Arginyl and histidyl groups are essential for organic anion exchange in renal brush-border membrane vesicles.
The effect of side chain modification on the organic anion exchanger in the renal brush-border membrane was examined to identify what amino acid residues constitute the substrate binding site. One histidyl-specific reagent, diethyl pyrocarbonate (DEPC), and 2 arginyl-specific reagents, phenylglyoxal and 2,3-butanedione, were tested for their effect on the specifically mediated transport of p-amino[3H]hippurate (PAH), a prototypic organic anion. The specifically mediated transport refers to the difference in the uptake of [3H]PAH in the absence and presence of a known competitive inhibitor, probenecid, and was examined in brush-border membrane vesicles isolated from the outer cortex of canine kidneys. The experiments were performed utilizing a rapid filtration assay. DEPC, phenylglyoxal, and 2,3-butanedione inactivated the specifically mediated PAH transport, i.e. probenecid inhibitable transport with IC50 values of 160, 710, and 1780 microM, respectively. The rates of PAH inactivation by DEPC and phenylglyoxal were suggestive of multiple pseudo first-order reaction kinetics and were consistent with a reaction mechanism whereby more than 1 arginyl or histidyl residue is inactivated. Furthermore, PAH (5 mM) did not affect the rate of phenylglyoxal inactivation. In contrast, PAH (5 mM) affected the rate of DEPC inactivation. The modification by DEPC was specific for histidyl residues since transport could be restored by treatment with hydroxylamine. The results demonstrate that histidyl and arginyl residues are essential for organic anion transport in brush-border membrane vesicles. We conclude that the histidyl residue constitutes the cationic binding site for the anionic substrate, whereas the arginyl residue(s) serves to guide the substrate to or away from the histidyl site.
Sokol
P P
PP
Department of Pharmacology, State University of New York, Health Science Center, Syracuse 13210.
Holohan
P D
PD
Ross
C R
CR
D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D001120:Arginine; D002074:Butanones / Q000494:pharmacology*; D003931:Diacetyl / Q000494:pharmacology*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D004285:Dogs; D005561:Formates / Q000494:pharmacology*; D006639:Histidine; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007672:Kidney Cortex / Q000378:metabolism*; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism*; D010658:Phenylglyoxal / Q000494:pharmacology*; D010130:p-Aminohippuric Acid / Q000378:metabolism
D000447:Aldehydes; D002074:Butanones; D005561:Formates; D006898:Hydroxylamines; D019811:Hydroxylamine; D006639:Histidine; D001120:Arginine; D003931:Diacetyl; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal; D004229:Dithiothreitol; D010130:p-Aminohippuric Acid