Behavior of cholesterol and spin-labeled cholestane in model bile systems studied by electron spin resonance and synchrotron x-ray.

The behavior of mixed bile salt micelles consisting of sodium taurocholate, egg phosphatidylcholine, and cholesterol has been studied by ESR spin labeling and synchrotron x-ray scattering. Consistent with published phase diagrams, pure and mixed bile salt micelles have a limited capacity to incorporate and, hence, solubilize cholesterol. Excess cholesterol crystallizes out, a process that is readily detected both by ESR spin labeling using 3-doxyl-5 alpha-cholestane as a probe for cholesterol and synchrotron x-ray scattering. Both methods yield entirely consistent results. The crystallization of cholesterol from mixed bile salt micelles is indicated by the appearance of a magnetically dilute powder spectrum that is readily detected by visual inspection of the ESR spectra. Both the absence of Heissenberg spin exchange and the observation of a magnetically dilute powder spectrum provide evidence for the spin label co-crystallizing with cholesterol. In mixed bile salt micelles containing egg phosphatidylcholine, the solubility of cholesterol is increased as detected by both methods. With increasing content of phosphatidylcholine and increasing mole ratio cholesterol/phosphatidylcholine, the anisotropy of motion of the spin probe increases. The spin label 3-doxyl-5 alpha-cholestane is a useful substitute for cholesterol provided that it is used in dilute mixtures with excess cholesterol: the cholesterol/spin label mole ratio in these mixtures should be greater than 100. Despite the structural similarity between the two compounds, there are still significant differences in their physico-chemical properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron spin resonance studies of membrane proteins in erythrocytes in myotonic muscular dystrophy.

A goat antibody produced against bovine adrenal ferredoxin has been employed to establish immunochemically the involvement of adrenal ferredoxin in the cholesterol side-chain cleavage reaction catalyzed by mammalian adrenal mitochondria. When added to preparations of bovine adrenocortical mitochondria, this antibody was found to inhibit the conversion of cholesterol to pregnenolone and progesterone, the 11β-hydroxylation of deoxycorticosterone and the NADPH-dependent reduction of cytochrome c. These observations demonstrate that, similar to the NADPH-cytochrome c reductase and steroid 11β-hydroxylase reactions, adrenal ferredoxin is also required for the oxidative cleavage of the cholesterol side-chain catalyzed by bovine adrenocortical mitochondria.The goat antibody to bovine adrenal ferredoxin was also found to interact with the comparable iron-sulfur proteins present in mitochondria prepared from sheep, rat, mouse, cat, dog, guinea pig, rabbit, and human adrenals. The interaction of the antibody with these iron-sulfur proteins resulted in the inhibition of both the cholesterol side-chain cleavage and NADPH-cytochrome c reductase activities catalyzed by these adrenal mitochondria. The NADH-dependent reduction of cytochrome c catalyzed by mammalian adrenal mitochondria was not inhibited by the goat antibody to adrenal ferredoxin. These results demonstrate the immunochemical similarity existing among mammalian adrenal ferredoxins and their involvement in the adrenal cholesterol side-chain cleavage reaction.     

Influence of calcium, cholesterol, and unsaturation on lecithin monolayers

Surface pressures and potentials of mixed monolayers of dicetyl phosphate-cholesterol, dipalmitoyl lecithin-cholesterol, egg lecithin-cholesterol, and phosphatidic acid-cholesterol were measured. The surface potential is shown to be a more reliable parameter for the study of interactions in monolayers than the surface pressure. Monolayers of dicetyl phosphate-cholesterol follow the additivity rule for area/molecule whereas lecithin-cholesterol monolayers deviate from it. The reverse is true for the additivity rule with regard to surface potential/molecule. Thus, the surface potential indicates that there is no interaction (or complex formation) between lecithin and cholesterol, but that there is ion-dipole interaction between dicetyl phosphate and cholesterol, as well as between phosphatidic acid and cholesterol. The apparent condensation of mixed monolayers of lecithin when cholesterol is added is explained by a consideration of molecular cavities or vacancies caused by thermal motion of the fatty acyl chains, the size of these cavities being influenced by the length and degree of saturation (especially the proportion of monounsaturation) of the fatty acyl chains and the extent of compression of the monolayer. The cholesterol molecules occupy these cavities and therefore cause no proportional increase in area/molecule in the mixed monolayers. Monolayers are liquefied by the presence of cholesterol as well as of unsaturated fatty acyl chains; in contrast, Ca(++)tends to solidify lecithin monolayers. The available evidence suggests that cholesterol can both impart fluidity to the monolayer and occupy the molecular cavities caused by the fatty acyl chains.     

