Substrate Uptake and Subcellular Compartmentation of Anoxic Cholesterol Catabolism in Sterolibacterium denitrificans

Cholesterol catabolism by actinobacteria has been extensively studied. In contrast, the uptake and catabolism of cholesterol by Gram-negative species are poorly understood. Here, we investigated microbial cholesterol catabolism at the subcellular level. ¹³C metabolomic analysis revealed that anaerobically grown Sterolibacterium denitrificans, a β-proteobacterium, adopts an oxygenase-independent pathway to degrade cholesterol. S. denitrificans cells did not produce biosurfactants upon growth on cholesterol and exhibited high cell surface hydrophobicity. Moreover, S. denitrificans did not produce extracellular catabolic enzymes to transform cholesterol. Accordingly, S. denitrificans accessed cholesterol by direction adhesion. Cholesterol is imported through the outer membrane via a putative FadL-like transport system, which is induced by neutral sterols. The outer membrane steroid transporter is able to selectively import various C₂₇ sterols into the periplasm. S. denitrificans spheroplasts exhibited a significantly higher efficiency in cholest-4-en-3-one-26-oic acid uptake than in cholesterol uptake. We separated S. denitrificans proteins into four fractions, namely the outer membrane, periplasm, inner membrane, and cytoplasm, and we observed the individual catabolic reactions within them. Our data indicated that, in the periplasm, various periplasmic and peripheral membrane enzymes transform cholesterol into cholest-4-en-3-one-26-oic acid. The C₂₇ acidic steroid is then transported into the cytoplasm, in which side-chain degradation and the subsequent sterane cleavage occur. This study sheds light into microbial cholesterol metabolism under anoxic conditions.     

Structural requirements of cholesterol for binding to Vibrio cholerae hemolysin

Cholesterol is necessary for the conversion of Vibrio cholerae hemolysin (VCH) monomers into oligomers in liposome membranes. Using different sterols, we determined the stereochemical structures of the VCH-binding active groups present in cholesterol. The VCH monomers are bound to cholesterol, diosgenin, campesterol, and ergosterol, which have a hydroxyl group at position C-3 (3betaOH) in the A ring and a C-C double bond between positions C-5 and C-6 (C-C Delta(5)) in the B ring. They are not bound to epicholesterol and dihydrocholesterol, which form a covalent link with a 3alphaOH group and a C-C single bond between positions C-5 and C-6, respectively. This result suggests that the 3betaOH group and the C-CDelta(5) bond in cholesterol are required for VCH monomer binding. We further examined VCH oligomer binding to cholesterol. However, this oligomer did not bind to cholesterol, suggesting that the disappearance of the cholesterol-binding potential of the VCH oligomer might be a result of the conformational change caused by the conversion of the monomer into the oligomer. VCH oligomer formation was observed in liposomes containing sterols with the 3betaOH group and the C-C Delta(5) bond, and it correlated with the binding affinity of the monomer to each sterol. Therefore, it seems likely that monomer binding to membrane sterol leads to the assembly of the monomer. However, since oligomer formation was induced by liposomes containing either epicholesterol or dihydrocholesterol, the 3betaOH group and the C-C Delta(5) bond were not essential for conversion into the oligomer.     

Effects of different sterols on the inhibition of cell culture growth caused by the growth retardant tetcyclacis

Cell division in cell suspension cultures can be completely blocked by the growth retardant tetcyclacis at a concentration of 10^-4 mol l^-1. In rice cells it has been demonstrated that the growth inhibition can be completely overcome by application of cholesterol independent of the duration of pretreatment with tetcyclacis. In suspension cultures of maize and soybean, too, the effect of tetcyclacis on cell division was neutralized by adding cholesterol. Other plant sterols, stigmasterol, campesterol and sitosterol were active in a decreasing order. Modifications in the cholesterol perhydro-cyclopentanophenanthrene-ring system indicate that the hydroxyl group at C-3 and the double bond between C-5 and C-6 in ring B are required for the activity. In contrast, gibberellic acid as well as ent-kaurenoic acid could not compensate retardant effects. Likewise, tetcyclasis did not change the level of gibberellins in rice cells as shown by radioimmunoassay. Thus, it is concluded that in cell suspension cultures sterols play a more important role in cell division than gibberellins.Abbreviation GA_x gibberelin A_x     

Effect of sterols on the permeability of alcohol-treated red beet tissue

Alcohols and hydrogen peroxide altered the permeability of membranes of Beta vulgaris root cells. Generally alcohols increased the permeability of membranes without going through an induction period except methanol which required a 10- to 15-hour induction period. The membrane effect of methanol could be inhibited with CaCl₂, cholesterol, β-sitosterol, and stigmasterol. Cholesterol was the most effective inhibitor, followed by β-sitosterol and stigmasterol; and at the same concentration, the sterols were more effective than CaCl₂, the classic membrane stabilizer.     

