Multilayer planar membranes of sarcoplasmic reticulum.

Multilayer planar membranes were constructed between a pair of cellulose sheets from fragmented sarcoplasmic reticulum (FSR) as well as a mixture of egg yolk lecithin and the Ca2+-ATPase purified from FSR. Since sodium deoxycholate was used instead of organic solvents in order to dissolve phospholipids in the process of the membrane preparation, the total activity of the Ca2+-ATPase was still preserved in the planar membrane of FSR. It was also indicated using a spin label technique that the orientation of phospholipids in the planar membrane of FSR was considerably disturbed by the presence of proteins such as the Ca2+-ATPase included in FSR.     

A special lipid mixture for membrane fluidization

The potency for membrane fluidization of mixtures containing neutral lipids (NL), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from hen egg yolk was tested on human erythrocytes and lymphocytes. A specific mixture consisting of 70% NL, 20% PC and 10% PE was found to be a potent membrane fluidizer operating almost exclusively by extracting membrane cholesterol. Spectral results and electron micrographs indicate that aqueous dispersion of this mixture consists of chylomicron-like assemblies where the neutral lipids provide the hydrophobic core on the surface of which phospholipids are spread as a monolayer.     

Transfer of cholesterol between liposomal membranes.

The transfer of cholesterol between liposomal membranes was examined. On incubation of liposomes compsoed of egg yolk phosphatidylcholine, phosphatidic acid and cholesterol (molar percentage, 65.8 : 1.3 : 32.9 or 65.5 : 6.3 : 31.2), almost complete equilibration of the cholesterol pools was achieved within 6 to 8 h at 37 degrees C. The rate of transfer of cholesterol from the liposomes, in which cholesterol was introduced by 'the exchange reaction', was not significantly different from that from liposomes prepared in the presence of cholesterol, in which the cholesterol was distributed homogenously. These findings indicate that half life for 'flip-flop' of cholesterol molecules in egg yolk phosphatidylcholine liposomes is less than 6 h at 37 degrees C. The transfer of cholesterol between liposomes was strongly dependent on temperature and was affected by the fatty acid composition of the phospholipid, suggesting that the 'fluidity' of the membranes strongly influences the transfer rate. A preferential distribution of cholesterol molecules was observed in heterogeneous liposomes with different classes of phospholipids. The 'affinity order' of cholesterol for phospholipid deduced from the present experiments is as follows: beef brain sphingomyelin greater than dipalmitoylglycerophosphocholine = dimyristoylglycerophosphocholine greater than egg yolk phosphatidylcholine.     

Interaction of amphotericin B with monolayers of egg lecithin and cholesterol: polorized absorption spectra

Polarized absorption spectra have been obtained of the antibiotic polyene, amphotericin B, interacting with monolayers of egg lecithin, cholesterol and equimolar egg lecithin-cholesterol at low and high surface pressures. An expression is derived which enables the determination from the polarization data of the orientations of the transition moments of the polyene absorption bands at 4077 and 3645 Å. For some of the systems the 3645-Å band is replaced by a previously unreported band appearing between 3610 and 3632 Å. The orientation of the 4077-Å transition moment (parallel to the long molecular axis) is found to vary from an angle of 64 ° with the surface for the low-pressure monolayers of cholesterol to 21 ° for the high-pressure films of egg lecithin-cholesterol. For the band between 3610 Å and 3645 Å, the angle varies from 90 ° for cholesterol to 18 ° for the high-pressure mixed-lipid film. It is found that a large increase in surface pressure of the cholesterol and egg lecithin-cholesterol monolayers causes a decrease in the angle of the 4077-Å moment for both films and that of the higher energy moment for the mixed film. Increasing the content of cholesterol in these monolayers rotates the orientation of the transition moments for both bands toward the surface normal, the change being greatest for the low-pressure films. The effectiveness of amphotericin B in lowering the surface tension of these lipid monolayers is related to its binding, orientation and extent of penetration. For low-pressure cholesterol films where the surface interaction with the polyene is greatest, the binding and penetration are large and the polyene molecule is oriented with its long dimension nearly perpendicular to the surface.     