The influence of cholesterol on molecular motion in egg lecithin bilayers--a variable-frequency electron spin resonance study of a cholestane spin probe

Electron spin resonance spectra at 9.5, 24. and 35 GHz were obtained for a cholestane spin probe in oriented multibilayers of egg lecithin of varying cholesterol content. In agreement with earlier studies, cholesterol induced a higher degree of spectral anisotropy in the multibilayers—the variation of the hyperfine separations with cholesterol content was in agreement with the model of Lapper et al. (Can. J. Biochem.50, 969 (1972)) where the amplitude of anisotropic probe motion decreased with increasing cholesterol content. Analysis of the electron spin resonance line shapes was done using the relatively simple modified Bloch equation approach, and correlation times for anisotropic probe motion were extracted from the spectra at three frequencies. The data demonstrate that increasing cholesterol content results in a decreased rate of anisotropic motion of the probe, providing further insight into the molecular mechanism of the condensing effect of cholesterol.     

Stability of mixed micellar bile models supersaturated with cholesterol.

The maximal equilibrium solubility of cholesterol in mixtures of phosphatidylcholine (PC)1 and bile salts depends on the cholesterol/PC ratio (Rc) and on the effective ratio (Re) between nonmonomeric bile salts and the sum (CT) of PC and cholesterol concentrations (Carey and Small, 1978; Lichtenberg et al., 1984). By contrast, the concentration of bile salts required for solubilization of liposomes made of PC and cholesterol does not depend on Rc (Lichtenberg et al., 1984 and 1988). Thus, for Rc greater than 0.4, solubilization of the PC-cholesterol liposomes yields PC-cholesterol-bile salts mixed micellar systems which are supersaturated with cholesterol. In these metastable systems, the mixed micelles spontaneously undergo partial revesiculation followed by crystallization of cholesterol. The rate of the latter processes depends upon Rc, Re, and CT. For any given Rc and Re, the rate of revesiculation increases dramatically with increasing the lipid concentration CT, reflecting the involvement of many mixed micelles in the formation of each vesicle. The rate also increases, for any given CT and Re, upon increasing the cholesterol to PC ratio, Rc, probably due to the increasing degree of supersaturation. Increasing the cholate to lipid effective ratio, Re, by elevation of cholate concentration at constant Rc and CT has a complex effect on the rate of the revesiculation process. As expected, cholate concentration higher than that required for complete solubilization at equilibrium yields stable mixed micellar systems which do not undergo revesiculation, but for lower cholate concentrations decreasing the degree of supersaturation (by increasing [cholate]) results in faster revesiculation. We interpret these results in terms of the structure of the mixed micelles; micelles with two or more PC molecules per one molecule of cholesterol are relatively stable but increasing the bile salt concentration may cause dissociation of such 1:2 cholesterol:PC complexes, hence reducing the stability of the mixed micellar dispersions. The instability of PC-cholesterol-cholate mixed systems with intermediary range of cholate to lipids ratio may be significant to gallbladder stone formation as: (a) biliary bile contains PC-cholesterol vesicles which may be, at least partially, solubilized by bile salts during the process of bile concentration in the gallbladder, resulting in mixtures similar to our model systems; and (b) the bile composition of cholesterol gallstone patients is within an intermediary range of bile salts to lipids ratio.     

Enzyme-catalyzed oxidation of cholesterol in mixed phospholipid monolayers reveals the stoichiometry at which free cholesterol clusters disappear.