Substrate and inhibitor specificity of the cholesterol oxidase in bovine adrenal cortex

1. Cholesteryl 3β-sulphate is oxidized in vitro by preparations of bovine adrenal-cortex mitochondria to pregnenolone sulphate and isocaproic acid (4-methyl-pentanoic acid) without hydrolysis of the ester linkage. 2. Free cholesterol is the preferred substrate for adrenal-cortex cholesterol oxidase; the apparent K_m for cholesteryl sulphate is 500μm and for free cholesterol 50μm under the same conditions. 3. Cholesteryl 3β-acetate is hydrolysed by bovine adrenal-cortex mitochondria in vitro to free cholesterol, which is subsequently oxidized to more polar steroids and isocaproic acid. Evidence was obtained that other cholesterol esters behave similarly. Cholesterol esters may thus act as precursors of steroid hormones. 4. Cholest-4-en-3-one is only poorly oxidized to isocaproic acid and more polar steroids and thus is probably not a significant precursor of steroid hormones. 5. Cholesteryl esters inhibit the oxidation of cholesterol competitively (K_i for cholesteryl phosphate 28μm, for cholesteryl sulphate 110μm, for cholesteryl acetate 65μm) but pregnenolone esters do not inhibit this system. 6. Pregnenolone and 20α-hydroxycholesterol (both metabolites of cholesterol in this system) inhibit the oxidation of cholesterol non-competitively. K_i for pregnenolone is 130μm and K_i for 20α-hydroxycholesterol is 17μm. 7. 25-Oxo-27-norcholesterol inhibits cholesterol oxidation non-competitively (K_i16μm). A number of other Δ⁵-3β-hydroxy steroids inhibit cholesterol oxidation and evidence was obtained that the 3β-hydroxyl group was necessary for inhibitory activity. 8. Pregnenolone, 20α-hydroxycholesterol and 25-oxo-27-norcholesterol inhibit oxidation of cholesteryl sulphate by this system but their sulphates do not. 9. 3β-Hydroxychol-5-enoic acid, 3α-hydroxy-5β-cholanic acid and 3β-hydroxy-22,23-bisnorchol-5-enoic acid stimulated formation of isocaproic acid from cholesterol. 10. No evidence was obtained that phosphorylation or sulphation are obligatory steps in cholesterol oxidation by adrenal-cortex mitochondria. 11. The cholesteryl 3β-sulphate sulphatase of bovine adrenal cortex was found mostly in the microsomal fraction and was inhibited by inorganic phosphate.     

Quantitative dietary requirements of the beetle Trogoderma for cholesterol and choline

The quantitative dietary requirements for cholesterol and choline of Trogoderma granarium were investigated. The ‘cholesterol-sparing’ activity of several sterols and substituents for choline were also studied. Cholesterol in amounts of 0·031 and 0·062 mg/g diet did not support growth. Two mg cholesterol/g diet gave optimal growth. Lanosterol, isocholesterol, 6-ketocholesterol, and cholecalciferol did not act as sparing sterols in the presence of 0·015 and 0·03 mg cholesterol/g diet. Cholesteryl methyl ether and cholesteryl chloride inhibited the growth of Trogoderma larvae in the presence of normally sufficient amounts of cholesterol. Choline was shown to be essential in the diet and the growth of the larvae was directly proportional to the amount of choline. Optimal growth was obtained at a level of 50 μg choline/g diet. Choline could be only poorly substituted by triethanol amine, γ-butyrobetaine, and carnitine.     

