Monitoring of phospholipid vesicle fusion by fluorescence energy transfer between membrane-bound dye labels.
A sensitive method which utilizes fluorescence energy transfer to assay Ca2+ -or Mg2+ -mediated fusion of phospholipid vesicles is reported. More than 85% quenching results when phosphatidylserine vesicles labelled with dansyl phosphatidylethanolamine (donor) are fused with vesicles labelled with rhodamine phosphatidylethanolamine (acceptor) in the presence of 5 mM CaCl2 or 10 mM MgCl2. Higher concentrations of divalent cations are required to obtain maximal quenching when phosphatidylserine is partially replaced with phosphatidylethanolamine or phosphatidylcholine. The rate of vesicle fusion is dependent upon the concentrations of both cation and vesicles. Maximum quenching occurs within 5 min using phosphatidylserine vesicles and 5 mM Ca2+, but quenching is incomplete even after 20 h with 0.8--2 mM Ca2+. This probably reflects the heterogeneous size distribution of these vesicles, since the extent of fusion was found to correlated with vesicle size. Binding of antibody to membrane-localized phenobarbital hapten effectively blocks Ca2+ -mediated vesicle fusion. This effect can be inhibited by preincubation of the antibody with phenobarbital. Leakage of tempocholine from intact vesicles induced by 5 mM Ca2+ occurs even when fusion is prevented by bound antibody. This demonstrates that fusion is not a necessary requirement for Ca2+ -induced leakage.
D000906:Antibodies; D002118:Calcium; D004396:Coloring Agents; D003619:Dansyl Compounds; D006241:Haptens; D005779:Immunodiffusion; D007700:Kinetics; D008274:Magnesium; D008567:Membranes, Artificial; D008823:Micelles; D010634:Phenobarbital; D010743:Phospholipids; D011485:Protein Binding; D012235:Rhodamines; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D000906:Antibodies; D004396:Coloring Agents; D003619:Dansyl Compounds; D006241:Haptens; D008567:Membranes, Artificial; D008823:Micelles; D010743:Phospholipids; D012235:Rhodamines; D008274:Magnesium; D002118:Calcium; D010634:Phenobarbital