In this study, we have used cholesterol oxidase as a probe to study cholesterol/phospholipid interactions in mixed monolayers at the air/water interface. Mixed monolayers, containing a single phospholipid class and cholesterol at differing cholesterol/phospholipid molar ratios, were exposed to cholesterol oxidase at a lateral surface pressure of 20 mN/m (at 22 degrees C). At equimolar ratios of cholesterol to phospholipid, the average rate of cholesterol oxidation was fastest in unsaturated phosphatidylcholine mixed monolayers (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and egg yolk phosphatidylcholine), intermediate in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and slowest in sphingomyelin monolayers (egg yolk or bovine brain sphingomyelin). The average oxidation rate in mixed monolayers was not exclusively a function of monolayer packing density, since egg yolk and bovine brain sphingomyelin mixed monolayers occupied similar mean molecular areas even though the measured average oxidation rate was different with these two phospholipids. This suggests that the phospholipid acyl chain composition influenced the oxidation rate. The importance of the phospholipid acyl chain length on influencing the average oxidation rate was further examined in defined phosphatidylcholine mixed monolayers. The average oxidation rate decreased linearly with increasing acyl chain lengths (from di-8:0 to di-18:0). When the average oxidation rate was examined as a function of the cholesterol to phospholipid (C/PL) molar ratio in the monolayer, the otherwise linear function displayed a clear break at a 1:1 stoichiometry with phosphatidylcholine mixed monolayers, and at a 2:1 C/PL stoichiometry with sphingomyelin mixed monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular packing in steroid-lecithin monolayers part III: Mixed films of 3-doxyl cholestane and 3-doxyl-17-hydroxyl-androstane with dipalmitoylphosphatidylcholine

A comparison is made of the condensing capability of 3-doxyl cholestane and 3-doxyl-17-hydroxyl-androstane in mixed monomolecular film systems with dipalmitoyl phosphatidylcholine. 3-doxyl cholestane closely resembled cholesterol in its mixed film behavior while 3-doxyl-17-hydroxyl-androstane did not. This was taken to indicate that the ability of cholesterol to condense expanded host lipids results primarily from hydrophobic interactions and does not involve specific direct polar interactions.A general model is set up based on steroid tilt angle in the mixed film, which indicates that the greater the tilt angle, the more ineffective the condensing capability. A quantitative comparison is made of these properties for cholesterol, cholestanol, epicholesterol, cholestone, 3-doxyl cholestane and coprostanol.     

Effects of lutein and cholesterol on alkyl chain bending in lipid bilayers: a pulse electron spin resonance spin labeling study.

A short pulse saturation recovery electron spin resonance technique has been used to study the effects of polar carotenoid-lutein and cholesterol on interactions of 14N:15N stearic acid spin-label pairs in fluid-phase phosphatidylcholine (PC) membranes. Bimolecular collisions for pairs consisting of various combinations of [14N]-16-, [14N]-10-, [14N]-7-, or [14N]-5-doxylstearate and [15N]-16-doxylstearate in dimyristoyl-PC (DMPC) or egg yolk PC (EYPC) membranes were measured at 27 degrees C. In the absence and presence of lutein or cholesterol for both lipid systems, the collision rates were ordered as 16:5 < 16:7 < 16:10 < 16:16. For all spin-label pairs studied, interaction frequencies were greater in DMPC than in EYPC. Polar carotenoid-lutein reduces the collision frequency for all spin-label pairs, whereas cholesterol reduces the collision frequency for 16:5 and 16:7 pairs and increases the collision frequency in the membrane center for 16:10 and 16:16 pairs. The presence of unsaturated alkyl chains greatly reduces the effect of lutein but magnifies the effect of cholesterol in the membrane center. The observed differences in the effects of these modifiers on alkyl chain bending result from differences in the structure of cholesterol and polar carotenoid and from their different localization within the lipid bilayer membrane. These studies further confirm the occurrence of vertical fluctuations of alkyl chain ends toward the bilayer surface.     

Structural isomers of tetradecenol discriminate between the lipid fluidity and phase transition theories of anesthesia

The long-chain unsaturated alcohols, cis- and trans-9,10-tetradecenol were found to be equipotent both as general anesthetics and local anesthetics. Their effects on the phase transition temperature of dipalmitoylphosphatidylcholine/water dispersions were measured spectroscopically by partitioning of the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl. Also spectroscopic order parameters were obtained from spin labeled egg lecithin/cholesterol vesicles, in the absence and presence of the two alcohols. Whereas the egg lecithin/cholesterol membranes were fluidised by both alcohols to a similar extent, cis-tetradecenol lowered the transition temperature of dipalmitoylphosphatidylcholine, while the trans-isomer elevated the transition temperature. These results are thus inconsistent with the phase transition model of anesthetic action. The possible application of these alcohols to diagnose the origin of discontinuities in Arrhenius plots is discussed.     

