Transbilayer movement of phosphatidylserine in erythrocytes. Inhibitors of aminophospholipid transport block the association of photolabeled lipid to its transporter.
The ability to cross-link [125I]iodo-azido-phosphatidylserine (125I-N3-PS) to the putative 32-kDa aminophospholipid transporter of human red blood cells (RBC) has been examined by SDS-PAGE. In the absence of transport inhibitors, 125I-N3-PS preferentially labeled the 32-kDa polypeptide, whereas treatment of the cells with pyridyldithioethylamine (PDA), a potent inhibitor of the aminophospholipid translocase, abrogated the association of the probe to this protein. ATP-depletion, low temperature, and diamide or 5,5'-dithiobis(2-nitrobenzoic acid), inhibitors that oxidize an endofacial sulfhydryl distinct from the PDA-sensitive site (Connor, J. and Schroit, A.J. (1990) Biochemistry 29, 37-43), also blocked association of the PS analogue to the protein. Once 125I-N3-PS became associated with the transporter, however, only PDA was able to partially displace it. These data suggest that sulfhydryl reactive reagents inhibit PS transport by blocking the association of PS with its transporter, a process that is also ATP- and temperature-dependent.
Connor
J
J
Department of Cell Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Schroit
A J
AJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D004220:Disulfides / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D010455:Peptides / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins; D010782:Photolysis; D011725:Pyridines / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013696:Temperature
D002352:Carrier Proteins; D004220:Disulfides; D008565:Membrane Proteins; D010455:Peptides; D010718:Phosphatidylserines; D049294:Phospholipid Transfer Proteins; D011725:Pyridines; D013438:Sulfhydryl Compounds; C055639:pyridyldithioethylamine; D000255:Adenosine Triphosphate