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. and Keppler, D. (1991) FEBS Lett. 279, 83-86]. The following inhibitors served to characterize further this transport system in plasma membrane vesicles from mastocytoma cells: Probenecid, an inhibitor of organic anion transport, induced half-maximal inhibition of the ATP-dependent LTC(4) transport at 71 mu M. Cyclosporin A and its non-immunosuppressive analog PSC 833 inhibited the ATP-dependent transport with K-i values of 4.5 mu M and 30 mu M, 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 mu M. The transport inhibitor MK 571 served as competitor in the photoaffinity labeling of LTC(4)-binding membrane proteins using [H-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 [H-3]LTC(4) labeling was observed in the molecular mass range of 100 kDa. The [H-3]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-azido[alpha-P-32]ATP predominantly labeled the LTC(4)-binding 35-kDa protein. The [H-3]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.