Cholesterol displacement from membrane phospholipids by hexadecanol

Adding cholesterol to monolayers of certain phospholipids drives the separation of liquid-ordered from liquid-disordered domains. The ordered phases appear to contain stoichiometric complexes of cholesterol and phospholipid. Furthermore, it has been suggested that the cholesterol in these complexes has a low chemical activity compared to that of the free sterol; i.e., that in excess of the phospholipid binding capacity. We have now tested the hypothesis that the membrane intercalator 1-hexadecanol (HD) similarly associates with phospholipids and thereby displaces the complexed cholesterol. HD introduced into monolayers of pure dimyristoylphosphatidylcholine generated highly condensed (stable and solid) domains. In contrast, the phase behavior of mixed monolayers of the phospholipid, sterol, and alcohol suggested that HD could substitute for cholesterol mole for mole in promoting liquid-ordered domains. We also found that the transfer of cholesterol from mixed monolayers to aqueous cyclodextrin was greatly stimulated by the presence of HD, but only at levels sufficient to competitively displace the sterol from the phospholipid. This enhanced efflux was interpreted to reflect an increase in uncomplexed cholesterol. We conclude that HD forms complexes with dimyristoylphosphatidylcholine that are surprisingly similar to those of cholesterol. HD competitively displaces cholesterol from the phospholipid and thereby increases its chemical activity.     

Plasmodium berghei: Electron spin resonance and lipid analysis of infected mouse erythrocyte membranes

Red blood cells from mice infected with Plasmodium berghei and from uninfected mice were labeled with stable, free radical derivatives of stearic acid. Electron spin resonance spectra of these samples showed that the degree of molecular order in these membranes decreased, and the rate of motion of the probe increased, with increasing levels of parasitemia. Parasitemia increased the ratio of unsaturated to saturated 18-carbon fatty acids, and decreased the percentage of arachidonic acid and of cholesterol. The effects of parasitemia on the membrane properties correlated with decreases in cholesterol/fatty acid ratios.     

Phase behavior of stratum corneum lipids in mixed Langmuir-Blodgett monolayers.

The lipids found in the bilayers of the stratum corneum fulfill the vital barrier role of mammalian bodies. The main classes of lipids found in stratum corneum are ceramides, cholesterol, and free fatty acids. For an investigation of their phase behavior, mixed Langmuir-Blodgett monolayers of these lipids were prepared. Atomic force microscopy was used to investigate the structure of the monolayers as a function of the monolayer composition. Three different types of ceramide were used: ceramide extracted from pigskin, a commercially available ceramide with several fatty acid chain lengths, and two synthetic ceramides that have only one fatty acid chain length. In pigskin ceramide-cholesterol mixed monolayers phase separation was observed. This phase separation was also found for the commercially available type III Sigma ceramide-cholesterol mixed monolayers with molar ratios ranging from 1:0.1 to 1:1. These monolayers separated into two phases, one composed of the long fatty acid chain fraction of Sigma ceramide III and the other of the short fatty acid chain fraction of Sigma ceramide III mixed with cholesterol. Mixtures with a higher cholesterol content consisted of only one phase. These observations were confirmed by the results obtained with synthetic ceramides, which have only one fatty acid chain length. The synthetic ceramide with a palmitic acid (16:0) chain mixed with cholesterol, and the synthetic ceramide with a lignoceric acid (24:0) chain did not. Free fatty acids showed a preference to mix with one of these phases, depending on their fatty acid chain lengths. The results of this investigation suggest that the model system used in this study is in good agreement with those of other studies concerning the phase behavior of the stratum corneum lipids. By varying the composition of the monolayers one can study the role of each lipid class in detail.     

Interactions of the cholesterol side-chain with egg lecithin a spin label study

The effect in egg lecithin liposomes of cholesterol and cholesterol analogues with side-chains of reduced length on the order parameters of two steroid spin labels has been studied. Analogoues with side-chains shorter than cholesterol by more than three carbons cause significantly less ordering than cholesterol. Liposomes containing a cholesterol analogue in which the side-chain is absent cause very little increase in the ordering of a new sterol spin label in which the nitroxide is incorporated into the side-chain. The results suggest that the sterol side-chain exerts a great influence on membrane rigidity within its immediate environment.     

Studies on mixed monolayers of phospholipids and fusogenic lipids.

