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The thermotropic phase behavior of a series of 1,2-diacylphosphatidylcholines containing linear saturated acyl chains of 10-22 carbons was studied by differential scanning calorimetry. When fully hydrated and thoroughly equilibrated by prolonged incubation at appropriate low temperatures, all of the compounds studied form an apparently stable subgel phase (the Lc phase). The formation of the stable Lc phase is a complex process which apparently proceeds via a number of metastable intermediates after being nucleated by incubation at appropriate low temperatures. The process of Lc phase formation is subject to considerable hysteresis, and our observations indicate that the kinetic limitations become more severe as the length of the acyl chain increases. The kinetics of Lc phase formation also depend upon whether the acyl chains contain an odd or an even number of carbon atoms. The Lc phase is unstable at higher temperatures and upon heating converts to the so-called liquid-crystalline state (the L alpha phase). The conversion from the stable Lc to the L alpha phase can be a direct, albeit a multistage process, as observed with very short chain phosphatidylcholines, or one or more stable gel states may exist between the Lc and L alpha states. For the longer chain compounds, conversions from one stable gel phase to another become separated on the temperature scale, so that discrete subtransition, pretransition, and gel/liquid-crystalline phase transition events are observed.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the effects of cholesterol on the thermotropic phase behavior of aqueous dispersions of a homologous series of linear saturated phosphatidylcholines, using high-sensitivity differential scanning calorimetry and an experimental protocol which ensures that broad, low-enthalpy phase transitions are accurately monitored. We find that the incorporation of small amounts of cholesterol progressively decreases the temperature and the enthalpy, but not the cooperativity, of the pretransition of all phosphatidylcholines exhibiting such a pretransition and that the pretransition is completely abolished at cholesterol concentrations above 5 mol % in all cases. The incorporation of increasing quantities of cholesterol also alters the main or chain-melting phase transition of these phospholipid bilayers in both hydrocarbon chain length-dependent and hydrocarbon chain length-independent ways. At cholesterol concentrations of from 1 to 20-25 mol %, the DSC endotherms of all phosphatidylcholines studied consist of a superimposed sharp and broad component, the former ascribed to the melting of cholesterol-poor and the latter to the melting of the cholesterol-rich phosphatidylcholine domains. The temperature and cooperativity of the sharp component are reduced only slightly and in a chain length-independent manner with increasing cholesterol concentration, an effect we ascribe to the colligative effect of the presence of small quantities of cholesterol at the domain boundaries. Moreover, the enthalpy of the sharp component decreases and becomes zero at 20-25 mol % cholesterol for all of the phosphatidylcholines examined.(ABSTRACT TRUNCATED AT 250 WORDS)

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We have investigated the effects of cholesterol on the thermotropic phase behavior of annealed and unannealed aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) using high-sensitivity differential scanning calorimetry (DSC), concentrating particularly on the cholesterol concentration range from 0 to 20 mol%. We find that the incorporation of cholesterol into low-temperature annealed DPPC bilayers decreases the enthalpy of the subtransition without affecting the transition temperature, such that the subtransition is abolished by 20 mol% cholesterol. Similarly, the incorporation of cholesterol progressively decreases the temperature and enthalpy of the pretransition and abolishes it entirely at cholesterol concentrations above 5 mol%. The incorporation of increasing quantities of cholesterol also alters the main or chain-melting phase transition. At cholesterol concentrations of 2 to 20 mol% cholesterol, the DSC endotherm arising from the main transition consists of superimposed sharp and broad components, the former due to the melting of cholesterol-poor and the latter to the melting of the cholesterol-rich DPPC domains. The temperature and cooperativity of the sharp component decreases slightly with increasing cholesterol concentration whereas the enthalpy decreases markedly, becoming zero at 20-25 mol% cholesterol. In contrast, the temperature and enthalpy of the broad component increases, and the cooperativity decreases markedly over this same range of cholesterol concentrations. An apparent increase in cooperativity of the overall DPPC endotherm at 7 mol% cholesterol is shown to arise because of a convergence in the transition temperatures of the sharp and broad components of the DSC endotherms. Some of our experimental findings, particularly the absence of any evidence for the existence of a triple point near 7.5 mol% cholesterol, do not accord with a recently proposed DPPC/cholesterol phase diagram derived from DSC and 2H-NMR data (see Vist, M.R. and Davis, J.H. (1990) Biochemistry 29, 451-464). In addition, we examined the effect of cholesterol on phosphatidylcholines (PCs) of different chain lengths and confirm that a eutectic point does not exist for any of these PC/cholesterol mixtures. We then propose a new, more complete DPPC/cholesterol phase diagram based on our high-sensitivity DSC data as well as some recent spectroscopic data on PC/cholesterol mixtures and explore some of its biological implications.

