TRANSBILAYER MOVEMENT OF FLUORESCENT AND SPIN-LABELED PHOSPHOLIPIDS IN THE PLASMA-MEMBRANE OF HUMAN FIBROBLASTS - A QUANTITATIVE APPROACH

All phospholipids in the plasma membrane of eukaryotic cells are subje ct to a slow passive transbilayer movement. In addition, aminophosphol ipids are recognized by the so-called aminophospholipid translocase, a nd are rapidly moved from the exoplasmic to the cytoplasmic leaflet of the plasma membrane at the expense of ATP hydrolysis. Though these pr incipal pathways of transbilayer movement of phospholipids probably ap ply to all eukaryotic plasma membranes, studies of the actual kinetics of phospholipid redistribution have been largely confined to non-nucl eated cells (erythrocytes). Experiments on nucleated cells are complic ated by endocytosis and metabolism of the lipid probes inserted into t he plasma membrane. Taking these complicating factors into account, we performed a detailed kinetic study of the transbilayer movement of sh ort-chain fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ; NBD) an d, for the first time, spin-labeled analogues of phosphatidylcholine ( PC), -ethanolamine (PE), -serine (PS), and sphingomyelin (SM) in the p lasma membrane of cultured human gingival fibroblasts. At 20 degrees C , the passive transbilayer diffusion of NBD analogues was very slow, a nd the choline-containing NBD analogues were internalized predominantl y by endocytosis. Spin-labeled analogues of PC and SM showed higher pa ssive transbilayer diffusion rates, and probably entered the cell by b oth passive transbilayer movement and endocytosis. In contrast, the ra pid uptake of NBD- and spin-labeled aminophospholipid analogues could be mainly ascribed to the action of the aminophospholipid translocase, since it was inhibited by ATP depletion and N-ethylmaleimide pretreat ment. The initial velocity of NBD-aminophospholipid translocation was eight to ten times slower than that of the corresponding spin-labeled lipid, and the half-times of redistribution of NBD-PS and spin-labeled PS were 7.2 and 3.6 minutes, respectively. Our data indicate that in human fibroblasts the initial velocity of aminophospholipid translocat ion is at least one order of magnitude higher than that in human eryth rocytes, which should be sufficient to maintain the phospholipid asymm etry in the plasma membrane.