The ATP-binding site of the human placental hydrogen ion 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)

Sulfonyl fluorides as privileged warheads in chemical biology

“…80 p -Nitrobenzenesulfonyl fluoride (NBSF) is a highly reactive electrophilic reagent described as a tyrosine-specific reagent to map reactive and functionally important tyrosine residues. Examples of its use in this manner include DNase, 81 estradiol 17β-dehydrogenase, 82 glucocorticoid receptor, 83 the placental taurine transporter and H + pump, 84,85 histones, 86 dopamine receptors, 87 avidin and streptavidin, 88 and snake venom phospholipases, 89 although there are no recent examples of its use in the literature, presumably due to the development of less reactive and more selective reagents as described above.…”

Amiloride and harmaline are potent inhibitors of NhaB a Na+/H+ antiporter from Escherichia coli

“…Two other organic compounds that are known to inhibit mammalian Na+/H ÷ antiporters are clonidine and cimetidine [18,19]. When ApH driven 22Na+ uptake was tested in the presence of clonidine, a concentration-dependent inhibition was observed although at concentrations higher than those observed for amiloride and harmaline with a K0.5 of 200 #M (data not shown).…”

“…The hallucinogenic drug, harmaline, was reported to inhibit mammalian Na+/H + exchanger [18,19]. The effect of harmaline on the Na ÷ efflux reaction from NhaB proteliposomes was tested.…”

Human placental syncytiotrophoblast expresses two pharmacologically distinguishable types of Na+-H+ exchangers, NHE-1 in the maternal-facing (brush border) membrane and NHE-2 in the fetal-facing (basal) membrane

“…The Na+-H+ exchanger is a transport system which has been described in the plasma membrane of various cell types [5][6][7][8][9][10]. In recent years, however, evidence has been accumulating to indicate that there are two types of Na+-H+ exchangers [11][12][13][14][15][16]. The housekeeping type (NHE-1) is present in the plasma membrane of non-polarized cells such as fibroblasts, and the epithelial or apical type (NHE-2) is present in the brush border membrane of polarized epithelia such as intestinal and renal tubular cells.…”

“…Even though non-polarized cells always appear to express only NHE-1, polarized epithelial cells may possess either NHE-2 only or both NHE-l and NHE-2 [11][12][13][14]. These two types of Na+-H+ exchangers can be distinguished by their sensitivities to inhibition by amiloride and its analogues [11,12,[14][15][16]. Recently we have demonstrated that the relative sensitivity to inhibition by clonidine and cimetidine can also be useful to differentiate between the two types [15,16].…”