THE ATP-BINDING SITE OF THE HUMAN PLACENTAL-H+ PUMP CONTAINS ESSENTIAL TYROSYL RESIDUES

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 ΔpH 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 > tetranitromethane > p-nitrobenzenesulfonyl fluoride > 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-(chloro-mercuri)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. The H+ pump was protected from the N-acetylimidazole-induced inhibition by its substrate, ATP. ADP, a reversible inhibitor of the pump, also offered protection, whereas adenosine and adenine did not. The protection with ATP was demonstrable not only in the case of N-acetylimidazole but also in the cases of NBD-Cl, tetranitromethane, and p-nitrobenzenesulfonyl fluoride. Kinetic analysis of the ATP-driven H+ pump in control and N-acetyl-imidazole-treated vesicles revealed that the inhibition of the pump by TV-acetylimidazole was due to a decrease in the affinity of the pump for ATP as well as a decrease in its maximal velocity. These results strongly suggest that the human placental H+ pump contains tyrosyl residues that are critical for its catalytic activity and that the location of these essential tyrosyl residues and the ATP-binding site interact with each other sterically and/or allosterically. © 1990, American Chemical Society. All rights reserved.