Outside-inside translocation of aminophospholipids in the human erythrocyte membrane is mediated by a specific enzyme.
When human erythrocytes are incubated with spin-labeled analogues of sphingomyelin, phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine, with a short beta chain (C5) bearing a doxyl group at the fourth carbon position, the labeled lipids incorporate readily in the outer monolayer. The incorporation is followed in fresh erythrocytes by a selective inward diffusion of the amino derivatives. This observation led us to postulate the existence of a selective ATP-dependent system that would flip aminophospholipids from the outer to the inner monolayer [Seigneuret, M., & Devaux, P. F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3751-3755]. This study further examines the nature of this selective transport and demonstrates that it is mediated by a specific membrane protein. By measurement of the initial rate of transverse diffusion of spin-labeled lipids incorporated at various concentrations in the membrane outer leaflet of packed erythrocytes, apparent Km values were determined for the phosphatidylserine and phosphatidylethanolamine analogues. A ratio of approximately equal to 1/9.4 [corrected] was obtained (KmPS/KmPE). Using spin-labels bearing either a 14N or a 15N isotope, we have carried out competition experiments allowing us to measure simultaneously the transport of two different phospholipids. By this procedure, we show that phosphatidylserine and phosphatidylethanolamine compete for the same transport site but that phosphatidylserine has a higher affinity, in agreement with a lower apparent Km. On the other hand, the slow diffusion of the phosphatidylcholine or sphingomyelin analogues has no influence on the transport of phosphatidylserine or phosphatidylethanolamine. Experiments carried out in ghosts loaded with ATP enabled us to determine the activation energies for phosphatidylserine and phosphatidylcholine transverse diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)
D001692:Biological Transport; D002352:Carrier Proteins / Q000097:blood*; D004578:Electron Spin Resonance Spectroscopy; D004910:Erythrocyte Membrane / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids / Q000097:blood*; D013113:Spin Labels; D013816:Thermodynamics
D002352:Carrier Proteins; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D013113:Spin Labels; D005033:Ethylmaleimide