1. The behaviour of mixed monolayers of 14 different lipids with preparations of erythrocyte lipids, purified natural and synthetic phospholipids, cholesterol and galactosylceramide was investigated. 2. The mean areas occupied per molecule in mixed films containing lipids that are fusogenic for hen erythrocytes were compared with those for corresponding films containing lipids that are inactive as fusogens. 3. Fusogenic lipids were found to exhibit interactions, which were not shown by non-fusogenic lipids, in mixed monolayers with several species of phospholipid, particularly those containing a choline head group. 4. Heterogeneity in the hydrophobic chains of phosphatidylcholine, their degree of unsaturation and the presence of cholesterol had little effect on the interaction of phosphatidylcholine with fusogenic lipids. 5. Fusogenic lipids showed little specific interaction with natural or synthetic preparations of phosphatidylethanolamine. 6. The possible significance of these observations in relation to the action of fusogenic lipids on biological membranes is discussed in the light of the asymmetrical distribution of phospholipids in erythrocyte membranes.     

Abrupt modifications of phospholipid bilayer properties at critical cholesterol concentrations.

The fluorescence generalized polarization (GP) of 2-dimethylamino-6-lauroylnaphthalene (Laurdan) reveals different effects of cholesterol on the phase behavior of phospholipid bilayers. Phospholipid vesicles composed of gel, liquid-crystalline, and coexisting domains of the two phases have been studied at temperatures from 1 to 65 degrees C, without cholesterol and with cholesterol concentrations of 3-50 mol %. Laurdan GP measurements show the general effect of cholesterol of increasing the molecular dynamics of the gel and of decreasing the molecular dynamics of the liquid-crystalline phase. In the liquid-crystalline phase, the increased order yields Laurdan GP values close to those obtained in the gel phase. At cholesterol concentrations > 15 mol % a phase transition cannot be detected. Using the wavelength dependence of the excitation and emission GP spectra we determine that differences between the two phospholipid phases cannot be detected. In particular, in vesicles composed of coexisting gel and liquid-crystalline phases the GP wavelength dependence characteristic of coexisting domains cannot be observed at cholesterol concentrations > or = 15 mol %. Cholesterol causes the decrease in both the polarity and the dipolar relaxation effects on the neighborhood of the fluorescent naphthalene moiety of Laurdan. Probably because of a cholesterol-induced increase in the bilayer packing, these effects do not occur continuously with the increase of cholesterol concentration in the bilayer. Cholesterol concentrations inducing higher Laurdan GP values have been determined at about 5, 10, 15, 30, and 45 mol % with respect to phospholipids. We propose that the formation of ordered molecular microdomains at critical cholesterol concentrations can explain the occurrence of the observed discontinuities.     

A structurally relevant coarse-grained model for cholesterol

Detailed atomistic computer simulations are now widely used to study biological membranes, including increasingly mixed lipid systems that involve, for example, cholesterol, which is a key membrane lipid. Typically, simulations of these systems start from a preassembled bilayer because the timescale on which self-assembly occurs in mixed lipid systems is beyond the practical abilities of fully atomistic simulations. To overcome this limitation and study bilayer self-assembly, coarse-grained models have been developed. Although there are several coarse-grained models for cholesterol reported in the literature, these generally fail to account explicitly for the unique molecular features of cholesterol that relate to its function and role as a membrane lipid. In this work, we propose a new coarse-grained model for cholesterol that retains the molecule's unique features and, as a result, can be used to study crystalline structures of cholesterol. In the development of the model, two levels of coarse-graining are explored and the importance of retaining key molecular features in the coarse-grained model that are relevant to structural properties is investigated.     

Dynamic imaging of lateral diffusion by electron spin resonance and study of rotational dynamics in model membranes. Effect of cholesterol.

The effects of cholesterol on the dynamics and the structural properties of two different spin probes, the sterol type CSL and the phospholipid type 16-PC, in POPC/cholesterol oriented multilayer model membranes were examined. Our results are consistent with a nonideal solution containing cholesterol-rich clusters created by the self association of cholesterol in POPC model membranes. The lateral diffusion coefficient D of the spin probes was measured over the temperature range of 15 to 60 degrees C and over the concentration range of 0 to 30 mol% of cholesterol in the model membrane by the electron spin resonance (ESR) imaging method. The rotational diffusion coefficients (including R perpendicular) and the order parameter S were determined utilizing a nonlinear least square ESR spectral simulation method. D, R perpendicular and S of CSL deviate considerably from linear dependence on mole percent cholesterol. The D of CSL was decreased by a factor of four at 15 degrees C and a factor of two at 60 degrees C for concentrations of cholesterol over 10 mol %, whereas those of 16-PC were hardly affected. Cholesterol decreased R perpendicular by a factor of 10 at 30 mol % of cholesterol, but it increased slightly that of 16-PC. A significant increase of S for CSL due to the presence of cholesterol was observed. It is shown how the difference in variation of S for CSL vs. 16-PC with composition may be interpreted in terms of their respective activity coefficients, and how a single universal linear relation is obtained for the S of both probes in terms of a scaled temperature. Simple but general correlations of D and of R perpendicular with S were also found, which aid in the interpretation of these diffusion coefficients.     