Interaction of the fluorescent probe diS-C3-(5) with lecithin-cholesterol membranes

Behaviour of fluorescent carbocyanine probe disS-C3(5) in the egg lecithin-cholesterol membrane suspension was studied in relation to the lecithin/cholesterol ratio. The partition coefficient of the probe between aqueous and lipid phases decreases unlinearly with increase of cholesterol molar part in a bilayer. This parameter over molar part units was estimated to be (2.4 +/- 0.1) X 10(6) for egg lecithin membranes and (1.8 +/- 0.2) X 10(6) for 10 mol% cholesterol, (1.2 +/- 0.1) X 10(6) for 20, (0.8 +/- 0.1) X 10(6) for 30, and (0.48 +/- 0.02) X 10(6) for 50 mol% cholesterol. It is suggested that the probe partition coefficient value consists of two components: one caused by pure lecithin bilayer regions and another by local lecithin concentration fluctuations in the mixed lecithin-cholesterol regions.     

A rapid radioassay procedure for plasma lecithin-cholesterol acyltransferase

A rapid and accurate single step procedure is described for the assay of lecithin-cholesterol acyltransferase activity. After incubation, using radiolabeled cholesterol as the substrate, an ethanolic solution of digitonin is added directly to the incubation mixture to extract the lipids. Excess cholesterol is then added, and the labeled cholesterol-digitonide along with denatured proteins are sedimented by low speed centrifugation, leaving the labeled esterified cholesterol in solution. An aliquot of the supernatant is counted in an aqueous scintillation mixture. The method correlates well with the established thin-layer chromatographic procedure using either lecithin-cholesterol vesicles or heat-inactivated plasma as the substrate for lecithin-cholesterol acyltransferase.     

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.     

Carbon-13 and proton nuclear magnetic resonance of unsonicated model and mitochondrial membranes

Proton nuclear magnetic resonance (PMR) spectra at 270 MHz of aqueous dispersions of nonsonicated egg lecithin, dipalmitoyl lecithin, egg lecithin-cholesterol (1 : 1) and dipalmitoyl lecithin-cholesterol (1 : 1), together with PMR spectra of mitochondrial membranes and their extracted lipids, have been obtained.Carbon-13 nuclear magnetic resonance (CMR) spectra at 25.2 MHz of egg lecithin, egg lecithin-cholesterol (1 : 1) and sphingomyelin, together with CMR spectra of chloroplast and mitochondrial membranes, and erythrocyte ghosts, have also been obtained. The results obtained using CMR appear very promising for further study of intact membrane structure.It is suggested, on the basis of CMR evidence, that the proteins in mitochondrial membranes may be considerably less mobile than the lipids.     

Separation and Formation of Egg Yolk Oil by Solubilizing the Lipoproteins of Spray-dried Egg Yolk into Polypeptides

Egg yolk oil was formed from a hen’s egg without using the traditional charring method and organic solvents. By treating a spray-dried egg yolk suspension with a commercial crude enzyme preparation having protease and lipase activities, the lipids (egg yolk oil) could be easily separated, and a transparent solution of soluble polypeptides was obtained. When the enzyme preparation (4 mg) was added to a 5% spray-dried egg yolk (100 mg) suspension, the reaction mixture became transparent, and the egg yolk oil floated to the surface of the reaction mixture within 3 h. In the case of a 10% spray-dried egg yolk suspension, a transparent solution and egg yolk oil could be successfully obtained within 4 to 5 h by using a larger reaction vessel and a 0.2M lactate buffer. About 87% of the total polypeptides in the initial reaction mixture was recovered from the transparent solution, while about 83% of the total phospholipids was recovered from the floating egg yolk oil.     

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.     