Microsomal synthesis of cholesterol from squalene, Lanosterol, and desmosterol. Evidence for the presence of two noncatalytic activator proteins in the 105,000 g supernatant fraction from brain, heart, and kidney

The biosynthesis of C₂₇ sterols (used as a generic term for 3 β-hydroxysterols containing 27 carbon atoms) from squalene and lanosterol, of cholesterol from desmosterol, and of lanosterol from squalene by microsomal fractions from adult rat heart, kidney, and brain was investigated. These conversions required the presence of 105,000g supernatant fraction. Heat treatment of the supernatant fractions resulted in a significant loss of their capacity to stimulate the conversion of squalene to sterols, but the capacity to stimulate conversion of lanosterol to C₂₇ sterols and desmosterol to cholesterol was unaffected. The stimulatory activity (for the conversion of all three substrates) of both the heated and unheated supernatant fractions was lost on treatment with trypsin. Thus the soluble fraction appears to contribute at least two essential protein components for the overall conversion of squalene to cholesterol; one a heat labile protein, which functions in the squalene to lanosterol sequence, and the other a heat-stable protein, which is operative in the pathway between lanosterol and cholesterol. Hepatic supernatant factors required for cholesterol synthesis by liver microsomal enzymes function with heart, kidney, and brain microsomal enzymes in stimulating sterol synthesis from squalene and sterol precursors. Moreover, heart, kidney, and brain supernatant fractions prepared in 100 mm phosphate buffer stimulated cholesterol synthesis from squalene and other sterol precursors by liver microsomes. The supernatant fractions of the extrahepatic tissues prepared in 20 mm phosphate buffer lacked the ability to stimulate the biosynthesis of lanosterol from squalene by liver microsomes but were able to stimulate the conversion of lanosterol to C₂₇ sterols or conversion of desmosterol to cholesterol. These findings indicate that the heat-stable protein factor present in the supernatant fractions from extrahepatic tissues is perhaps identical to that in liver, but that the heat-labile factor in extrahepatic tissues, which catalyzes the cyclization of squalene to lanosterol, differs in some respect from that in liver.     

Partial purification and specificity of triacylglycerol: Sterol acyltransferase from Sinapis alba

Triacylglycerol: sterol acyltransferase is present in roots of Sinapis alba seedlings. The enzyme is located predominantly in the cell membrane structures sedimenting at 300–16 000 g but can be solubilized by acetone treatment and buffer extraction. During gel filtration on Sephadex G-100 the acyltransferase activity was separated into two peaks corresponding to MW 1.8 × 10¹⁴ and MW ? 10⁵, respectively. A number of natural 3β-hydroxysterols can be esterified by the solubilized acyltransferase. The rate of esterification is much higher for sterols containing a planar ring system. The number and position of double bonds, as well as the structure of the side chain at C- 17 of the sterol molecule, are of secondary importance. Triacylglycerols containing fatty acids C, C₆-C₂₂ can be utilized as acyl donors. Among triacylglycerols containing saturated fatty acids, tripalmitoylglycerol (C_16:0) is the best acyl donor. For triacylglycerols containing C₁₈-fatty acids the following sequence was observed: trioleoylglycerol (C_18:1) > trilinoleoylglycerol (C_18:2) > trilinolenoylglycerol (C_18:3) > tristearoylglycerol (C_18:0).     

Implications of cholesterol autoxidation products in the pathogenesis of inflammatory diseases

There is rising interest in non-enzymatic cholesterol oxidation because the resulting oxysterols have biological activity and can be used as non-invasive markers of oxidative stress in vivo. The preferential site of oxidation of cholesterol by highly reactive species is at C₇ having a relatively weak carbon–hydrogen bond. Cholesterol autoxidation is known to proceed via two distinct pathways, a free radical pathway driven by a chain reaction mechanism (type I autoxidation) and a non-free radical pathway (type II autoxidation). Oxysterols arising from type II autoxidation of cholesterol have no enzymatic correlates, and singlet oxygen (¹ΔgO₂) and ozone (O₃) are the non-radical molecules involved in the mechanism. Four primary derivatives are possible in the reaction of cholesterol with singlet oxygen via ene addition and the formation of 5α-, 5β-, 6α- and 6β-hydroxycholesterol preceded by their respective hydroperoxyde intermediates. The reaction of ozone with cholesterol is very fast and gives rise to a complex array of oxysterols. The site of the initial ozone reaction is at the Δ_5,6 –double bond and yields 1,2,3-trioxolane, a compound that rapidly decomposes into a series of unstable intermediates and end products. The downstream product 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (sec-A, also called 5,6-secosterol), resulting from cleavage of the B ring, and its aldolization product (sec-B) have been proposed as a specific marker of ozone-associated tissue damage and ozone production in vivo. The relevance of specific ozone-modified cholesterol products is, however, hampered by the fact sec-A and sec-B can also arise from singlet oxygen via Hock cleavage of 5α-hydroperoxycholesterol or via a dioxietane intermediate. Whatever the mechanism may be, sec-A and sec-B have no enzymatic route of production in vivo and are reportedly bioactive, rendering them attractive biomarkers to elucidate oxidative stress-associated pathophysiological pathways and to develop pharmacological agents.     

