The passive transbilayer movement-flip-flop-was investigated on planar bilayer lipid membranes (BLMs), containing myristic, stearic, or linoleic long-chain fatty acids (FA). In response to a transbilayer pH gradient, a difference in the surface charges between inner and outer leaflets appeared. Because the BLM was formed from FA and neutral lipid, a surface potential difference was originated solely by a concentration difference of the initially equally distributed ionized FA. As revealed by zeta-potential measurements, the corresponding surface potential difference DeltaPhi(s) was at least twice the value expected from a titration of the FA alone. The additional surface charge was attributed to FA flip-flop induced by the transbilayer pH gradient. DeltaPhi(s) was derived from capacitive current measurements carried out with a direct current (dc) bias and was corrected for changes of membrane dipole potential Phi(d). Dual-wavelength ratiometric fluorescence measurements have shown that Phi(d) values of the pure DPhPC bilayers and BLMs containing 40 mol % FA differ by less than 6%. It is concluded that fast FA flip-flop is not restricted to membranes with high curvature. The role of pH gradient as an effective driving force for the regulation of FA uptake is discussed.

Ricin is a promising candidate for the treatment of cancer because it can be selectively targeted to tumor cells via linkage to monoclonal antibodies. Biochemical evidence suggests that escape of ricin or its ribosome-inactivating subunit from an intracellular compartment is mediated by retrograde transport to the endoplasmic reticulum and subsequent direction into the ER-associated degradation pathway. Alternatively, lipase activity of ricin may facilitate leakage from endocytic vesicles. We have observed ricin-mediated release of macromolecular dyes from lipid vesicles that mimic the composition of endosomal membranes. Release of small molecules occurs to the same extent, suggesting an all-or-none mechanism due to bilayer destabilization. The level of accompanying membrane fusion depends on vesicle composition. Since it takes 24 h of incubation before the first traces of lysolipids are detectable by matrix-assisted laser desorption/ionization mass spectrometry, membrane destabilization is not due to the lipase activity of ricin.

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