602 Biochimica et Biopt~vsica A cta 856 (1986)602-609 Elsevier BBA 73063 Inhibition of phosphate transport in human erythrocytes by 7-chloro-4-nitrobenzo-2-oxa- 1,3-diazole (NBD-CI) James D. Craik, Kenseelan Gounden and Reinhart A.F. Reithmeier Department of Biochemistry, Universi(v of Alberta, Edmonton, Alberta, T6G 2H7 (Canada) (ReceivedOctober28th, 1985) Key words: Phosphate transport; Band3; Tyrosine modification; (Erythrocyte membrane) Treatment of intact human erythrocytes with 7-chioro-4-nitrobenzo-2-oxa-l,3-diazole (NBD-CI) leads to inhibition of anion transport as measured by [32Plphosphate exchange for intracellular chloride. Inhibition is rapid at 37°C (80% inhibition, 1.7 mM NBD-CI, 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-CI. Transport inhibition caused by brief incubations of erythrocytes with NBD-CI could be almost completely reversed with dithiothreitol or /~-mercaptoethanol. Prolonged incubation (60 min, 37°C, pH 6.4, sucrose-citrate buffer) following NBD-CI 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/~-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 rain, 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-CI 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. Introduction The Band 3 polypeptide, a major integral glyco- protein of the human erythrocyte membrane, mediates the rapid transmembrane exchange of anions necessary for efficient respiration [1,2]. Treatment of erythrocytes with a wide variety of chemical reagents indicates that lysine [3,4], arginine [5] and carboxylic acid [6,7] but not cy- steine [8] residues are essential for anion trans- port. Transport inhibition by carbodiimides [6,7] could be caused in part by modification of tyro- Abbreviations: NBD-CI. 7-chloro-4-nitrobenz-2-oxa-l,3-di- azole; otvr-NBD, O-tyrosyl reactionproduct of NBD-C1. sine residues so the effect of tyrosine-selective reagents on anion exchange in intact human erythrocytes was studied. Preliminary experiments indicated that NBD-C1 was the most potent of the reagents examined. Materials and Methods Materials [32p]phosphoric acid, carrier free, from New England Nuclear; [U-14C]NBD-C1 (109 mCi/ mmol) from Research Products International Corp., Mount Prospect, IL. Unlabelled NBD-C1 from Pfaltz & Bauer Inc., Waterbury, CT; 4,4'-di- nitrostilbene-3,Y-disulfonic acid (DNDS) and 2- 0005-2736/86/$03.50 ~ 1986 ElsevierSciencePublishers B.V.(BiomedicalDivision)