Inhibition of phosphate transport in human erythrocytes by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl).
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 degrees 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 beta-mercaptoethanol. Prolonged incubation (60 min, 37 degrees 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 beta-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 degrees C, pH 8.3 in sucrose-citrate medium) and p-nitrobenzene sulfonyl fluoride (31% inhibition, 1.8 mM, 3 min, 37 degrees C, pH 8.1 in sucrose-citrate medium) but not by N-acetylimidazole (10% inhibition, 37.5 mM, 30 min, 37 degrees 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.
D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism / Q000494:pharmacology*; D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000838:Anions / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002250:Carbon Radioisotopes; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D010069:Oxadiazoles / Q000494:pharmacology*; D010710:Phosphates / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014443:Tyrosine / Q000502:physiology*
D000838:Anions; D002250:Carbon Radioisotopes; D010069:Oxadiazoles; D010710:Phosphates; D013439:Sulfhydryl Reagents; D014443:Tyrosine; D002731:4-Chloromercuribenzenesulfonate; D009327:4-Chloro-7-nitrobenzofurazan; D005033:Ethylmaleimide