NOVEL RADIOACTIVE PHOSPHOLIPID PROBES AS A TOOL FOR MEASUREMENT OF PHOSPHOLIPID TRANSLOCATION ACROSS BIOMEMBRANES

In an attempt to develop a new method to measure transbilayer phospholipid translocation, with a higher sensitivity and higher temporal resolution, novel radioactive phospholipid probes (*C5-PC, *C5-PE, and *C5-PS) with a short acyl chain at thc 2-position were synthesized. The *C5-PC probe was made by coupling lysophosphatidylcholine with [C-14]pentanoic acid, using N,N-carbonyldiimidazole as a coupling agent (yield 37%), and *C5-PE and *C5-PS were synthesized by exchanging the choline moiety of *C5-PC for ethanolamine and L-serine, respectively, as catalyzed by phospholipase D. The usefulness of the probes was confirmed by measuring phospholipid translocation across the human erythrocyte plasma membrane, in which thc presence of aminophospholipid translocase was revealed using EPR techniques (Zachowski, A., Farve, E., Cribier, S., Herve, P. and Devaux, P.F. (1986) Biochemistry 25, 2585-2590). Using the present probes, ATP-dependent and SH-reagent-inhibitable translocation of *C5-PS and *C5-PE from outer to inner leaflets, which is characteristic to the translocation mediated by aminophospholipid translocase, was detected with a higher sensitivity than seen with the EPR technique. These radioactive phospholipid probes will be useful to measure phospholipid translocation with a high sensitivity and have the potential for application in measurements of transbilayer lipid-translocation for a wide variety of membranes.