Flow cytometric analysis of transmembrane phospholipid movement in bull sperm

Fluorescent phospholipids are useful to investigate phospholipid dynamics in biological membranes. We used flow cytometry to investigate transbilayer phospholipid movement in live sperm cells. Acyl-labeled N-4-nitrobenzo-2-oxa-1,3-diazole (NBD) -phosphatidylcholine (-PC), -phosphatidylethanolamine (-PE), or -phosphatidylserine (-PS) were incorporated into sperm cells, and the transbilayer location was determined by extraction of probe from cell with excess bovine serum albumin (BSA) or by chemical destruction of probe by sodium dithionite. Using these methods, we have measured the head group specific outer leaflet to inner leaflet movement (flip) of the aminophospholipids NBD-PS and NBD-PE. The fluorescent phospholipids moved inward across the plasma membrane with half-times of 1.8, 2.5, and 11.2 min, for NBD-PS, NBD-PE, and NBD-PC and reached apparent equilibrium levels of 88%, 94%, and 32% inside, respectively. The inward movement of NBD-PE was inhibited by sulfhydryl reagents, elevated intracellular Ca2+, and depletion of cellular ATP. Analysis of the kinetics of NBD-PE and -PS extraction by BSA allows determination of the rates for outward movement (flop) across the plasma membrane. Half-times for flop were 4.7 and 4.5 min for NBD-PS and -PE, respectively. Based on these measurements, a simple model of NBD-phospholipid equilibria was developed and fit to the kinetic data. Computer-generated fits reflected major features of the experimental data and provide a potential tool for predicting the dynamics of endogenous lipids.