Studies on sickled erythrocytes provide evidence that the asymmetric distribution of phosphatidylserine in the red cell membrane is maintained by both ATP-dependent translocation and interaction with membrane skeletal proteins.
In order to study factors which are involved in maintenance of phosphatidylserine (PS) asymmetry within the human red cell membrane, we measured the effect of ATP-depletion and of membrane skeleton/lipid bilayer uncoupling induced by sickling on the distribution of PS within the membrane bilayer of sickle cells. Trace amounts of radiolabeled PS were introduced into the outer membrane leaflet of both fresh and ATP-depleted reversibly sickled cells (RSCs), using a non-specific lipid transfer protein purified from bovine liver. The equilibration of the newly introduced PS over the two halves of the bilayer was monitored by treatment of the cells with phospholipase A2 which selectively hydrolyzes only those molecules present in the outer membrane leaflet. Within 1 h after insertion into fresh RSCs, only 10% of the labeled PS was accessible to the action of phospholipase A2. This fraction was markedly increased when the cells were subsequently deoxygenated. Prolonged deoxygenation of RSCs, deprived of their ATP after incorporation of radiolabeled PS, caused enhanced phospholipase A2-induced hydrolysis of radiolabeled PS. Similarly, phospholipase A2-induced hydrolysis of endogenous PS in intact RSCs was markedly enhanced when ATP-depleted, but not when fresh cells, were incubated under nitrogen for 3.5 h. Deoxygenated ATP-depleted RSCs markedly enhanced the rate of thrombin formation in the presence of purified coagulation factors Xa, Va, prothrombin and Ca2+. This enhancement appeared to be dependent on the duration of incubation under nitrogen. This phenomenon, indicating the presence of increasing amounts of endogenous PS in the outer membrane leaflet, was not observed when either fresh RSCs or ATP-depleted normal erythrocytes were incubated under nitrogen. Our present observations provide evidence that, in addition to the interaction of PS with the skeletal proteins, an ATP-dependent translocation of PS is required to maintain its absolute asymmetric distribution in the human erythrocyte membrane.
Middelkoop
E
E
Department of Biochemistry, State University of Utrecht, The Netherlands.
Lubin
B H
BH
Bevers
E M
EM
Op den Kamp
J A
JA
Comfurius
P
P
Chiu
D T
DT
Zwaal
R F
RF
van Deenen
L L
LL
Roelofsen
B
B
D000255:Adenosine Triphosphate / Q000097:blood*; D000755:Anemia, Sickle Cell / Q000097:blood*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008051:Lipid Bilayers / Q000097:blood*; D008565:Membrane Proteins / Q000097:blood*; D010718:Phosphatidylserines / Q000097:blood*; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010743:Phospholipids / Q000097:blood; D013925:Thromboplastin / Q000097:blood
D008051:Lipid Bilayers; D008565:Membrane Proteins; D010718:Phosphatidylserines; D010743:Phospholipids; D000255:Adenosine Triphosphate; D013925:Thromboplastin; D010741:Phospholipases A; D054467:Phospholipases A2