Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers.

A technique for the production of supported phospholipid bilayers by adsorption and fusion of small unilamellar vesicles to supported phospholipid monolayers on quartz is described. The physical properties of these supported bilayers are compared with those of supported bilayers which are prepared by Langmuir-Blodgett deposition or by direct vesicle fusion to plain quartz slides. The time courses of vesicle adsorption, fusion and desorption are followed by total internal reflection fluorescence microscopy and the lateral diffusion of the lipids in the adsorbed layers by fluorescence recovery after photobleaching. Complete supported bilayers can be formed with phosphatidylcholine vesicles at concentrations as low as 35 microM. However, the adsorption, fusion and desorption kinetics strongly depend on the used lipid, NaCl and Ca2+ concentrations. Asymmetric negatively charged supported bilayers can be produced by incubating a phosphatidylcholine monolayer with vesicles composed of 80% phosphatidylcholine and 20% phosphatidylglycerol. Adsorbed vesicles can be removed by washing with buffer. The measured fluorescence intensities after washing are consistent with single supported bilayers. The lateral diffusion experiments confirm that continuous extended bilayers are formed by the monolayer-fusion technique. The measured lateral diffusion coefficient of NBD-labeled phosphatidylethanolamine is (3.6 +/- 0.5) x 10(-8) cm2/s in supported phosphatidylcholine bilayers, independent of the method by which the bilayers were prepared.

Binding and calcium-induced aggregation of laminin onto lipid bilayers.

Direct binding of laminin in the form of its complex with nidogen to planar lipid bilayers was demonstrated with total internal reflection fluorescence microscopy. Binding occurred equally well to zwitterionic (phosphatidylcholine) and negatively charged (phosphatidylglycerol) lipids and was enhanced by sulfatides but only at nonphysiological molar ratios higher than 30 mol %. Strong interactions with lipid bilayers were also observed for bovine serum albumin. This explains a strong inhibition of laminin binding by this protein. However, binding of laminin to sulfatide-rich bilayers was not completely inhibited. Observable by the microscopic technique was the formation of laminin clusters on the surface of the bilayer which occurred concomitantly with binding. Both processes were strongly enhanced by the presence of calcium. These results show that calcium-induced laminin self-assembly is enhanced at lipid surfaces.

Membrane interaction of 'peptide P' derived from the repeating motif of properdin.

A 24 amino acid residue peptide corresponding to the central part of the 'thrombospondin-repeat' motif of the human serum protein properdin was synthesized. The peptide, termed 'peptide P', contains three tryptophans near the N-terminus and an arginine cluster close to the C-terminus. Its sequence closely matches a consensus sequence which has been claimed to characterize a sulfatide binding motif. Membrane binding of peptide P was analyzed using changes in its tryptophan emission upon adding small unilamellar vesicles. The peptide bound to the membranes in a way suggesting simple water/membrane partitioning. Analysis of electrostatic effects at different ionic strengths indicated small electrostatic contributions upon interaction with zwitterionic lipid, despite the large charge number (z = +4) of the peptide. Membrane affinity was increased by one order of magnitude if the bilayers contained 20% of negatively charged lipid. No difference could be detected whether the charged lipid was sulfatide or phosphatidylglycerol. Strong and rapid vesicle aggregation was evident as the peptide associated with the negatively charged vesicles. In addition, a fluorescent energy transfer assay with vesicles and internal total reflection fluorescence microscopy on supported bilayers were used to study membrane interaction of whole human properdin. No sulfatide specificity could be detected.

Binding of proteins to specific target sites in membranes measured by total internal reflection fluorescence microscopy.

