Asymmetric exchange of vesicle phospholipids catalyzed by the phosphatidylcholine exhange protein. Measurement of inside--outside transitions.

Abstract

Purified phosphatidylcholine exchange protein was used to exchange phosphatidylcholine between homogeneous single-walled phosphatidylcholine vesicles and human erythrocyte ghosts. When excess ghosts were present, it was found that only 70% of the vesicle phosphatidylcholine was available for exchange. This fraction corresponds closely to the amount of phosphatidycholine in the outer monolayer of these vesicles, indicating that only the outer surface of the vesicle is accessible to the exchange protein. Also, it was found that all phosphatidylcholine introduced into vesicles by the exchange protein was available for subsequent exchange. Using the exchange protein, asymmetrical vesicles were prepared in which the outer monolayer was either enriched or depleted in radioactive phosphatidylcholine as compared to the inner monolayer. Re-equilibration of the radioactivity between the two surfaces of the vesicle (flip-flop) could not be detected, even after 5 days at 37degrees. It is estimated that the half-time for flip-flop is in excess of 11 days at 37degrees. These results indicate that the properties of the exchange protein can be expolited to measure phosphatidylcholine flip-flop rates and possible phosphatidylcholine asymmetry in biological and model membranes, without altering the structure of the membrane.