Characterization of the ATP‐dependent leukotriene C 4 export carrier in mastocytoma cells

The biosynthesis of leukotriene C 4 (LTC 4 ) must be followed by an export of this mediator into the extracellular space where it interacts with receptors. Using mastocytoma cells we have demonstrated the existence of a primary‐active, ATP‐dependent transport mediating this export of LTC 4 [Schaub, T., Ishikawa, T. & Keppler, D. (1991) FEBS Lett. 279 , 83–86]. The following inhibitors served to characterize further this transport system in plasma membrane vesicle from mastocytoma cells: Probenecid, an inhibitor of organic anion transport, induced half‐maximal inhibition of the ATP‐dependent LTC 4 transport at 71 μ. Cyclosporin A and its non‐immunosuppressive analog PSC 833 inhibited the ATP‐dependent transport with K i values of 4.5 μM and 30μM, respectively. The LTD 4 receptor antagonist 3‐([{3‐(2‐[7‐chloro‐2‐quinolinyl]ethenyl)phenyl}‐{(3‐dimethylamino‐3‐oxopropyl)‐thio}methyl]thio)propanoic acid (MK 571) was the most potent competitive inhibitor of the export carrier with a K i value of 0.8 μM. The transport inhibitor MK 571 served as competitor in the photoaffinity labeling of LTC 4 ‐binding membrane proteins using [ 3 ]LTC 4 as the photolabile ligand. Proteins with molecular masses of about 190 kDa and 35 kDa were predominantly labeled. In addition, a minor [ 3 H]LTC 4 labeling was observed in the molecular mass range of 100kDa. The [ 3 H]LTC 4 labeling of the 190‐kDa protein was competed for by MK 571. The labeled proteins resisted extraction from the membrane with 2% sodium taurocholate suggesting that they are integral membrane proteins. Treatment of the membrane proteins with peptide N‐glycosidase F resulted in the appearance of an additional labeled polypeptide of about 140 kDa suggesting that the 190‐kDa protein is a glycoprotein. Photoaffinity labeling with 8‐azio[α‐ 32 P]ATP predominantly labeled the LTC 4 ‐binding 35‐kDa protein. The [ 3 H]LTC 4 ‐labeled 190‐kDa protein showed a mean isoelectric point at pH 6.3 with a range of pH 5.8–6.7, while the 35‐kDa protein had an isoelectric point at pH 6.8. Specific labeling of a 190‐kDa membrane glycoprotein by the glutathione conjugate LTC 4 , which is competed for by a potent inhibitor of the ATP‐dependent LTC 4 export carrier, pinpoints its involvement in the ATP‐dependent transport of LTC 4 and related conjugates.