Esterification of side-chain oxysterols by lysosomal phospholipase A2

Side-chain oxysterols produced from cholesterol either enzymatically or non-enzymatically show various bioactivities. Lecithin-cholesterol acyltransferase (LCAT) esterifies the C3-hydroxyl group of these sterols as well as cholesterol. Lysosomal phospholipase A2 (LPLA2) is related to LCAT but does not catalyze esterification of cholesterol. First, esterification of side-chain oxysterols by LPLA2 was investigated using recombinant mouse LPLA2 and dioleoyl-PC/sulfatide/oxysterol liposomes under acidic conditions. TLC and LC-MS/MS showed that the C3 and C27-hydroxyl groups of 27-hydroxycholesterol could be individually esterified by LPLA2 to form a monoester with the C27-hydroxyl preference. Cholesterol did not inhibit this reaction. Also, LPLA2 esterified other side-chain oxysterols. Their esterifications by mouse serum containing LCAT supported the idea that their esterifications by LPLA2 occur at the C3-hydroxyl group. N-acetylsphingosine (NAS) acting as an acyl acceptor in LPLA2 transacylation inhibited the side-chain oxysterol esterification by LPLA2. This suggests a competition between hydroxycholesterol and NAS on the acyl-LPLA2 intermediate formed during the reaction. Raising cationic amphiphilic drug concentration or ionic strength in the reaction mixture evoked a reduction of the side-chain oxysterol esterification by LPLA2. This indicates that the esterification could progress via an interfacial interaction of LPLA2 with the lipid membrane surface through an electrostatic interaction. The docking model of acyl-LPLA2 intermediate and side-chain oxysterol provided new insight to elucidate the transacylation mechanism of sterols by LPLA2. Finally, exogenous 25-hydroxycholesterol esterification within alveolar macrophages prepared from wild-type mice was significantly higher than that from LPLA2 deficient mice. This suggests that there is an esterification pathway of side-chain oxysterols via LPLA2.     

Acyl CoA:Cholesterol acyl transferase activity in human placental microsomes: Inhibition by progesterone

The characteristics of acyl CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) in microsomes prepared from human term placenta were studied and the rate of incorporation of [1-¹⁴C] oleoyl CoA into cholesteryl esters was measured. The apparent K_m of the enzyme for [1-¹⁴C] oleoyl CoA was 38 ± 9 μm and the V for the reaction was 15 ± 6 pmol × mg^? protein × min^?1. The Hill coefficient for the reaction was 1.2, indicative of some degree of positive cooperativity. Cholesterol, added to the incubation mixture, did not influence ACAT activity, indicating that endogenous microsomal cholesterol served as an effective substrate for the placental ACAT enzyme. However, [1,2-³H]cholesterol in the presence of oleoyl CoA was incorporated into cholesteryl esters by placental microsomes. When progesterone was present in the incubation mixture at a concentration of 20 μm, ACAT activity was inhibited 50%. Pregnenolone, 5α-dihydroprogesterone, 17α-hydroxyprogesterone, deoxycorticosterone, dehydroisoandrosterone, androstenedione, testosterone, and estradiol-17β also inhibited ACAT activity, whereas corticosterone, cortisol, and estriol had little effect. These results are supportive of the view that ACAT activity in human placenta may be regulated by endogenously synthesized steroid hormones.     

