Multiple routes and regulation by tyrosine phosphorylation characterize the ATP-dependent transport of 2,4-dinitrophenyl S-glutathione in inside-out vesicles from human erythrocytes.
ATP-dependent efflux routes for 2,4-dinitrophenyl S-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 micro;m indicated that multiple transport routes were operative. Sensitivity to naphthyl glucuronide separated [3H]DNP-SG transport into two low-affinity components which by computer analysis exhibited Km values of 330 and 1400 micro;m, respectively. At low substrate concentrations, two high-affinity routes were observed. The predominant activity (hMOAT3a) exhibited a Km of 0.18 micro;m (Vmax = 22 pmol/min/mg protein), whereas the second activity (MOAT3b) had a Km of 0.58 micro;m (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 micro;m genistein showed that increased transport resulted from the appearance of a new activity (hMOAT3c) with a Km of 0.85 micro;m and a substantially elevated Vmax (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 substrate Km value 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.
Saxena
M
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Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California, 92037, USA.
Henderson
G B
GB
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000097:blood*; D000818:Animals; D027321:Anion Transport Proteins; D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002474:Cell-Free System; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism*; D019833:Genistein; D005965:Glucuronates / Q000378:metabolism / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology; D007939:Leukemia L1210; D019000:Phosphotyrosine / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D013379:Substrate Specificity
D027321:Anion Transport Proteins; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D005965:Glucuronates; D007529:Isoflavones; D019000:Phosphotyrosine; C030190:S-(2,4-dinitrophenyl)glutathione; C029091:naphthyl glucuronide; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases; D000251:Adenosine Triphosphatases; C068387:S-(dinitrophenyl)glutathione ATPase; D005978:Glutathione