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Abstract

Transient exposure of human placental brush-border membrane vesicles to cholate reorients the ATP-driven H⁺ pump, enabling the pump to transport H⁺ into the vesicles upon addition of ATP to the external medium. H⁺ uptake can be measured in these vesicles by following the decrease in the absorbance of acridine orange, a delta pH indicator. We investigated the role of tyrosyl residues in the catalytic function of the H⁺ pump by studying the effects of tyrosyl group specific reagents on ATP-driven H⁺ uptake in cholate-pretreated membrane vesicles. The reagents tested were 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl), N-acetylimidazole, tetranitromethane, and p-nitrobenzenesulfonyl fluoride. Treatment of the membrane vesicles with these reagents resulted in the inhibition of the ATP-driven H⁺ uptake, and the inhibitory potency was in the following order: NBD-Cl greater than tetranitromethane greater than p-nitrobenzenesulfonyl fluoride greater than N-acetylimidazole. The inhibition of the H⁺ pump by NBD-Cl was reversible by 2-mercaptoethanol, and the inhibition by N-acetylimidazole was reversible by hydroxylamine. Since these reagents are not absolutely specific for tyrosyl groups and can also react with thiol groups, we studied the interaction of N-acetylimidazole with the H⁺ pump whose triol groups were masked by reaction with p-(chloromercuri)benzenesulfonate. The SH-masked pump was totally inactive, but the activity could be restored by dithiothreitol. On the contrary, the activity of the SH-masked H⁺ pump which was subsequently treated with N-acetylimidazole could not be restored by dithiothreitol, suggesting that thiol groups were not involved in the inhibition of the H⁺ pump by N-acetylimidazole.