Comparison of the interaction of tomatine with mixed monolayers containing phospholipid, egg sphingomyelin, and sterols

The interaction of the glycoalkaloid tomatine with monolayers of a phospholipid (dimyristoylphosphatidylcholine, DMPC), and sphingolipid (egg sphingomyelin), and cholesterol is compared. Using measurements of the surface pressure response as a function of the subphase concentration of tomatine, interfacial binding constants are estimated for mixed monolayers of DMPC and cholesterol and for those of egg sphingomyelin and cholesterol of mole ratio 7:3. The binding constants obtained suggest a stronger interaction of tomatine with DMPC and cholesterol mixed monolayers, reflecting easier displacement of cholesterol from its interaction with DMPC than from its interaction with egg sphingomyelin. Mixtures of tomatine and cholesterol are found to spread directly at the water-air interface and form stable monolayers, suggesting that cholesterol holds tomatine at the interface despite the absence of observed monolayer behavior for tomatine alone. The interaction of tomatine with DMPC and cholesterol monolayers is found to exhibit a pH dependence in agreement with previously reported results for its interaction with liposomes; in particular, the interaction is much less at pH 5 than at pH 7 or pH 9. It is found that while tomatine interacts strongly with monolayers containing sitosterol, it does not interact with monolayers containing sitosterol glucoside. The response of monolayers of varying composition of DMPC and cholesterol to tomatine is also examined. Brewster angle microscopy (BAM) reveals further evidence for formation of suspected islands of tomatine + cholesterol complexes upon interaction with mixed monolayers of lipid and sterol.     

The chemical constitution of carcinolipin

1. The neutral portion of the molecule of carcinolipin was found to be cholesterol by comparison of mixed melting points with cholesterol, its dibromide and its acetate. 2. The fatty acid present in carcinolipin was subjected to oxidative degradation by chromic acid and permanganate. Butan-2-one was the main neutral degradation product resulting from both these procedures. A mixture of dibasic acids was obtained after the oxidation with chromic acid. Permanganate oxidation yielded a complete homologous series of branched-chain C(5)-C(17) fatty acids. 3. The mass spectrum of the acid was characteristic for a saturated C(17) acid. The alcohol prepared by lithium aluminium hydride reduction of the original acid showed a mass spectrum typical for an anteiso compound. 4. Comparison of mixed melting points, gas-liquid-chromatographic behaviour and mass spectra of the fatty acid isolated from carcinolipin with an authentic sample of 14-methylhexadecanoic acid demonstrated the identity of these compounds. Cholesterol esters synthesized from authentic cholesterol and the fatty acid isolated from carcinolipin or synthetic 14-methylhexadecanoic acid showed an identical stimulating effect on the incorporation of labelled algal-protein hydrolysate into rat liver transfer RNA in vitro. 5. Mass spectra, results of oxidative degradations and comparisons with an authentic sample, as well as biological activity of the synthetic cholesterol 14-methylhexadecanoate, provided good evidence that carcinolipin is cholesterol (+)-14-methylhexadecanoate.     

Cholestane spin label study of filipin action on lipid planar multibilayers.

EPR spectra of a cholestane probe dissolved in egg yolk lecithin and lecithin-cholesterol planar multibilayers were observed as a function of the filipin dose. The probe is structurally similar to cholesterol; its normal position when dissolved is with the long axis approximately along the bilayer normal. Both cholesterol-containing and cholesterol-free samples showed spectral components characteristic of bilayer fragmentation (tilted domains) which increased with dose. Furthermore, the cholesterol-free spectra indicated that some of the probe was frozen with the long molecular axis perpendicular to the slide normal. The frozen spectral component increased with dose. Spectra from a fatty acid probe did not have this feature. We interpret this as due to probe complexed with filipin (in place of cholesterol) in accordance with the filipin-cholesterol aggregate model of deKruijff and Demel. An ultraviolet study of filipin-probe interaction indicates that the probe is capable of complexing in just such a manner but has less affinity for the drug than cholesterol. Spectra from the cholesttane probe in liposomes were also observed.