Rapid flip-flop of phospholipids in endoplasmic reticulum membranes studied by a stopped-flow approach

The transbilayer movement of short-chain spin-labeled and fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) phospholipid analogs in rat liver microsomes is measured by stopped-flow mixing of labeled microsomes with bovine serum albumin (BSA) solution. Extraction of analogs from the outer leaflet of microsomes to BSA can be directly monitored in conjunction with electron paramagnetic resonance or fluorescence spectroscopy by taking advantage of the fact that the signal of spin-labeled or fluorescent analogs bound to BSA is different from that of the analogs inserted into membranes. From the signal kinetics, the transbilayer movement and the distribution of analogs in microsomal membranes can be derived provided the extraction of analogs by BSA is much faster in comparison to the transbilayer movement of analogs. Half-times of the back-exchange for spin-labeled and fluorescent analogs were <3.5 and <9.5 s, respectively. The unprecedented time resolution of the assay revealed that the transbilayer movement of spin-labeled analogs is much faster than previously reported. The half-time of the movement was about 16 s or even less at room temperature. Transmembrane movement of NBD-labeled analogs was six- to eightfold slower than that of spin-labeled analogs.