A new quantitative technique for measuring the binding of proteins to membranes is described. The method is based on a combination of total internal reflection fluorescence microscopy and the preparation of supported planar bilayers. Specific and reversible binding of a fluorescence-labeled monoclonal antibody to lipid haptens that were embedded in supported bilayers has been measured by this technique and compared to binding experiments that were conducted on membrane vesicles in solution. Equilibrium binding constants and kinetic parameters have been determined and used to expand the picture of the antibody-lipid hapten reaction. Estimates demonstrate that this technique is capable of measuring a broad range of binding constants (down to about 10(4) M-1) using only small amounts of ligand and receptor.

Fluorescence lifetime distributions of diphenylhexatriene-labeled phosphatidylcholine as a tool for the study of phospholipid-cholesterol interactions.

Fluorescence lifetimes of 1-palmitoyl-2-diphenylhexatrienylpro-pionyl-phosphatidylc hol ine in vesicles of palmitoyloleoyl phosphatidylcholine (POPC) (1:300, mol/mol) in the liquid crystalline state were determined by multifrequency phase fluorometry. On the basis of statistic criteria (chi 2red) the measured phase angles and demodulation factors were equally well fitted to unimodal Lorentzian, Gaussian, or uniform lifetime distributions. No improvement in chi 2red could be observed if the experimental data were fitted to bimodal Lorentzian distributions or a double exponential decay. The unimodal Lorentzian lifetime distribution was characterized by a lifetime center of 6.87 ns and a full width at half maximum of 0.57 ns. Increasing amounts of cholesterol in the phospholipid vesicles (0-50 mol% relative to POPC) led to a slight increase of the lifetime center (7.58 ns at 50 mol% sterol) and reduced significantly the distributional width (0.14 ns at 50 mol% sterol). Lifetime distributions of POPC-cholesterol mixtures containing greater than 20 mol% sterol were within the resolution limit and could not be distinguished from monoexponential decays on the basis of chi 2red. Cholesterol stabilizes and rigidifies phospholipid bilayers in the fluid state. Considering its effect on lifetime distributions of fluorescent phospholipids it may also act as a membrane homogenizer.

Influence of plasmalogen deficiency on membrane fluidity of human skin fibroblasts: a fluorescence anisotropy study.

The influence of plasmalogen deficiency on membrane lipid mobility was determined by measuring fluorescence anisotropy of trimethylammoniumdiphenylhexatriene (TMA-DPH) and diphenylhexatrienylpropanoylhydrazylstachyose (glyco-DPH) inserted in the plasma membranes of human skin fibroblasts deficient in plasmalogens. The cells used were from patients affected with cerebrohepatorenal (Zellweger) syndrome (CHRS) or rhizomelic chondrodysplasia punctata. Their plasmalogen content (0-5% of total phospholipid) is significantly reduced compared with that of control cells from healthy donors (13-15% of total phospholipid) or of CHRS fibroblasts supplemented with the plasmalogen precursor, hexadecylglycerol. Plasmalogen-deficient cells consistently showed lower fluorescence anisotropies of membrane-bound DPH fluorophores corresponding to higher membrane lipid mobilities as compared to controls. However, very similar lipid mobilities were found for sonicated aqueous dispersions of phospholipids extracted either from CHRS or control cells. Therefore, the differences observed with living cells are not due to differences in the overall physical properties of the membrane lipid constituents. Other phenomena such as lipid asymmetry and/or plasmalogen-protein interactions may be responsible for the effects observed in the biomembranes.

Behavioral self-help materials as an adjunct to nicotine gum.

The present study assessed the incremental effectiveness of behavioral self-help materials specifically written to accompany nicotine gum. Subjects (187 women and 117 men) were randomly assigned either behavioral self-help booklets or a factual information pamphlet. All subjects received prescriptions for Nicorette from study physicians. Contrary to prediction, subjects provided self-help booklets fared no better than did subjects provided comparison pamphlets. Overall abstinence levels were encouraging, however. Nicotine gum users were far more likely than nonusers to maintain abstinence through 6-month follow-up. Perhaps self-help materials could be improved by condensing and simplifying content and by adopting a more attractive multicolored pictorial format.