Sulfhydryl groups are essential for organic anion exchange in canine renal brush-border membranes

The effect of several sulfhydryl-modifying reagents (HgCl2, p-chloromercuribenzenesulfonic acid (PCMBS), N-ethylmaleimide) on the renal organic anion exchanger was studied. The transport of p-amino[3H]hippurate, a prototypic organic anion, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. HgCl2, PCMBS and N-ethylmaleimide inactivated p-aminohippurate transport with IC50 values of 38, 78 and 190 microM. The rate of p-aminohippurate inactivation by N-ethylmaleimide followed apparent pseudo-first-order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the N-ethylmaleimide concentration with a slope of 0.8. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of N-ethylmaleimide inactivates one essential sulfhydryl group per active transport unit. Substrate (1 mM p-aminohippurate) affected the rate of the N-ethylmaleimide (1.3 mM) inactivation: the t1/2 values for inactivation in the presence and absence of p-aminohippurate were 7.4 and 3.7 min, respectively. The results demonstrate that there are essential sulfhydryl groups for organic anion transport in the brush-border membrane. Moreover, the ability of substrate to alter sulfhydryl reactivity suggests that the latter may play a dynamic role in the transport process.