Essential tyrosine residues in transport of organic cations in renal BBMV

The effects of tyrosine modifying agents on organic cation transport in brush-border membrane vesicles prepared from rabbit renal cortex were investigated. Treatment of membranes with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and N-acetylimidazole reduced the initial rate of transport of N'-methylnicotinamide (NMN) significantly. The inactivation of NMN transport by NBD-Cl was concentration and time dependent. The maximal transport rate (Tmax) of NMN transport (18.4 +/- 4.3 pmol X s-1 X mg protein-1) in vesicles treated with NBD-Cl (0.15 mM) was reduced to 56% of the Tmax in the control vesicles (32.6 +/- 8.4 pmol X s-1 X mg protein-1, P less than 0.05); whereas the Km was not changed. Treatment with 2-mercaptoethanol reversed the reaction of NBD-Cl with sulfhydryl groups but did not significantly change the transport of NMN in the control or the treated membranes, suggesting that tyrosine but not sulfhydryl residues are involved. The overshoot of NMN uptake in the presence of a proton gradient was 3.38 +/- 0.67 pmol/mg protein in the untreated membranes and was reduced to 2.05 +/- 0.71 pmol/mg protein in the NBD-Cl-treated membranes (P less than 0.05). Studies with the pH-sensitive dye, acridine orange, demonstrated that NBD-Cl-treated vesicles were not leakier with respect to protons, suggesting that NBD-Cl may have specifically affected the organic cation-proton transporter.(ABSTRACT TRUNCATED AT 250 WORDS)