Characterization of the ATP-dependent leukotriene C4 export carrier in mastocytoma cells.
The biosynthesis of leukotriene C4 (LTC4) 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 LTC4 [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 vesicles from mastocytoma cells: Probenecid, an inhibitor of organic anion transport, induced half-maximal inhibition of the ATP-dependent LTC4 transport at 71 microM. Cyclosporin A and its non-immunosuppressive analog PSC 833 inhibited the ATP-dependent transport with Ki values of 4.5 microM and 30 microM, respectively. The LTD4 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 Ki value of 0.8 microM. The transport inhibitor MK 571 served as competitor in the photoaffinity labeling of LTC4-binding membrane proteins using [3H]LTC4 as the photolabile ligand. Proteins with molecular masses of about 190 kDa and 35 kDa were predominantly labeled. In addition, a minor [3H]LTC4 labeling was observed in the molecular mass range of 100 kDa. The [3H]LTC4 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-32P]ATP predominantly labeled the LTC4-binding 35-kDa protein. The [3H]LTC4-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 LTC4, which is competed for by a potent inhibitor of the ATP-dependent LTC4 export carrier, pinpoints its involvement in the ATP-dependent transport of LTC4 and related conjugates.
Leier
I
I
Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Jedlitschky
G
G
Buchholz
U
U
Keppler
D
D
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D017997:Leukotriene C4 / Q000378:metabolism*; D012515:Mast-Cell Sarcoma / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D017997:Leukotriene C4; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate