Influence of the bovine seminal plasma protein PDC-109 on cholesterol in the presence of phospholipids

The interaction of the major bovine seminal plasma protein PDC-109 with cholesterol was studied by employing spin-labelled analogues. It could be shown that PDC-109 does not interact directly with cholesterol molecules. However, in the presence of phospholipids we found a strong reduction of cholesterol motion by PDC-109. The fraction of immobilized cholesterol was largest for phosphorylcholine-containing lipids. This is consistent with the preferential interaction between PDC-109 and phosphatidylcholine. It is concluded that a stronger association and interaction of PDC-109 with phosphatidylcholine leads to an enhanced fraction of immobilized cholesterol analogues, but not to a phospholipid-dependent specific interaction between the protein and cholesterol. Moreover, the interaction of PDC-109 with various spin-labelled analogues of phosphatidylcholine (lysoPC, diacylPC) was investigated. In membranes of lipid vesicles the protein caused an immobilization of the phosphatidylcholine analogues mainly in the outer membrane leaflet, with no differences between diacylPC and lysoPC. The results are of relevance for understanding the physiological role of PDC-109 in the genesis of sperm cells.     

Role of cell surface mannose residues in host cell invasion by Trypanosoma cruzi

The cholesterol content of human erythrocyte membranes has been modified by incubation of intact cells with sonicated egg phosphatidylcholine/cholesterol vesicles and with egg phosphatidylcholine vesicles. (Na+ + K+)-ATPase ATP hydrolyzing activity was measured as a function of membrane cholesterol content. High membrane cholesterol inhibits the ATPase activity of the enzyme and low membrane cholesterol activates that enzyme activity. The most likely mechanism of inhibition is suggested to comprise direct cholesterol-protein interactions which lead to a low activity conformation. Ouabain binding studies show that the inhibition is not due to a loss of enzyme from the membrane.     

Effect of cholesterol on the valinomycin-mediated uptake of rubidium into erythrocytes and phospholipid vesicles

Human erythrocytes have been treated with lipid vesicles in order to alter the cholesterol content of the cell membrane. Erythrocytes have been produced with cholesterol concentrations between 33 and 66 mol% of total lipid. The rate of valinomycin-mediated uptake of rubidium into the red cells at 37°C was lowered by increasing the cholesterol concentration of the cell membrane. Cholesterol increased the permeability to valinomycin at 20°C of small (less than 50 nm), unilamellar egg phosphatidylcholine vesicles formed by sonication. Cholesterol decreased the permeability to valinomycin at 20°C of large (up to 200 nm) unilamellar egg phosphatidylcholine vesicles formed by freezethaw plus brief sonication. It is concluded that cholesterol increases the permeability of small membrane vesicles to hydrophobic penetrating substances while above the transition temperature but has the opposite effect on large membrane vesicles and on the membranes of even larger cells.     

Phospholipid hydrolysis caused by Clostridium perfringens α-toxin facilitates the targeting of perfringolysin O to membrane bilayers

Clostridium perfringens causes gas gangrene and gastrointestinal disease in humans. These pathologies are mediated by potent extracellular protein toxins, particularly α-toxin and perfringolysin O (PFO). While α-toxin hydrolyzes phosphatidylcholine and sphingomyelin, PFO forms large transmembrane pores on cholesterol-containing membranes. It has been suggested that the ability of PFO to perforate the membrane of target cells is dictated by how much free cholesterol molecules are present. Given that C. perfringens α-toxin cleaves the phosphocholine headgroup of phosphatidylcholine, we reasoned that α-toxin may increase the number of free cholesterol molecules in the membrane. Our present studies reveal that α-toxin action on membrane bilayers facilitates the PFO?cholesterol interaction as evidenced by a reduction in the amount of cholesterol required in the membrane for PFO binding and pore formation. These studies suggest a mechanism for the concerted action of α-toxin and PFO during C. perfringens pathogenesis.     

Haptenic activity of galactosyl ceramide and its topographical distribution on liposomal membranes. I. Effect of cholesterol incorporation

The relation between the immune-reaction of phosphatidylcholine liposomes containing spin-labeled galactosyl ceramide with or without cholesterol and the topographical distribution of the glycolipid in membranes was studied. In egg yolk phosphatidylcholine liposomes, both immune agglutination and antibody binding occurred, irrespectively of the presence of cholesterol, though the motion of the fatty acyl chain of spin-labeled galactosyl ceramide was restricted by cholesterol. In dipalmitoyl phosphatidylcholine liposomes, unlike in egg yolk phosphatidylcholine liposomes, the immune-reaction depended on the cholesterol content. The electron spin resonance (ESR) spectra of spin-labeled galactosyl ceramide in dipalmitoyl phosphatidylcholine liposomes indicated that cholesterol affected the topographical distribution of spin-labeled galactosyl ceramide in the liposomes. Without cholesterol, most of the spin-labeled galactosyl ceramide was clustered on the dipalmitoyl phosphatidylcholine membrane, but with increase of cholesterol, random distribution of hapten on the membrane increased. The cholesterol-dependent change in the topographical distribution of hapten on the membranes was parallel with that of immune reactivity. 'Aggregates' composed solely of galactosyl ceramide did not show any binding activity with antibody. The findings suggest that the recognition of galactosyl ceramide by antibody depended on the topographical distribution of hapten molecules. Phosphatidylcholine and/or cholesterol may play roles as 'spacers' for the proper distribution of 'active' haptens on the membranes. The optimum density of haptens properly distributed on liposomal membranes is discussed.     

Molecular interactions in the hydrogen belts of membranes glucose-6-phosphatase,lysophosphatidylcholine, and cholesterol

Microsomal glucose-6-phosphatase from rat liver is activated by phosphatidylcholine but inhibited by lysophosphatidylcholine. Inhibition occurs not by membrane lysis but in an intact bilayer; it is reversible; and it is overcome by addition of cholesterol but not if the cholesterol-hydroxyl group is blocked. An analog of lysophosphatidylcholine deprived of hydrogen bonding sites, 1-ether-2-deoxylysophosphatidylcholine, is a partial activator, and its effect on the enzyme in a phosphatidylcholine bilayer is not modulated by cholesterol. It appears to be one of the functions of cholesterol to buffer the lysophospholipids in membranes by complexing with them through hydrogen bonding in the hydrogen belt region. Lysophosphatidylcholine/cholesterol association is favored over phosphatidylcholine/cholesterol association.     

Molecular interactions in the hydrogen belts of membranes. Glucose-6-phosphatase, lysophosphatidylcholine, and cholesterol

Microsomal glucose-6-phosphatase from rat liver is activated by phosphatidylcholine but inhibited by lysophosphatidylcholine. Inhibition occurs not by membrane lysis but in an intact bilayer; it is reversible; and it is overcome by addition of cholesterol but not if the cholesterol-hydroxyl group is blocked. An analog of lysophosphatidylcholine deprived of hydrogen bonding sites, 1-ether-2- deoxylysophosphatidylcholine , is a partial activator, and its effect on the enzyme in a phosphatidylcholine bilayer is not modulated by cholesterol. It appears to be one of the functions of cholesterol to buffer the lysophospholipids in membranes by complexing with them through hydrogen bonding in the hydrogen belt region. Lysophosphatidylcholine/cholesterol association is favored over phosphatidylcholine/cholesterol association.     

Plant sterol inhibition of abscisic acid-induced perturbations in phospholipid bilayers

Abscisic acid (ABA)-induced phospholipid bilayer perturbations (permeability and lipid vesicle aggregation) are shown to be reversed by incorporation of a commercially available mixture of plant sterols (60% beta-sitosterol, 27% campesterol and 13% dihydrobrassicasterol) into the membranes. As little and 5 membrane mol% plant sterol inhibits ABA-stimulated permeability of both saturated and unsaturated mixed phosphatidylcholine/phosphatidylethanolamine bilayers to the fluorescent anion carboxyfluorescein by more than 50%. The same conclusion was reached by an osmotic swelling technique for the uncharged permeant solute erythritol. Hormone-induced carboxyfluorescein permeability to mixed acyl chain phosphatidylcholine bilayers was similarly inhibited by the sterols, but only if the membranes were tested at a temperature where liquid crystal and gel states coexist. The plant sterols were also shown to prevent the ABA-induced fusion of mixed phosphatidylcholine/phosphatidylethanolamine bilayers. The ABA effect on membranes is inhibited equally by plant sterols as well as cholesterol. From these experiments a possible role is suggested for plant sterols in controlling the mode of action of ABA.     

Melittin-lipid bilayer interactions and the role of cholesterol

The membrane-destabilizing effect of the peptide melittin on phosphatidylcholine membranes is modulated by the presence of cholesterol. This investigation shows that inclusion of 40 mol % cholesterol in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes reduces melittin's affinity for the membrane. It is significant that the presence of cholesterol does not increase the amount of membrane-associated melittin needed to cause maximum leakage from, or major structural rearrangements of, the liposomes. Furthermore, comparison of microscopy and leakage data suggests that melittin-induced leakage occurs via different mechanisms in the cholesterol-free and cholesterol-supplemented systems. In the absence of cholesterol, leakage of carboxyfluorescein takes place from intact liposomes in a manner compatible with the presence of small melittin-induced pores. In the presence of cholesterol, on the other hand, adsorption of the peptide causes complete membrane disruption and the formation of long-lived open-bilayer structures. Moreover, in the case of cholesterol-supplemented systems, melittin induces pronounced liposome aggregation. Cryotransmission electron microscopy was used, together with ellipsometry, circular dichroism, turbidity, and leakage measurements, to investigate the effects of melittin on phosphatidylcholine membranes in the absence and presence of cholesterol. The melittin partitioning behavior in the membrane systems was estimated by means of steady-state fluorescence spectroscopy measurements.     

Spin label studies of erythrocytes with abnormal lipid composition: comparison of red cells in a hereditary hemolytic syndrome and lecithin: Cholesterol acyltransferase deficiency

Erythrocytes from patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency have been shown to exhibit an increase in membrane fluidity which is surprisingly small in view of the extensive alterations both in membrane lipicl composition (namely, an elevation in cholesterol and phosphatidylcholine contents as well as a decrease in phosphatidylethanolamine) and in the functional properties of these cells. In the hope of deriving some information concerning the interrelationship between the structural and functional abnormalities, we have used the spin probe 5-doxyl stearic acid to investigate the temperature-dependent fluidity properties of red cells from two patients with a hereditary hemolytic syndrome (HHS) whose red cells are also characterized by qualitatively similar alterations in phosphatidylcholine and phosphatidylethanolamine but, unlike those in LCAT deficiency, have relatively normal levels of membrane cholesterol. A small increase in membrane fluidity of HHS erythrocytes equivalent to that previously observed in LCAT deficiency was found, indicating that membrane cholesterol level does not exert an important modulatory influence on membrane fluidity in these cells. It is concluded that while the distinct patterns of structural and functional erythrocyte alterations in these two disorders cannot be explained on the basis of differences in bulk membrane fluidity, the marginally increased fluidity may underlie the abnormalities in osmotic fragility and membrane p-nitrophenylphosphatase activity which are shared in common by both types of modified red cells.     

Influence of cholesterol on electroporation of bilayer lipid membranes: chronopotentiometric studies

This paper presents the results of constant-current (chronopotentiometric) measurements of the egg yolk phosphatidylcholine (PC) bilayer membrane without and with cholesterol. The experiments were performed on planar bilayer lipid membrane (BLM) formed by the Mueller-Rudin method. It is demonstrated that the constant-intensity current flow through bilayer membranes generated fluctuating pores in their structure. The presence of cholesterol in the membrane caused an increase in the value of the breakdown potential. It is postulated that greater stability of the bilayer with cholesterol can result from an increased critical pore radius (at which the bilayer would undergo irreversible rupture). This confirms that cholesterol has a stabilizing effect on BLM. Besides, our results suggest that addition of cholesterol causes shift in the distribution of pore conductance towards a smaller value. It is suggested that this can be connected with the phenomenon of domain formation in the membranes containing high concentration of cholesterol. Moreover, it is shown that chronopotentiometry with programmable current intensity is a promising method for observation of the membrane recovery process.     

Influence of cholesterol on electroporation of bilayer lipid membranes: chronopotentiometric studies

This paper presents the results of constant-current (chronopotentiometric) measurements of the egg yolk phosphatidylcholine (PC) bilayer membrane without and with cholesterol. The experiments were performed on planar bilayer lipid membrane (BLM) formed by the Mueller-Rudin method. It is demonstrated that the constant-intensity current flow through bilayer membranes generated fluctuating pores in their structure. The presence of cholesterol in the membrane caused an increase in the value of the breakdown potential. It is postulated that greater stability of the bilayer with cholesterol can result from an increased critical pore radius (at which the bilayer would undergo irreversible rupture). This confirms that cholesterol has a stabilizing effect on BLM. Besides, our results suggest that addition of cholesterol causes shift in the distribution of pore conductance towards a smaller value. It is suggested that this can be connected with the phenomenon of domain formation in the membranes containing high concentration of cholesterol. Moreover, it is shown that chronopotentiometry with programmable current intensity is a promising method for observation of the membrane recovery process.     

Studies on the lipid composition of the rat liver endoplasmic reticulum after induction with phenobarbitone and 20-methylcholanthrene

1. The cholesterol content, proportions of different phospholipids and fatty acid components of phosphatidylcholine and phosphatidylethanolamine were studied in rat liver endoplasmic-reticulum membrane, after a single injection of 20-methylcholanthrene or injections of phenobarbitone for 5 days. 2. A marked decrease in the proportion of cholesterol occurred 5 days after injection of 20-methylcholanthrene or phenobarbitone. 3. The proportion of phosphatidylcholine was increased by injection of phenobarbitone and minor changes occurred in other phospholipids. 4. Phenobarbitone caused the proportion of linoleic acid in phosphatidylcholine and phosphatidylethanolamine to increase to 120-125% of the control and the proportion of oleic acid, arachidonic acid and docosahexaenoic acid to decrease. 5. 20-Methylcholanthrene caused an increase in the proportion of oleic acid in phosphatidylcholine and ethanolamine to 125-140% of the control, 1 day after injection. 6. The increased proportion of linoleic acid in phosphatidylcholine after phenobarbitone injection occurs simultaneously with the increase of cytochrome P-450 concentration, the rate of oxidative demethylation of aminopyrine and the rate of hydroxylation of biphenyl. It is therefore considered that distinct species of phosphatidylcholine or phosphatidylethanolamine containing linoleic acid in the beta position are essential in the endoplasmic-reticulum membrane for optimal activity of oxidative demethylation.     

Immunological studies on the localization of phosphatidylglycerol in the membranes of Mycoplasma hominis.

Phosphatidylglycerol is the main component (87%) of the membrane phospholipids of Mycoplasma hominis. It is immunologically active. Antibodies directed against phosphatidylglycerol were detected in rabbits intravenously immunised with native M. hominis or isolated M. hominis membranes. The intravenous method of immunisation was chosen in order to select for a response to surface antigenic determinants. Anti-phosphatidylglycerol antibodies were induced in rabbits by intravenously injecting the flocculated complexes of methylated bovine serum albumin and a phosphatidylglycerol/phosphatidylcholine/cholesterol mixture. These antibodies were specifically bound to intact M. hominis, as shown by complement fixation and Coombs tests. Native M. hominis were not agglutinated by anti-phosphatidylglycerol antibodies; but after partial digestion of the membrane proteins with Pronase, the mycoplasmas were heavily agglutinated by the anti-phosphatidylglycerol antibodies. The same amount of anti-phosphatidylglycerol antibodies was bound to intact M. hominis, containing 600 mug of phosphatidylglycerol as to 6 mug of phosphatidylglycerol in the optimal configurational arrangement of a mixed phosphatidylglycerol/phosphatidylcholine/cholesterol micelle. It is concluded that the major part of the phosphatidylglycerol in native M. hominis membranes is masked, probably by membrane proteins, and is not accessible to the anti-phosphatidylglycerol antibodies.     

The replacement of cholesterol by phytosterols and the increase of total sterol content in model erythrocyte membranes

The activity of phytosterols on human organism includes the ability of these compounds to incorporate into membranes. In the consequence the plant sterols are able to increase total sterol concentration in membrane or/and to replace cholesterol molecules. The aim of this work was to compare the influence of both these effects on the properties of model erythrocyte membranes. Moreover, the interactions between the plant sterols (β-sitosterol and stigmasterol) and saturated–monounsaturated phosphatidylcholine were investigated and the condensing and ordering potency of these phytocompounds on membrane phospholipids were thoroughly analyzed. It was found that the addition of the plant sterols into model membrane modifies the condensation, ordering and interactions in the system. Moreover, the replacement of mammalian sterol by phytosterol more strongly influences the model system than even a 10% increase of total sterol concentration induced by the incorporation of the plant sterol, at constant content of cholesterol. The investigated plant sterols at their lower concentration in the mixed system are of similar effect on its properties. At higher content stigmasterol was found to modify the properties of model membrane more strongly than β-sitosterol.     

Model Plasma Membrane Exhibits a Microemulsion in Both Leaves Providing a Foundation for “Rafts”

We consider a model lipid plasma membrane, one that describes the outer leaf as consisting of sphingomyelin, phosphatidylcholine, and cholesterol and the inner leaf of phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and cholesterol. Their relative compositions are taken from experiment; the cholesterol freely interchanges between leaves. Fluctuations in local composition are coupled to fluctuations in the local membrane curvature, as in the Leibler-Andelman mechanism. Structure factors of components in both leaves display a peak at nonzero wavevector. This indicates that the disordered fluid membrane is characterized by structure of the corresponding wavelength. The scale is given by membrane properties: its bending modulus and its surface tension, which arises from the membrane’s connections to the cytoskeleton. From measurements on the plasma membrane, this scale is on the order of 100 nm. We find that the membrane can be divided into two different kinds of domains that differ not only in their composition but also in their curvature. The first domain in the outer, exoplasmic leaf is rich in cholesterol and sphingomyelin, whereas the inner, cytoplasmic leaf is rich in phosphatidylserine and phosphatidylcholine. The second kind of domain is rich in phosphatidylcholine in the outer leaf and in cholesterol and phosphatidylethanolamine in the inner leaf. The theory provides a tenable basis for the origin of structure in the plasma membrane and an illuminating picture of the organization of lipids therein.     

EPR investigation of clomipramine interaction with phosphatidylcholine membranes in presence and absence of cholesterol

The effects of tricylic antidepressant clomipramine (CLO) on the membrane properties of saturated dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine as well as on unsaturated egg yolk phosphatidylcholine liposomes were investigated by the electron paramagnetic resonance spin-labeling technique, in combination with the simulation of the spectra, taking into account that the membrane is heterogeneous and composed of the regions with different fluidity characteristics. Different spin labels, monitoring membrane properties in the upper and inner parts of the membrane, were used. In general, two spectral components, having different motional characteristics, were detected in all liposomes investigated. In liposomes with saturated chains, CLO decreased the phase-transition temperature, disordered the membrane, and increased polarity in the upper part of the membrane. However, less impact was observed in liposomes with unsaturated chains. In dipalmitoyl phosphatidylcholine liposomes, it also induced molecular rearrangements near the pretransition temperature. The presence of 30 mol% cholesterol increased the fluidizing effect of CLO and modified the lateral diffusion of nitroxide in the inner part of the membrane. A unique anomalous increase in diffusion of nitroxide, dependent on CLO concentration, was detected in the temperature region where the phosphatidylcholine membrane without cholesterol experiences the phase transitions. Since the changes in the central part of the membrane were even more pronounced than in the upper part of the membrane, it could be concluded that CLO incorporates into the membrane with its hydrophobic ring parallel to the phospholipid chains.     

Cholesterol attenuates and prevents bilayer damage and breakdown in lipoperoxidized model membranes. A spin labeling EPR study

The stabilizing effect of cholesterol on oxidized membranes has been studied in planar phospholipid bilayers and multilamellar 1-palmitoyl-2-linoleoyl-phosphatidylcholine vesicles also containing either 1-palmitoyl-2-glutaroyl-phosphatidylcholine or 1-palmitoyl-2-(13-hydroxy-9,11-octadecanedienoyl)-phosphatidylcholine oxidized phosphatidylcholine in variable ratio. Lipid peroxidation-dependent membrane alterations in the absence and in the presence of cholesterol were analyzed using Electron Paramagnetic Resonance spectroscopy of the model membranes spin labelled with either cholestane spin label (3-DC) or phosphatidylcholine spin label (5-DSPC). Cholesterol, added to lipid mixtures up to 40% final molar ratio, decreased the inner bilayer disorder as compared to cholesterol-free membranes and strongly reduced bilayer alterations brought about by the two oxidized phosphatidylcholine species. Furthermore, Sepharose 4B gel-chromatography and cryo electron microscopy of aqueous suspensions of the lipid mixtures clearly showed that cholesterol is able to counteract the micelle forming tendency of pure 1-palmitoyl-2-glutaroyl-phosphatidylcholine and to sustain multilamellar vesicles formation. It is concluded that membrane cholesterol may exert a beneficial and protective role against bilayer damage caused by oxidized phospholipids formation following reactive oxygen species attack to biomembranes.     

Changes in plasma membrane properties and phosphatidylcholine subspecies of insect Sf9 cells due to expression of scavenger receptor class B, type I, and CD36

In mammalian cells scavenger receptor class B, type I (SR-BI), mediates the selective uptake of high density lipoprotein (HDL) cholesteryl ester into hepatic and steroidogenic cells. In addition, SR-BI has a variety of effects on plasma membrane properties including stimulation of the bidirectional flux of free cholesterol (FC) between cells and HDL and changes in the organization of plasma membrane FC as indicated by increased susceptibility to exogenous cholesterol oxidase. Recent studies in SR-BI-deficient mice and in SR-BI-expressing Sf9 insect cells showed that SR-BI has significant effects on plasma membrane ultrastructure. The present study was designed to test the range of SR-BI effects in Sf9 insect cells that typically have very low cholesterol content and a different phospholipid profile compared with mammalian cells. The results showed that, as in mammalian cells, SR-BI expression increased HDL cholesteryl ester selective uptake, cellular cholesterol mass, FC efflux to HDL, and the sensitivity of membrane FC to cholesterol oxidase. These activities were diminished or absent upon expression of the related scavenger receptor CD36. Thus, SR-BI has fundamental effects on cholesterol flux and membrane properties that occur in cells of evolutionarily divergent origins. Profiling of phospholipid species by electrospray ionization mass spectrometry showed that scavenger receptor expression led to the accumulation of phosphatidylcholine species with longer mono- or polyunsaturated acyl chains. These changes would be expected to decrease phosphatidylcholine/cholesterol interactions and thereby enhance cholesterol desorption from the membrane. Scavenger receptor-mediated changes in membrane phosphatidylcholine may contribute to the increased flux of cholesterol and other lipids elicited by these receptors.     

Membrane-damaging action of staphylococcal alpha-toxin on phospholipid-cholesterol liposomes

The mechanism of membrane damage by staphylococcal alpha-toxin was studied using carboxyfluorescein (internal marker)-loaded multilamellar liposomes prepared from various phospholipids and cholesterol. Liposomes composed of phosphatidylcholine or sphingomyelin and cholesterol bound alpha-toxin and released carboxyfluorescein in a dose dependent manner, when they were exposed to alpha-toxin of concentrations higher than 1 or 8 micrograms/ml, respectively. In contrast, the other liposomes composed of phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol or phosphatidylinositol plus cholesterol were not susceptible to the toxin even at high concentrations up to 870 micrograms/ml. The insensitive liposomes containing either phosphatidylserine or phosphatidylglycerol were made sensitive to alpha-toxin by inserting phosphatidylcholine into the liposomal membranes. In addition, phosphorylcholine inhibited the toxin-induced marker release from liposomes. These results indicated that the choline-containing phospholipids are required for the interaction between alpha-toxin and liposomal membranes. Susceptibility of liposomes containing phosphatidylcholine or sphingomyelin increased with the increase in cholesterol contents of the liposomes. Based on these results, we propose that the choline-containing phospholipids are possible membrane components or structures responsible for the toxin-membrane interaction, which leads to damage of membranes. Furthermore, cholesterol may facilitate the interaction between alpha-toxin and membrane as a structural component of the membrane.