Inverse effects of dansylation of red blood cell membrane on band 3 protein‐mediated transport of sulphate and chloride

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Abstract
Dansylation of the [human] red cell membrane produces inverse effects on SO42- and Cl- equilibrium exchange. The former is enhanced by several orders of magnitude; the latter is inhibited. Both effects are potentiated after dansylation in the presence of 2-(4-amino-3-sulfophenyl)-6-methyl-7-benzothiazol sulfonic acid (APMB), a disulfonic acid that combines noncovalently with the 4,4''-diisothiocyanate dihydrostilbene-2,2''-disulfonic acid (H2DIDS) binding site of the anion transport protein. After dansylation, the maximum of the pH dependence of SO42- exchange near pH 6.3 is replaced by a plateau. When dansylation is performed in the presence of APMB, the plateau is reached at a much higher level, at around pH 7.0, and resembles that observed by Funder and Wieth for Cl-. The mutual interactions between the transfer site, the H2DIDS binding site and the as yet unidentified danysl chloride binding sites were studied in detail. Occupation of the H2DIDS binding site by the noncovalently binding agents 4,4''-dinitrostilbene-2,2''-disulfonate (DNDS), 4,4''-bis(acetamido) stilbene-2,2''-disulfonate (DAS) or APMB inhibit the enhanced SO42- exchange across the previously dansylated membrane. The apparent KI [inhibition constant] value remains the same as in untreated membranes for DNDS, is reduced to 1/3 for DAS and to 1/60 for APMB. When dansylation is carried out while the H2DIDS binding site is occupied by DNDS, APMB or DAS, the enhancemnt of SO42- exchange (as measured after removal of excess dansyl chloride and the additional agent) is prevented by DNDS, augmented by APMB and not affected by DAS. The agents apparently stabilize different conformations of the H2DIDS binding site that are associated with different accessibilities of the dansyl chloride binding sites. The SO42- equilibrium exchange as measured at a fixed Cl- concentration is enhanced when the Cl- concentration at which dansylation is carried out is increased, indicating allosteric interactions between anion binding and the exposure of the dansyl chloride binding sites. The enhanced K+ efflux from dansylated red cells is independent of the described modifications of the dansylation reaction by APMB, DAS or DNDS, demonstrating that there exists no simple correlation between the changes of anion and cation movements that are induced by dansylation.

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