Binding of sterols affects membrane functioning and sphingolipid composition in wheat roots

The present work was devoted to the exploration of the role of sterols in the functioning of membranes in root cells. Membrane characteristics and composition of the membrane lipids in the roots of wheat (Triticum aestivum L.) seedlings treated with exogenous cholesterol and antibiotic nystatin, which specifically binds with endogenous sterols, were analyzed. Cholesterol caused a fall of membrane potential, acidification of the incubation medium, decrease in potassium leakage of roots, and increase in the level of exogenous superoxide radical. Similarly to cholesterol, the application of nystatin also induced the depolarization of the plasma membrane, but in contrast with cholesterol it was accompanied by alkalinization of the incubation medium and decrease in the level of exogenous superoxide radical. Analysis of membrane lipids showed that following nystatin treatment the total sterol content in roots did not change, while the level of complex sphingolipids represented mainly by glycoceramides became higher. Using mass spectrometry with electrospray ionization (+ESI-MS) for the analysis of the glycoceramide composition, we showed that nystatin induced changes in the ratios of molecular species of glycoceramides. It was suggested that the modification of the sterol component of plasma membrane could influence membrane functioning by changing the sphingolipid composition of lipid rafts.     

Microsomal cholesterol ester hydrolase of rat brain: lipids as effector of enzymatic activity

Evidence is presented that lipid plays an important role in the function of the microsomal cholesterol ester hydrolase of rat brain. The catalytic activity was almost completely lost when most of cholesterol and up to 70% of phospholipids were removed from lyophilized microsomes by extraction with chloroform at ?20 °C. The activity was completely restored when the chloroform-extracted lipid was added back to the assay mixture in the amount equal to the original concentration. Cholesterol or individual phospholipid alone was not effective in reconstituting the lost enzymatic activity. Effective restoration of the activity required addition of cholesterol and a phospholipid. Among the phospholipids tested, phosphatidylserine was the most effective, followed by ethanolamine phospholipids and phosphatidylcholine. The apparent V was dependent on the amount of the lipid added, while the K_m for the substrate, cholesteryl oleate, remained relatively constant, indicating that the effect of the added lipid was primarily on the reaction rate and not on the affinity of the enzyme to the substrate. The similar lipid dependence was observed with the Triton X-100-solubilized enzyme preparation. When the lipid phase of the microsomal membrane was perturbed, the enzyme became unstable when heated at 50 °C and its activity showed a discontinuity in the Arrhenius plots. Therefore, not only the concentration of the added lipid but also the physical state of the lipid phase around the enzyme appeared to be important for the activity of the rat brain microsomal cholesterol ester hydrolase.     

Metabolism of 24-dihydrolanosterol in Ochromonas malhamensis and Chlorella ellipsoidea

24-Dihydrolanosterol-[2-³H] was converted to cholesterol in Chlorella ellipsoidea but ergost-5-enol, poriferasterol, clionasterol were not labelled. The absence of the necessary 24(25) double bond precursor eliminates the possibility of C₂₈ and C₂₉ sterol synthesis. However, it was confirmed that 24-dihydrolanosterol was metabolized by Ochromonas malhamensis to give cholesterol, brassicasterol, and poriferasterol.     

Cholesterol modulation of β-adrenergic receptor characteristics

Cholesterol, a major structural component of plasma membranes, has a profound influence on cell surface receptor characteristics and on adenylate cyclase activity. β-Adrenergic receptor number, adenylate cyclase activity, and receptor-cyclase coupling were assessed in rat lung membranes following preincubation with cholesteryl hemisuccinate. β-Adrenergic receptor number increased by 50% without a change in antagonist affinity. However, β-adrenergic receptor affinity for isoproterenol increased 2-fold as a result of an increase in the affinity of the isoproterenol high-affinity binding site. This increase in agonist affinity did not potentiate hormone-stimulated adenylate cyclase activity, which decreased 3-fold following cholesterol incorporation. However, the ratio of isoproterenol to GTP-stimulated activity was unchanged with cholesterol. Stimulation distal to the receptor by GTP, NaF, GppNHp, Mn²⁺ and forskolin also demonstrated 50–80% reduced enzyme activity following cholesterol incorporation. These data suggest that membrane cholesterol incorporation decreases catalytic unit activity without affecting transduction of the hormone signal.     

Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers

Ostreolysin, a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), recognizes and binds specifically to membrane domains enriched in cholesterol and sphingomyelin (or saturated phosphatidylcholine). These events, leading to permeabilization of the membrane, suggest that a cholesterol-rich liquid-ordered membrane phase, which is characteristic of lipid rafts, could be its possible binding site. In this work, we present effects of ostreolysin on membranes containing various steroids. Binding and membrane permeabilizing activity of ostreolysin was studied using lipid mono- and bilayers composed of sphingomyelin combined, in a 1/1 molar ratio, with natural and synthetic steroids (cholesterol, ergosterol, β-sitosterol, stigmasterol, lanosterol, 7-dehydrocholesterol, cholesteryl acetate, and 5-cholesten-3-one). Binding to membranes and lytic activity of the protein are both shown to be dependent on the intact sterol 3β-OH group, and are decreased by introducing additional double bonds and methylation of the steroid skeleton or C17-isooctyl chain. The activity of ostreolysin mainly correlates with the ability of the steroids to promote formation of liquid-ordered membrane domains, and is the highest with cholesterol-containing membranes. Furthermore, increasing the cholesterol concentration enhanced ostreolysin binding in a highly cooperative manner, suggesting that the membrane lateral distribution and accessibility of the sterols are crucial for the activity of this new member of cholesterol-dependent cytolysins.     

Structural aspects of anthocyanin-flavonoid complex formation and its role in plant color

The complex formation of flavonoids with anthocyanins, resulting in increase in both absorbance and in a bathochromic shift of the visible absorption maximum of the latter, is based mainly on hydrogen bond formation between the carbonyl group of the anthocyanin anhydrobase and aromatic hydroxyl groups of the complex-forming flavonoids. The larger the number of hydroxyl groups in the flavonoid molecule, the stronger the complex formation. The presence of a 3-hydroxyl group in the flavonoid molecule has little effect on the complex-forming ability. The nature of the sugar substituent of the complex-forming flavonoid compound has no influence on the reaction. The 5-hydroxyl group of flavonoids is strongly bound by intramolecular hydrogen bond to the 4-carbonyl and does not participate in the complex formation. The most important hydroxyl group in the flavonoid molecule is the one in the 7-position. Unsaturation at C₂C₃ in the heterocyclic ring is an important factor for complex formation. Aromatic hydroxyl groups in the flavonoid system alone cannot account for all the complex-forming ability, suggesting additional involvement by electrostatic forces and configurational or steric effects.     

The neutral sterols of Megalotomus quinquespinosus (say) (hemiptera: Alydidae) and identification of makisterone a as the major free ecdysteroid

Last-stage nymphs of the broad-headed bug, Megalotomus quinquespinosus contain the C₂₈ ecdysteroid makisterone A as their major ecdysteroid. No ecdysone or 20-hydroxyecdysone was detected in whole body extracts analyzed by high performance liquid chromatography and radioimmune assay. Analyses of the neutral sterols of this phytophagous hemipteran revealed that the sterol composition of the nymphs was highly reflective of their dietary sterols. The most abundant nymphal sterols were sitosterol (46.6%), Δ⁷-stigmastenol (13.8%) and spinasterol (13.4%). Cholesterol accounted for only 0.2% of the total sterols and indicates that this species is incapable of converting 24-alkyl sterols to cholesterol.     

Influence du milieu de culture sur la composition en sterols de Leptosphaeria typhae

C₂₇, C₂₈ and C₂₉ sterols have been isolated from a Leptosphaeria typhae culture grown in vitro in light on a synthetic medium. These products were characterized by GLC and MS. Saturated and mono-, di- and tri-unsaturated sterols are present, both free and esterified. There are significant differences between these sterols and those in the same fungus grown on “oat water”. Unexpectedly, cholesterol was detected in the latter case.     

Interplay of unsaturated phospholipids and cholesterol in membranes: effect of the double-bond position

The structural and dynamical properties of lipid membranes rich in phospholipids and cholesterol are known to be strongly affected by the unsaturation of lipid acyl chains. We show that not only unsaturation but also the position of a double bond has a pronounced effect on membrane properties. We consider how cholesterol interacts with phosphatidylcholines comprising two 18-carbon long monounsaturated acyl chains, where the position of the double bond is varied systematically along the acyl chains. Atomistic molecular dynamics simulations indicate that when the double bond is not in contact with the cholesterol ring, and especially with the C18 group on its rough β-side, the membrane properties are closest to those of the saturated bilayer. However, any interaction between the double bond and the ring promotes membrane disorder and fluidity. Maximal disorder is found when the double bond is located in the middle of a lipid acyl chain, the case most commonly found in monounsaturated acyl chains of phospholipids. The results suggest a cholesterol-mediated lipid selection mechanism in eukaryotic cell membranes. With saturated lipids, cholesterol promotes the formation of highly ordered raft-like membrane domains, whereas domains rich in unsaturated lipids with a double bond in the middle remain highly fluid despite the presence of cholesterol.