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Gramicidin S (GS) is a cyclic decapeptide of primary structure [cyclo-(Val-Orn-Leu-D-Phe-Pro)(2)] secreted by Bacillus brevis. It is a powerful antimicrobial agent with potent cidal action on a wide variety of Gram-negative and Gram-positive bacteria as well as on several pathogenic fungi. Unfortunately, however, GS is rather non-specific in its actions and also exhibits a high hemolytic activity, limiting its use as an antibiotic to topical applications. In a wide variety of environments, the GS molecule exists as a very stable amphiphilic antiparallel beta-sheet structure with a polar and a non-polar surface. Moreover, the large number of structure-activity studies of GS analogs which have been carried out indicate that this 'sidedness' structure is required for its antimicrobial action. In this review, we summarize both published and unpublished biophysical studies of the interactions of GS with lipid bilayer model and with biological membranes. In general, these studies show that GS partitions strongly into liquid-crystalline lipid bilayers in both model and biological membranes, and seems to be located primarily in the glycerol backbone region below the polar headgroups and above the hydrocarbon chains. The presence of GS appears to perturb lipid packing in liquid-crystalline bilayers and GS can induce the formation of inverted cubic phases at lower temperatures in lipids capable of forming such phases at higher temperature in the absence of peptide. The presence of GS at lower concentrations also increases the permeability of model and biological membranes and at higher concentrations causes membrane destabilization. There is good evidence from studies of the interaction of GS with bacterial cells that the destruction of the integrity of the lipid bilayer of the inner membrane is the primary mode of the antimicrobial action of this peptide. The considerable lipid specificity of GS for binding to and destabilization of lipid bilayer model membranes indicates that the design of GS analogs with an improved antimicrobial potency and a markedly decreased toxicity for eukaryotic cell plasma membranes should be possible.

Previous vibrational spectroscopic studies of solid acyl-alkyl and diacyl phosphatidylcholines suggested that the sn1- and sn2-carbonyl stretching modes of 1,2-diacylglycerolipids have different absorption maxima. To address the assignment of sn1- and sn2-carbonyl stretching modes of hydrated 1,2-diacylglycerolipids, aqueous dispersions of 1-palmitoyl-2-hexadecyl phosphatidylcholine (PHPC), 1-hexadecyl-2-palmitoyl phosphatidylcholine (HPPC), 1,2-dipalmitoylphosphatidylcholine (DPPC), as well as hydrated samples of unlabeled, sn1-13C=O-labeled, sn2-13C=O-labeled, and doubly 13C=O-labeled dimyristoylphosphatidylcholine (DMPC) were examined by Fourier transform infrared spectroscopy. The ester carbonyl stretching (nu C=O) bands of HPPC and PHPC each exhibit maxima near 1726 cm-1 and appear to be a summation of three subcomponents with maxima near 1740 cm-1, 1725 and 1705-1711 cm-1. In contrast, the nu C=O band of DPPC exhibits its maximum near 1733 cm-1 and appears to be a summation of two components centered near 1742 and 1727 cm-1. Thus the ester carbonyl group of the acyl-alkyl PCs appears to reside in a more polar environment than the ester carbonyl groups of their diacyl analogue. This observation implies that the polar/apolar interfaces of hydrated bilayers formed by PHPC and by HPPC are significantly different from that of DPPC and raises the question of whether the acyl-alkyl PCs are suitable models of their diacyl analogue. The absorption maximum of the nu C=O band of the doubly 13C=O-labeled DMPC occurs near 1691 cm-1 and those of its subcomponents occur near 1699 and 1685 cm-1. These frequencies are consistent with a 12C=0/13C0 'isotopic shift' of 42-43cm-1. snl - and snY2-13C0O-labeled DMPC each exhibit well resolved 12C and 13C vc-0 bands with absorption maxima near 1734 and 1692 cm-1, respectively. With both specifically 13C=O-labeled lipids, the 12C and 13C vo bands each seem to be a summation of subcomponents with absorption maxima near 1742 and 1727 cm-1 (12C vc=o) and 1699 and 1685 cm-1 (13C VC_o),regardless of whether the 13C=O-labeled fatty acyl chain is esterified at the snl - or sn2- positions of the glycerol backbone.We conclude that in hydrated 1,2-diacyl PC bilayers, the patterns of infrared absorption exhibited by ester carbonyl groups located at the primary and secondary positions of the glycerol backbone are similar. Also, the resolvable subcomponents of their v0 bands are each a summation of comparable contributions from both ester carbonyl groups and therefore cannot be attributed to the inequivalent locations of the two ester carbonyl groups. This result differs from that of the vibrational spectroscopic studies alluded to above and raises the question of whether data obtained in studies of dry (or poorly hydrated) lipids are applicable to fully hydrated lipid bilayers. To address questions of why the results of the two studies differ, we have also examined the vc=o bands of solid samples of DPPC, HPPC, and PHPC. We find that the vc-0 bands of all solid lipids studied differ from those...