Abstract
The transbilayer mobility of phosphatidylcholine (PC) molecules in the membrane of homozygous reversible sickle cells (RSCs) was studied using a PC-specific exchange protein from beef liver. In deoxygenated RSCs, all of the PC present in the membrane of the intact cell is rapidly available for exchange, mediated by this protein. Since a substantial amount of the PC is present in the inner membrane leaflet of these cells, this observation implies that the PC molecules in their membranes do experience rapid transbilayer movements. To determine the actual rate of transbilayer movement of the PC, radioactive PC was introduced into the outer monolayer of oxygenated RSCs using the PC-specific exchange protein. Subsequently, the cells were incubated at 37 degrees C under oxy- and deoxygenating conditions to enable the PC to equilibrate within the bilayer. At various time intervals, samples were taken and treated with phospholipase A2, which selectively degrades the PC in the outer monolayer. Analysis of the specific radioactivities of the lyso-PC thus produced, as well as of the residual PC, enabled us to follow the fate of the radioactive PC previously introduced into the outer membrane layer. The half-time value for transbilayer equilibration of the PC in deoxygenated RSCs was determined to be 3.5 h, which is about four times lower than that for oxygenated RSCs. This increased transbilayer mobility of PC, observed in deoxygenated RSCs, is immediately restored to the normal low rate upon reoxygenation of the cells, indicating a complete reversibility of this phenomenon.
Highlights
From the $Department of Biochemistry, State Universityof Utrecht, Padualaan 8, 3584CH Utrecht, The Netherlands and the §Bruce Lyon Memorial Research Laboratory and Departmentof HematologylOncology, Children’s Hospital Medical Center, Oakland, California94609
Thetransbilayer mobility of phosphatidylcholine myelin are found in the outer monolayer, whereas the inner (PC) molecules in the membrane of homozygous re- layer comprises 80% of the PE anadll of the PS (1,2)
Sainscuebstantial amount of the PC is present in the inner membrane leaflet of these cells, this observation implies that the PC molecules in their membranes do experience rapid transbilayer movements
Summary
The introduction of "C-labeled PC into the outer membrane layer, a Erythrocytes-After obtaininginformedconsent, freshvenous 33% suspension of oxygenated RSCs was incubated for 1 h at 37 "C blood from normal (AA) controls and homozygous sickle cell (SS) in the presence of egg [14C]PC/cholesterolvesicles At various used as a donor system (35).PC/cholesterol vesicles, have time intervals, samples (comprisin3g10 p l of packed cells)were taken heen more thoroughly characterized, and sufficiently high amounts from theabove incubation mixtures.After collecting the erythrocytes of radioactive PC can be incorporated into the outer monolayer of by centrifugation, theywere resuspended in 6 volumes of the following the erythrocyte membrane within reasonably short times of incuba- buffer: 90 mM KCl, 45 mM NaCl, 10 mM CaC12,0.25 mM MgCl,, 22 tion, despite the slower exchange rate (35). After 1 h, the ghost membranes were spun down at 2500 X g for 20 min and stored a t
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