Abstract
We examined the effect of diethyl pyrocarbonate (DEPC), a histidine-specific reagent, on the H+/organic cation antiport system in brush-border membrane vesicles isolated from the rat renal cortex. Pretreatment of membrane vesicles with DEPC resulted in the inhibition of tetraethylammonium transport. This inhibition was reversed by subsequent treatment with hydroxylamine, but not with dithiotreitol. In contrast, the uptake of p-aminohippurate, a typical organic anion, was not inhibited by DEPC pretreatment. In the absence of an H+ gradient, pretreatment with DEPC inhibited the uptake of tetraethylammonium at pH 6.0-7.0, but not at pH 7.5. The Vmax value of tetraethylammonium uptake at pH 7.0 was decreased without any change in the Km value, but the kinetic parameters at pH 7.5 were unchanged. Unlabeled tetraethylamonium did not protect against the inhibition by DEPC. These results suggest that histidine residues in the organic cation carrier are essential for transport at acidic and neutral pH values, but not at alkaline pH values, and that histidine residues play an important role as regulatory sites in the H+/organic cation antiport system rather than as binding sites for organic cations.
Highlights
From the Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan
During the course of studiesontetraethylammoniumtransportinrenalbrushbordermembranes [3, 6,7]andinLLC-PKIapicalmembranes [8], we found that transport activity wasmarkedly inhibited by sulfhydryl reagents, which indicated that sulfhydryl groups were essential for the H+/organic cation antiport system ($6-8)
In the present studyw,e examined the organic cation carrier are essential for transport the effect of diethyl pyrocarbonate (DEPC) on the H+/organic cation antiport system, at acidic and neutral pH values, but not at alkalinepH in comparison with theeffects of other chemical modifiers
Summary
H + gradient by brush-border membrane vesicles pretreated with DEPC. The uptakoef tetraethylammonium for 10 s at p H 7.0 was decreased in the brush-border membrane vesicles pretreatedwithDEPCin a concentrationdependentmanner,andtheuptake was significantly decreased at 0.5 mM DEPC. These results indicate that DEPC directly inactivates the H+/organic cation antiposrytstem. The uptakeof p-aminohippurate, anorganic anion, for 10 s and 30 min was decreased when treated with DEPC at the concentrationosf 5 and 10mM (Fig. 2B).Judging from theuptakefor 30 min,theintravesicular spacedecreased when treated with 5 mM DEPC, and the pretreatment concentration of DEPC for subsequent studies was 1
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