The behaviors of two chemically well-defined sphingolipids, N-palmitoyl-sphingomyelin (C16:0-SM) and the corresponding ceramide (C16:0-Cer), in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) matrix were compared. Minor attenuation of lateral diffusion upon increasing the mole fraction of C16:0-SM (XSM, up to 0.25) was indicated by the slight decrement in the excimer/monomer intensity ratio (Ie/Im) for a trace amount (mole fraction X = 0.01) of a pyrene-labeled ceramide analogue (N-[(pyren)-1-yl]decanoyl-sphingosine, PDCer) in keeping with the miscibility of C16:0-SM in POPC. Increasing membrane order was revealed by the augmented polarization P for diphenylhexatriene (DPH). In contrast, when C16:0-Cer was substituted for C16:0-SM an approximately 1.6-fold increase in Ie/Im for PDCer was evident upon increasing Xcer, with parallel increment in DPH polarization. In agreement with our recent data on natural ceramides in dimyristoylphosphatidylcholine (DMPC) bilayers [Holopainen et al. (1997) Chem. Phys. Lipids 88, 1-13], we conclude that C16:0-Cer becomes enriched into microdomains in the fluid POPC membrane. Interestingly, enhanced formation of microdomains by ceramide was observed when the total sphingolipid content in tertiary alloys with POPC was maintained constant (Xcer + XSM = 0.25) and the SM/Cer stoichiometry was varied. Finally, when ceramide was generated enzymatically in POPC/C16:0-SM (3:1, molar fraction) LUVs by sphingomyelinase (SMase, Bacillus cereus), maximally approximately 85% of hydrolysis of sphingomyelin was measured within <3 min at 30 degreesC. The formation of ceramide was accompanied by a closely parallel increase in DPH polarization. There was also an increase in Ie/Im for PDCer; however, these changes in Ie/Im were significantly slower, requiring approximately 105 min to reach a steady state. These data show that the rapid enzymatic formation of ceramide under these conditions is followed by much slower reorganization process, resulting in the formation of microdomains enriched in this lipid.

Four new hybrid (bolaphile/amphiphile) ion-pairs were synthesized. Electron microscopy indicated that each of these forms bilayer membranes upon dispersion in aqueous media. Membrane properties have also been examined by differential scanning calorimetry, microcalorimetry, temperature-dependent fluorescence anisotropy measurements, and UV-vis spectroscopy. The T m values for the vesicular 1, 2, 3, 4, and 5 were 38, 12, 85, 31.3, and 41.6 °C, respectively. Interestingly the T m values for 1 and 3 were found to depend on their concentration. The entrapment of small solute and the release capability have also been examined to demonstrate that these bilayers form enclosed vesicles. X-ray diffraction of the cast films has been performed to understand the nature and the thickness of these membrane organizations. The membrane widths ranged from 33 to 47 Å. Finally, the above observations have been analyzed in light of the results obtained from molecular modeling studies. Thus we have demonstrated that membrane properties can be modulated by simple structural changes at the amphiphile level. It was shown that by judicious incorporation of central, isomeric, disubstituted aromatic units as structural anchors into different bolaphiles, one can modulate the properties of the resulting vesicles.

Membrane association and detachment of cytochrome c (cyt c) in millisecond to second time domain were investigated by stopped-flow fluorescence spectroscopy monitoring the efficiency of energy transfer from a pyrene-fatty acid containing phospholipid derivative, 1-palmitoyl-2-[10-(pyren-1-yl)-decanoyl]-sn-glycero-3-phosphoglyce rol (PPDPG, mole fraction X = 0.01) to the heme of the cyt c. Large unilamellar liposomes composed of egg phosphatidylcholine (eggPC) with varying content of the acidic phospholipid phosphatidylglycerol (eggPG) were employed. Unexpectedly, the rate of binding of cyt c to membranes was attenuated upon increasing the mole fraction of the acidic phospholipid (XPG). For example, at 50 microM phospholipid and 5 microM cyt c, when XPG was increased from 0.20 to 0.40 the half-time for the single-exponential decay in fluorescence increased from 4.7 to 8.6 ms. A similar observation was made for the membrane binding of another cationic protein, histone H1. We suggest that the formation of cooperative hydrogen-bonded networks by deprotonated and protonated PG in the vesicle surface retards the binding of cyt c to the liposome surface. However, once formed, the complex of cyt c with acidic phospholipids is stabilized by increasing XPG. Accordingly, significantly prolonged half-times of dissociation of cyt c from liposomes by NaCl, ATP, and different cationic proteins are measured upon increasing XPG. Differences between the latter cationic membrane binding ligands most likely reflect the varying relative contributions of hydrophobicity and Coulombic forces to their attachment to liposomes. Our data on the release and binding of cyt c to liposomes as a function of XPG and in the presence of ATP also provide the first direct experimental evidence for multiple lipid binding sites in cyt c.

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