Multiple Routes and Regulation by Tyrosine Phosphorylation Characterize the ATP-Dependent Transport of 2,4-DinitrophenylS-Glutathione in Inside-Out Vesicles from Human Erythrocytes

ATP-dependent efflux routes for 2,4-dinitrophenylS-glutathione (DNP-SG) were investigated using inside-out vesicles from human erythrocytes. Nonlinear double-reciprocal plots of transport at substrate concentrations ranging from 0.07 to 500 μmindicated that multiple transport routes were operative. Sensitivity to naphthyl glucuronide separated [3H]DNP-SG transport into two low-affinity components which by computer analysis exhibitedKmvalues of 330 and 1400 μm, respectively. At low substrate concentrations, two high-affinity routes were observed. The predominant activity (hMOAT3a) exhibited aKmof 0.18 μm(Vmax= 22 pmol/min/mg protein), whereas the second activity (MOAT3b) had aKmof 0.58 μm(Vmax= 16 pmol/min/mg protein). High-affinity transport of DNP-SG increased substantially (2.5-fold) in vesicles preincubated with genistein or other tyrosine kinase inhibitors. Kinetic analyses in vesicles pretreated with 50 μmgenistein showed that increased transport resulted from the appearance of a new activity (hMOAT3c) with aKmof 0.85 μmand a substantially elevatedVmax(80 pmol/min/mg protein). At varying concentrations of genistein, a progression was observed that was consistent with the conversion of hMOAT3b to hMOAT3a and hMOAT3a to hMOAT3c. Phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, produced an opposite progression. Specificity studies showed that hMOAT3a exhibited the highest affinity for various anionic conjugates, and had a notable binding preference for glutathione disulfide. The relative effectiveness of the various inhibitors was similar for hMOAT3a, hMOAT3b, and hMOAT3c, as well as for a corresponding mMOAT3 activity from L1210 mouse cells. The results show that human erythrocytes contain multiple ATP-dependent efflux systems for DNP-SG and that separation of these systems can be achieved on the basis of substrateKmvalue and inhibitor and activator specificity. High-affinity transport can proceed via three activities which appear to be subforms of a single system with differing levels of tyrosine phosphorylation. Multiple hMOAT3 subforms provide flexibility for extruding various anionic conjugates and may have evolved in erythrocytes to expedite the efflux of GS-SG.