonaethylene glycol lauryl ether (15).…”

“…Given MP = 100,000 (as in the case where there is one band 3 molecule per vesicle), vp = 0.73 ml/g, VL = 0.98 ml/g (18), and an error in density matching of 0.5 mg/ml, the Other Methods. The measurement of sulfate transport across lipid vesicle membranes, under exchange conditions, and its evaluation were performed as described earlier (12,21). In the evaluations, the anion efflux curves are fitted by the sum of a constant term (describing the contribution of protein-free vesicles) and two exponentials, representing efflux from vesicles that contain a reconstituted anion transport system [rate constant k1 (in the nomenclature of ref.…”

“…12, kieff)] and from those that show only protein-induced leak flux (rate constant k2), respectively. In the fit, a fixed value of k2 is used, which was determined from a separate efflux curve measured in the presence of high concentrations of the anion-transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS) (or H2DIDS plus flufenamate) (12,21). Turnover numbers, T, for the inhibitorsensitive flux component were calculated from k1, the sulfate concentration cs, the internal vesicle volume v; [as determined from the vesicle dimensions (19)], and the molar protein/lipid ratio, q, by applying Eq.…”

“…Electron microscopy of negatively stained samples, gel electrophoresis, and the quantification of phospholipid were done as described (12).…”

Reconstitution of the Torpedo californica nicotinic acetylcholine receptor into planar lipid bilayers

“…Experiments on the reconstitution of erythrocyte band 3 protein revealed that there too, protein function and aggregation depend on the lipid-detergent composition and on the way of vesicle bilayer formation [21].…”