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Abstract

The presence of an ATP-driven H⁺ pump as measured by H⁺ uptake upon addition of ATP was not demonstrable in human placental brush-border membrane vesicles when used in their native form, owing to their right-side-out orientation. However, the presence of the H⁺ pump in these membranes became evident when the membrane vesicles were transiently exposed to 1% cholate, with subsequent removal of the detergent to re-form the vesicles. Apparently, cholate pretreatment reoriented the H⁺ pump from an inward-facing configuration to outward-facing. Consequently, H⁺ uptake in response to externally added ATP was easily demonstrable in these cholate-pretreated vesicles by using the delta pH indicator Acridine Orange. In addition, bafilomycin A1-sensitive ATPase activity was measurable in cholate-pretreated vesicles, but not in native intact vesicles, indicating reorientation of the H⁺ pump. The reoriented H⁺ pump was electrogenic because H⁺ uptake was stimulated by an inside-negative anion-diffusion potential or when the vesicles were voltage-clamped. ATP supported H⁺ uptake with an apparent K_m of 260 microM. ITP and GTP supported the pump activity partially, whereas CTP and UTP did not. Mg²⁺ and Mn²⁺ were the most preferred bivalent cations. Co²⁺ and Zn²⁺ showed partial activity, whereas Ca²⁺ and Ba²⁺ showed little or no activity. The pump was inhibited by nanomolar concentrations of bafilomycin A₁ and micromolar concentrations of N-ethylmaleimide, p-chloromercuribenzenesulphonate, NN-dicyclohexylcarbodi-imide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but was relatively insensitive to oligomycin, vanadate and NaN₃. The inhibition by N-ethylmaleimide was protectable by ATP. It is concluded that human placental brush-border membranes possess an ATP-driven H⁺ pump and that, on the basis of its characteristics, it belongs to the class of vacuolar (V-type) H⁺ pumps.