Tyrosine residues are essential for the activity of the human placental taurine transporter.
Treatment of human placental brush-border membrane vesicles with four tyrosine group-specific reagents, N-acetylimidazole, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), tetranitromethane and p-nitrobenzesulfonyl fluoride, inhibited NaCl gradient-driven taurine uptake in these vesicles without affecting the vesicle integrity. The relative potency of these reagents to inhibit the transporter was in the following order: tetranitromethane greater than NBD-Cl greater than p-nitrobenzenesulfonyl fluoride greater than N-acetylimidazole. The inhibition by N-acetylimidazole was reversible with hydroxylamine and the inhibition by NBD-Cl was reversible with 2-mercaptoethanol. Kinetic analysis of taurine uptake in control and in N-acetylimidazole-treated membrane vesicles revealed that the inhibition was primarily due to a reduction in the maximal velocity. There was no change in the affinity of the transporter for taurine in control and treated vesicles. The transporter could be protected from the N-acetylimidazole-induced inhibition by Na+. The dependence of taurine uptake rate on extravesicular Na+ concentration was sigmoidal and analysis of the data revealed that two Na+ ions were involved per transport of one taurine molecule. It is concluded that tyrosine residues are essential for optimal transport function of the human placental taurine transporter and that these critical tyrosine residues are located at or near the Na+-binding site of the transporter.
Kulanthaivel
P
P
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Leibach
F H
FH
Mahesh
V B
VB
Ganapathy
V
V
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008871:Microvilli / Q000378:metabolism*; D009578:Nitrobenzenes / Q000494:pharmacology; D010920:Placenta / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011247:Pregnancy; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013654:Taurine / Q000378:metabolism*; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine
D002352:Carrier Proteins; D007093:Imidazoles; D009578:Nitrobenzenes; D013439:Sulfhydryl Reagents; D013654:Taurine; C034442:4-nitrobenzenesulfonyl fluoride; D014443:Tyrosine; D009327:4-Chloro-7-nitrobenzofurazan; D013774:Tetranitromethane; D011188:Potassium; C012160:N-acetylimidazole