The transport of exogenously supplied fluorescent analogues of aminophospholipids from the outer to inner leaflet in red blood cells (RBC) is dependent upon the oxidative status of membrane sulfhydryls. Oxidation of a sulfhydryl on a 32-kDa membrane protein by pyridyldithioethylamine (PDA) has been previously shown [Connor & Schroit (1988) Biochemistry 27, 848–851] to inhibit the transport of NBD-labeled phosphatidylserine (NBD-PS). In the present study, other sulfhydryl oxidants were examined to determine whether additional sites are involved in the transport process. Our results show that diamide inhibits the transport of NBD-PS via a mechanism that is independent of the 32-kDa site. This is shown by the inability of diamide to block labeling of the 32-kDa sulfhydryl with 125I-labeled PDA and to protect against PDA-mediated inhibition of NBD-PS transport. Diamide-mediated inhibition, but not PDA-mediated inhibition, could be reversed by reduction with cysteamine or endogenous glutathione. Similarly, treatment of RBC with 5,5′-dithiobis(2-nitrobenzoic acid), which depletes endogenous glutathione and induces oxidation of endofacial proteins [Reglinski et al. (1988) J. Biol. Chem. 263, 12360–12366], inhibited NBD-PS transport in a manner analogous to diamide. Once established, the asymmetric distribution of NBD-PS could not be altered by oxidation of either site. These data indicate that a second site critical to the transport of aminophospholipids resides on the endofacial surface and suggest that the transport of aminophospholipids across the bilayer membrane of RBC depends on a coordinated and complementary process between a cytoskeletal component and the 32-kDa membrane polypeptide; both must be operative for transport to proceed.
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