Nonmediated flip-flop of phospholipid analogues in the erythrocyte membrane as probed by palmitoylcarnitine: basic properties and influence of membrane modification.
The rules governing the transbilayer reorientation (flip-flop) of long-chain amphiphilic components in biological membranes were further elucidated by studying the flip-flop of palmitoylcarnitine in human erythrocytes. Flip rates were derived from the time-dependent decrease of extractability of palmitoylcarnitine by albumin after primary insertion of trace amounts of the labeled probe into the outer membrane layer. The flip rate (half time 2.6 hr at 37 degrees C in human erythrocytes) is fast enough to be measurable also in membranes exhibiting low flip rates such as that of ox erythrocytes. Flip rate constants for the inward and outward reorientation are similar and the probe equilibrates at a 1:1 ratio between the two layers. The flip is a simple, diffusion-like process. It is not inhibited but even enhanced by chemical modification of membrane proteins. It is also enhanced by insertion of channel-forming antibiotics into the membrane and by pre-exposure of the cells to temperatures exceeding 42 degrees C. The extent of this enhancement increases with the duration and the temperature of the pre-exposure. Since spectrin is denatured in this range of temperatures, the finding constitutes a new piece of evidence that the membrane skeleton is involved in the maintenance of bilayer stability and that a decrease of bilayer stability goes along with the formation of local defects acting as flip sites for phospholipids and related compounds. As a particularity, the flip is enhanced by lowering the pH and exhibits interindividual variability, phenomena not observed for the flip-flop of lysophosphatidylcholine. This suggests that generalizations on the kinetics of nonmediated flip-flop of membrane-intercalated amphiphiles may not be justified.
Classen
J
J
Department of Physiology, Medical Faculty, RWTH Aachen Pauwelsstrasse, Federal Republic of Germany.
Deuticke
B
B
Haest
C W
CW
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002331:Carnitine / Q000031:analogs & derivatives*; D002417:Cattle; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D015335:Molecular Probes; D010172:Palmitoylcarnitine / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011489:Protein Denaturation; D013049:Spectrin / Q000378:metabolism
D008051:Lipid Bilayers; D015335:Molecular Probes; D010743:Phospholipids; D013049:Spectrin; D010172:Palmitoylcarnitine; D000255:Adenosine Triphosphate; D002331:Carnitine