Identification of histidyl and thiol groups at the active site of rabbit renal dipeptide transporter.

摘要: Active transport of dipeptides in rabbit renal brush-border membrane vesicles is energized by an inward-directed H+ gradient rather than a Na+ gradient. We examined the effects treatment with diethylpyrocarbonate (DEP), reagent specific for histidyl groups, on this gradient-dependent dipeptide uptake. DEP inhibited uptake all three studied, Gly-sarcosine, Gly-Gly, and Gly-Pro (Ki = 0.6-0.9 mM), inhibition was noncompetitive. The transporter could be protected from presence substrates during inhibitor, whereas leucine plus failed to offer protection. Na+-dependent also 2.5 mM) amino acid Na+, but not dipeptides. Treatment thiol group-specific reagents, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole,3-bromopyruvate, p-chloromercuribenzenesulfonic acid, N-ethylmaleimide, potency their order: 7-chloro-4-nitrobenz-2-oxa-1,3-diazol greater 3-bromopyruvate N-ethylmaleimide. reversed some cases reducing agents such as 2,3-dimercaptopropanol following incubation inhibitors. Dipeptide protect inhibition. conclude that groups are present at or near substrate-binding site transporter.