Reincorporation of adenosine 5'-diphosphate/adenosine 5'-triphosphate carrier into phospholipid membranes. Phospholipid-protein interaction as studied by phosphorus-31 nuclear magnetic resonance and electron microscopy

Combined phosphorus-31 nuclear magnetic resonance (31P NMR) and electron microscopic studies were performed on the ADP/ATP carrier protein from beef heart mitochondria. The protein was incorporated into phospholipids by addition of Triton-protein micelles to a lipid suspension or to the dry lipid. All of the phospholipid (egg phosphatidylcholine or mixtures of egg phosphatidylcholine and egg phosphatidylethanolamine) that contributed to the observed 31P NMR signal under these conditions appeared to be in a bilayer configuration. Freeze-fracturing and negative-staining electron microscopy showed unilamellar vesicles and multilayers. An isotropic signal could be attributed to vesicle rotation, judging from its sensitivity to increasing viscosity. The presence of small vesicles was also noticeable in the 31P NMR spectra of planar oriented membranes. In the presence of phosphatidylethanolamine, aggregation of protein particles was observed. Gel chromatography of the protein-Triton-phospholipid mixture revealed that, before Triton removal, large amounts of protein are associated with multibilayers. Separation of loaded and unloaded membranes by centrifugation in D2O showed that, upon stepwise addition, protein incorporates preferentially into unloaded liposomes. From these findings a mechanism of protein reincorporation was deduced.     

Freeze-etch electron microscopy of erythrocytes,Acholeplasma laidlawii cells and liposomal membranes after the action of filipin and amphotericin B

Freeze-etch electron microscopy demonstrated that filipin induces the formation of aggregates 150–250Åin diameter, in the membranes of rat erythrocytes, in cholesterol-containing membranes ofAcholeplasma laidlawii cells and in egg lecithin-cholesterol liposomes. No change in fracture faces was observed when cholesterol was absent in the membranes ofA. laidlawii, and lecithin liposomes.Amphotericin B does not visibly affect the freeze-etch morphology of erythrocytes, cholesterol-containingA. laidlawii cells and lecithin-cholesterol liposomes.     

Interaction of rat lecithin-cholesterol acyltransferase with rat apolipoprotein A-I and with lecithin-cholesterol vesicles.

The interaction of rat plasma lecithin-cholesterol acyltransferase with lecithin-cholesterol vesicles and with rat apo-A-I was studied in comparison with that of human plasma lecithin-cholesterol acyltransferase to clarify the reaction mechanism of rat plasma lecithin-cholesterol acyltransferase. The interaction of both human and rat lecithin-cholesterol acyltransferase with lecithin-cholesterol vesicles was investigated by gel permeation chromatography on Superose 12. Both enzymes had almost the same affinity to the vesicles. The affinity of rat enzyme to rat apo-A-I was stronger than that of human enzyme to human apo-A-I when estimated on the apo-A-I-Sepharose 4B column. When human apo-A-I was added to the human enzyme/vesicle mixture which contained the enzyme-vesicle complex, the enzyme was effectively dissociated from the complex. But when rat apo-A-I was added to the rat enzyme/vesicle mixture, apo-A-I-enzyme-vesicle complex was still recognized by its elution pattern on gel permeation chromatography. This suggests that the mixture of rat enzyme, rat apo-A-I, and vesicles, which are the major components in the rat lecithin-cholesterol acyltransferase reaction, forms a stronger complex than do the components of the human reaction.     

Lutein and zeaxanthin as protectors of lipid membranes against oxidative damage: the structural aspects.

Two main xanthophyll pigments are present in the membranes of macula lutea of the vision apparatus of primates, including humans: lutein and zeaxanthin. Protection against oxidative damage of the lipid matrix and screening against excess radiation are the most likely physiological functions of these xanthophyll pigments in macular membranes. A protective effect of lutein and zeaxanthin against oxidative damage of egg yolk lecithin liposomal membranes induced by exposure to UV radiation and incubation with 2, 2'-azobis(2-methypropionamidine)dihydrochloride, a water-soluble peroxidation initiator, was studied. Both lutein and zeaxanthin were found to protect lipid membranes against free radical attack with almost the same efficacy. The UV-induced lipid oxidation was also slowed down by lutein and zeaxanthin to a very similar rate in the initial stage of the experiments (5-15 min illumination) but zeaxanthin appeared to be a better photoprotector during the prolonged UV exposure. The decrease in time of a protective efficacy of lutein was attributed to the photooxidation of the carotenoid itself. Both lutein and zeaxanthin were found to slightly modify mechanical properties of the liposomes in a very similar fashion as concluded on the basis of H(1) NMR and diffractometric measurements of pure egg yolk membranes and membranes pigmented with the xanthophylls. Linear dichroism analysis of the mean orientation of the dipole transition moment of the xanthophylls incorporated to the lipid multibilayers revealed essentially different orientation of zeaxanthin and lutein in the membranes. Zeaxanthin was found to adopt roughly vertical orientation with respect to the plane of the membrane. The relatively large orientation angle between the transition dipole and the axis normal to the plane of the membrane found in the case of lutein (67 degrees in the case of 2 mol% lutein in EYPC membranes) was interpreted as a representation of the existence of two orthogonally oriented pools of lutein, one following the orientation of zeaxanthin and the second parallel with respect to the plane of the membrane. The differences in the protective efficacy of lutein and zeaxanthin in lipid membranes were attributed to a different organization of zeaxanthin-lipid and lutein-lipid membranes.     

Molecular motion and order in oriented lipid multibilayer membranes evaluated by simulations of spin label ESR spectra. Effects of temperature,cholesterol and magnetic field

A simulation method to interpret electron spin resonance (ESR) of spin labelled amphiphilic molecules in oriented phosphatidylcholine multibilayers in terms of a restricted motional model is presented. Order and motion of the cholestane spin label (3-spiro-doxyl-5α-cholestane) incorporated into egg yolk phosphatidylcholine, dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine, pure and in mixture with cholesterol, were studied at various termperatures. With egg yolk phosphatidylcholine identical sets of motional parameters were obtained from simulations of ESR spectra obtained at three microwave frequencies (X-, K- and Q-band). With dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine analyses of the spectra show that phase transitions occur in samples containing up to 30 mol % cholesterol. The activation energy for the motion of the spin label is about three times larger above than below the phase transition, indicating a more collective motion in the liquid crystalline state than in the gel state. In the liquid crystalline state the activation energy is larger in the pure phosphatidylcholines than with cholesterol added. Additions of cholesterol to egg phosphatidylcholine induces a higher molecular order but does not appreciably affect correlation times. This is in contrast to dipalmitoylphosphatidylcholine where both order and correlation times are affected by the presence of cholesterol. The activation energies follow the same order as the transition temperatures: dipalmitoylphosphatidylcholine > dimyristoylphosphatidylcholine > egg yolk phosphatidylcholine, suggesting a similar order of the cooperativity of the motion of the lipid molecules. Magnetic field-induced effects on egg phosphatidylcholine multibilayers.     

Molecular motion and order in oriented lipid multibilayer membranes evaluated by simulations of spin label ESR spectra. Effects of temperature, cholesterol and magnetic field.

