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To better understand the interactions of saturated ceramides with unsaturated glycerophospholipids in bilayer membranes, we measured how palmitoyl ceramide (PCer) and dihydroceramide (dihydro-PCer, lacking the trans 4 double bond of the sphingoid base of ceramide) can interact with phosphatidylcholines (PCs) with palmitic acid in the sn-1 position and increasingly unsaturated acyl chains in the sn-2 position. The PCs were 16:0/18:1 (POPC), 16:0/18:2 (PLPC), 16:0/20:4 (PAPC), and 16:0(22:6 (PDPC). We also included di-18:1-PC (DOPC) to compare it with POPC. Because the ceramides were expected to segregate laterally to an ordered ceramide-rich phase, we determined the formation of the ordered phase using lifetime analysis of trans-parinaric acid (tPA) fluorescence. The presence of ordered domains, as indicated by tPA lifetime analysis, was verified by an analysis of tPA anisotropy as a function of temperature. The interaction between PCer and POPC was clearly more favored than interactions with DOPC, as seen from a more thermostable gel phase in POPC than in DOPC at equal ceramide content. The concentration needed for PCer gel phase formation was also lower in POPC than in the DOPC bilayers, suggesting that POPC had better miscibility in the ordered phase. The increased unsaturation of the sn-2 acyl chains of the PCs had more clear effects of dihydro-PCer segregation than on PCer segregation, and the dihydro-PCer gel phase became more thermostable as the unsaturation in the PC increased. We conclude that the interactions between ceramides and PCs were complex and affected both by the trans 4 double bond of PCer by the palmitoyl acyl in the sn-1 position and by the overall degree of unsaturation of the sn-2 acyl chain of the PCs. [ABSTRACT FROM AUTHOR]

Ceramide is a bioactive lipid with important roles in several biological processes including cell proliferation and apoptosis. Although 3-ketoceramides that contain a keto group in place of the 3-OH group of ceramide occur naturally, ceramide derivatives oxidized at the primary 1-OH group have not been identified to date. To evaluate how the oxidative state of the 1-OH group affects the physical properties of membranes, we prepared novel ceramide derivatives in which the 1-OH group was oxidized to a carboxylic acid (PCerCOOH) or methylester (PCerCOOMe) and examined the rigidity of their monolayers and the formation of gel domains in palmitoyloleoylphosphatidylcholine (POPC) or sphingomyelin (SM) bilayers. As a result, PCerCOOH and PCerCOOMe exhibited membrane properties similar to those of native ceramide, although the deprotonated form of PCerCOOH, PCerCOO-, exhibited markedly lower rigidity and higher miscibility with POPC and SM. This was attributed to the electrostatic repulsion of the negative charge, which hampered the formation of the ceramide-enriched gel domain. The similarities in the properties of PCerCOOMe and ceramide revealed the potential to introduce various functional groups onto PCerCOOH via ester or amide linkages; therefore, these derivatives will also provide a new strategy for developing molecular probes, such as fluorescent ceramides, and inhibitors of ceramide-related enzymes. [ABSTRACT FROM AUTHOR]

In this study, we examined the influence of bilayer thickness on the activity of the actinoporin toxins sticholysin I and II (StnI and StnII) at 25 °C. Bilayer thickness was varied using dimonounsaturated phosphatidylcholine (PC) analogues (with 14:1, 16:1, 18:1, 20:1, and 22:1 acyl chains). In addition, N-14:0-sphingomyelin (SM) was always included because StnI and StnII are SM specific. Cholesterol was also incorporated as indicated. In cholesterol-free large unilamellar vesicles (LUVs) the PC:SM molar ratio was 4:1, and when cholesterol was included, the complete molar ratio was 4:1:0.5 (PC:SM:cholesterol, respectively). Stn toxins promote bilayer leakage through pores formed by oligomerized toxin monomers. Initial calcein leakage was moderately dependent on bilayer PC acyl chain length (and thus bilayer thickness), with higher rates observed with di-16:1 and di-18:1 PC bilayers. In the presence of cholesterol, the maximum rates of calcein leakage were observed in di-14:1 and di-16:1 PC bilayers. Using isothermal titration calorimetry to study the Stn-LUV interaction, we observed that the bilayer affinity constant (Ka) peaked with LUVs containing di-18:1 PC, and was lower in shorter and longer PC acyl chain bilayers. The presence of cholesterol increased the binding affinity approximately 30-fold at the optimal bilayer thickness (di-18:1-PC). We conclude that bilayer thickness affects both functional and conformational aspects of Stn membrane binding and pore formation. Moreover, the length of the actinoporins' N-terminal α-helix, which penetrates the membrane to form a functional pore, appears to be optimal for the membrane thickness represented by di-18:1 PC. [ABSTRACT FROM AUTHOR]