Chemical modification of membrane proteins in relation to inhibition of anion exchange in human red blood cells

Abstract

Mono-, di-, and trisulfonic acids, including 4,4'-diacetamido stilbene-2,2'-disulfonic acid (DAS) and 2-(4'-amino phenyl)-6-methylbenzene thiazol-3',7-disulfonic acid (APMB) produce a reversible inhibition of sulfate equilibrium exchange in human red cells. A study of the sidedness of the action of a number of these sulfonic acids in red cell ghosts revealed that some, like DAS, inhibit only at the outer membrane surface while others, like APMB, inhibit at either surface. This finding suggests that at least two different types of membrane sites are involved in the control of anion permeability. The nature of the anion permeability controlling sites in the outer cell surface was investigated by studying the effects of DAS on the inhibition by dinitrofluorobenzene (DNFB) of anion equilibrium exchange and on the binding of DNFB to the proteins of the red blood cell membrane. After exposure to DNFB in the presence of DAS for a certain period of time, there was a reduction of both the inhibitory effect of DNFB on sulfate exchange and the binding of DNFB to the protein in band 3 of SDS polyacrylamide gel electropherograms (nomenclature of Steck, J. Cell. Biol., 62: 1, '74). Since binding to other membrane proteins was not affected, this observation supports the assumption that the protein in band 3 plays some role in anion transport. In accordance with the absence of an inhibitory effect at the inner membrane surface, internal DAS does not affect DNFB binding to the protein in band 3. DAS protected the anion exchange system not only against inhibition by DNFB but also by m-isothiocyanato benzene sulfonic acid. In contrast to DAS, the equally inhibitory phlorizin does not reduce the rate of dinitrophenylation of the protein in band 3. This suggests that either not all inhibitors of anion exchange exert their action by a combination with sites on the protein in band 3 or that in spite of the described evidence this protein is not involved in the control of anion movements.

Anion transport in red blood cells and arginine‐specific reagents

“…Its involvement was based on our finding that through differential labelling we were able to find a common binding site for the two anion transport inhibitors, SITS and DNFB [1,2]. The same conclusion was drawn in [3] using another anion transport inhibitor (DIDS).…”

“…Resealed ghosts were prepared essentially as in [2], Red cells were hemolysed at a cell : medium ratio of 1: 20 in a medium containing 4 mM MgSO4 and 1.45 mM acetic acid. Five min after hemolysis sufficient EDTA was added to obtain a final concentration of 20mM EDTA in the hemolysate.…”

Ionic-strength-dependent changes in the structure of the major protein of the human erythrocyte membrane

“…of 90000-100000. The structure of this protein has become of particular interest since it has been shown to be involved in the transport of anions across the erythrocyte membrane (Cabantchik & Rothstein, 1974;Zaki et al, 1975). Polypeptide 3 has been shown to traverse the membrane permeability barrier (Bretscher, 1971;Steck, 1972;Boxer et al, 1974), and unique regions of the protein are accessible at each face of the erythrocyte membrane (Boxer et al, 1974).…”

Erratum

“…Otherwise the data almost exactly superimpose. Measurements were made at 37°C in 5 mM phosphate buffer, pH 7.4, under argon. the data to equation (2). In this way we obtain DII = 1,296 ± 99 S-I and 1,492 ± 202 S-I for the control and crosslinked membranes, respectively.…”

“…Experimental points were obtained by averaging 32 signals. The solid lines were obtained by fitting the data to equation (2). For illustrative purposes the two curves have been artificially separated by a vertical displacement of the lower curve by 20%.…”