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Abstract

Brush-border membranes from rat kidney cortext are transiently exposed to cholate to reorient ATP-driven H⁺ pumps to the outside of the vesicles, The carboxyl group reagent, N,N'- dicyclohexylcarbodiimide (DCCD), inhibits ATP-driven H⁺ uptake into cholate-pretreated vesicles irreversibly, Complete inhibition requires treatment of vesicles with 0.2 mM DCCD for ⩾15 min, ATP and ADP do not protect the H⁺ pump from inactivation suggesting that DCCD modifies pump subunits involved in H⁺ translocation, but not those related to ATP hydrolysis, With [¹⁴C]DCCD a 16 kDa protein is strongly labeled in brush-border and endosomal membranes, but not in basolateral membranes, Molecular mass of this protein and distribution similar to H⁺-ATPases suggest a role as H⁺- conducting subunit of the H⁺ pumps, The SH-group reagent, N-ethylmaleimide (NEM), also inhibits ATP-driven H⁺ uptake irreversibly, As opposed to DCCD, ATP and ADP protect the pump from irreversible inhibition indicating that NEM modifies SH-groups in the proximity of ATP hydrolysis sites, Finally, 15 mM of a potent inhibitor of vacuolar ATPases, bafilomycin B₁, abolishes ATP-driven H⁺ uptake, Inactivation by DCCD and NEM, labeling of 16 kDa subunits by [¹⁴C]DCCD, and high sensitivity to bafilomycin indicate that the H⁺ pump (H⁺- ATPase) in rat renal brush-border membranes belongs to the class of vacuolar ATPases, Bafilomycin may prove a valuable tool for specific inhibition of the renal H⁺-ATPase in future studies.