Calcium/phosphate-induced immobilization of fluorescent phosphatidylserine in synthetic bilayer membranes: inhibition of lipid transfer between vesicles

Abstract

Resonance energy transfer between 4-nitro-2,1,3-benzoxadiazole (NBD) acyl chain labeled phospholipid analogues and (lissamine) rhodamine B labeled phosphatidylethanolamine was used to monitor the rate of NBD-labeled lipid transfer between a variety of small unilamellar donor vesicles and dioleoylphosphatidylcholine (DOPC) acceptor vesicles. In the presence of appropriate concentrations of Ca2+ and phosphate, the transfer rate of NBD-phosphatidylserine (NBD-PS) from vesicles composed of lipid extracts from human red blood cells was reduced by approximately 10-fold, while the transfer rates of NBD-phosphatidylcholine, -ethanolamine, -glycerol, -N-succinylethanolamine, and -phosphatidic acid were essentially unaffected. A systematic evaluation of the lipid composition needed to facilitate the Ca2+/phosphate-induced inhibition of NBD-PS transfer revealed that the process was dependent upon the inclusion of both cholesterol and phosphatidylethanolamine (PE) in the donor vesicle population. Inhibition of NBD-PS transfer required the sequential addition of phosphate and Ca2+ to the vesicles, indicating that the combined interaction of Ca2+ and phosphate at the membrane surface was a prerequisite for inhibition to occur. Parallel experiments designed to determine the possible mechanism of this phenomenon showed that inhibition of NBD-PS transfer was not due to Ca2+-mediated phase separations or vesicle-vesicle fusion. However, the addition of Ca2+ and phosphate to vesicles composed of total red blood cell lipids or cholesterol/PE did result in their aggregation. On the other hand, aggregation per se did not seem to be responsible for the inhibition of transfer since NBD-PS-containing vesicles composed of DOPC or DOPC/DOPE also aggregated, although NBD-PS transfer was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)