Inhibition of phosphate transport in human erythrocytes by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl)

Abstract

Treatment of intact human erythrocytes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) leads to inhibition of anion transport as measured by [ 32P]phosphate exchange for intracellular chloride. Inhibition is rapid at 37°C (80% inhibition, 1.7 mM NBD-Cl, 3 min, pH 6.9) and not reversed by washing the cells with 1% bovine serum albumin in isotonic sucrose citrate buffer. Pretreatment of cells with N- ethylmaleimide and p- chloromercuribenzenesulfonic acid enhanced transport inhibition by NBD-Cl. Transport inhibition caused by brief incubations of erythrocytes with NBD-Cl could be almost completely reversed with dithiothreitol or β-mercaptoethanol. Prolonged incubation (60 min, 37°C, pH 6.4, sucrose-citrate buffer) following NBD-Cl treatment leads to partial reversal of transport inhibition. The residual inhibition is then only partially reversed by dithiothreitol treatment. Reversal of transport inhibition of dithiothreitol or β-mercaptoethanol may be prevented by incubation of the erythrocytes with sodium dithionite. Phosphate transport was readily inhibited by other tyrosine-directed reagents, tetranitromethane (55% inhibition, 1.6 mM, 3 min, 37°C, pH 8.3 in sucrose-citrate medium) and p- nitrobenzene sulfonyl fluoride (31% inhibition, 1.8 mM, 3 min, 37°C, pH 8.1 in sucrose-citrate medium) but not by N- acetylimidazole (10% inhibition, 37.5 mM, 30 min, 37°C, pH 7.5). These results suggest that NBD-Cl inhibits anion exchange by two mechanisms; a rapid inhibition reversible by sulfhydryl reagents, possibly due to modification of a tyrosine residue(s), and a slower irreversible inhibition due to modification of an essential amino group in the transporter.