title: The ATP-binding site of the human placental H+ pump contains essential tyrosyl residues.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
authors: Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
authors: P
authors: P
authors: Kulanthaivel
authors: B J
authors: BJ
authors: Simon
authors: G
authors: G
authors: Burckhardt
authors: V B
authors: VB
authors: Mahesh
authors: F H
authors: FH
authors: Leibach
authors: V
authors: V
authors: Ganapathy
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D009578:Nitrobenzenes / Q000494:pharmacology; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine
chemical_list: D007093:Imidazoles; D009578:Nitrobenzenes; C034442:4-nitrobenzenesulfonyl fluoride; D014443:Tyrosine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan; D013774:Tetranitromethane; C012160:N-acetylimidazole