A simulation method to interpret electron spin resonance (ESR) of spin labelled amphiphilic molecules in oriented phosphatidylcholine multibilayers in terms of a restricted motional model is presented. Order and motion of the cholestane spin label (3-spiro-doxyl-5alpha-cholestane) incorporated into egg yolk phosphatidylcholine, dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine, pure and in mixture with cholesterol, were studied at various temperatures. With egg yolk phosphatidylcholine identical sets of motional parameters were obtained from simulations of ESR spectra obtained at three microwave frequencies (X-, K- and Q-band). With dipalmitoylphosphatidylcholine and dimyristoylphosphatidylcholine analyses of the spectra show that phase transitions occur in samples containing up to 30 mol % cholesterol. The activation energy for the motion of the spin label is about three times larger above than below the phase transition, indicating a more collective motion in the lipid crystalline state than in the gel state. In the liquid crystalline state the activation energy is larger in the pure phosphatidylcholines than with cholesterol added. Additions of cholesterol to egg phosphatidylcholine induces a higher molecular order but does not appreciably affect correlation times. This is in contrast to dipalmitoylphosphatidylcholine where both order and correlation times are affected by the presence of cholesterol. The activation energies follow the same order as the transition temperatures: dipalmitoylphosphatidylcholine greater than dimyristoylphosphatidylcholine greater than egg yokd phosphatidylcholine, suggesting a similar order of the cooperativity of the motion of the lipid molecules. Magnetic field-induced effects on egg phosphatidylcholine multibilayers were found at Q-band measurements above 40 degrees C. The cholestane spin label mimics order and motion of cholesterol molecule incorporated into the lipid bilayers. This reflects order and motion of the portions of the lipid molecules on the same depth of the bilayer as the rigid steroid portions of the intercalated molecules.     

The synthesis and properties of a functional fluorescent cholesterol analog

The synthesis of a new fluorescent cholesterol analog is described. The analog contains a cholesterol nucleus attached via a hydrophilic spacer to N-4-nitrobenzo-2-oxa-1,3-diazole. Since the cholesterol moiety is not perturbed this molecule probably interacts with lipid bilayers in much the same way as cholesterol itself does. The compound can be readily incorporated into small unilamellar vesicles by sonicating a mixture of it with egg yolk phosphatidylcholine in a buffer. Furthermore, the analog can be incorporated into preformed membranes either by exchange from vesicles containing the analog or by uptake from sonicated micelles of the analog. Thus this analog shows potential as a useful tool for studying the interactions of cholesterol with cell membranes.     

Cholesterol as activator of ADP-ATP exchange in reconstituted liposomes and in mitochondria

The influence of cholesterol on ADP-ATP exchange activity was measured in the reconstituted system, submitochondrial (sonic) particles and mitoplasts (isolated inner mitochondrial membranes). In the reconstituted system, cholesterol markedly enhanced the nucleotide-uptake rate, when added to membranes of various compositions i.e., pure phosphatidylcholine, phosphatidylcholine/phosphatidylethanolamine mixtures and crude egg yolk phospholipids. The stimulation was linearly dependent on the amount of incorporated cholesterol up to 7–13% added sterol, depending on the type of phospholipids. Cholesterol influenced neither the amount of actively reconstituted carrier proteins nor the affinity of the carrier towards nucleotides nor the breakpoint of temperature dependence in the Arrhenius plot. The stimulation could be correlated with an increase in the molecular activity of the carrier protein. The influence of cholesterol was also measured in the natural environment of the carrier protien, i.e., the inner mitochondrial membrane. Both with submitochondrial particles from beef heart and especially with mitoplasts from rat liver, incorporation of cholesterol by fusion with sterol-containing liposomes led to a stimulation of ADP-ATP exchange activity, comparable to the effect in the reconstituted system. These results are discussed in relation to the absence of cholesterol in the inner mitochondrial membrane and in the view of the generally accepted ordering effect of cholesterol on phospholipid bilayers.     

Yolk lipids.

The mature egg yolk of the domestic hen possesses remarkably constant lipid and lipoprotein composition despite much variation in dietary and environmental conditions. The greatest differences are seen in the fatty acid composition of the triacylglycerols which may show significant alterations in the content of the minor acids including certain polyunsaturated acids. The lipid class composition appears to be minimally affected by dietary influences, including the cholesterol content of the diet. The limited dietary influence on the yolk lipid composition extends to different strains of the hens. Genetic selection has led to some increase in the cholesterol content of the egg, but the desired lowering of the cholesterol content of egg yolk has not been realized. Likewise, production of a polyunsaturated fatty acid egg does not appear to be practical. As a result the egg yolk continues to provide a food product of nearly constant composition, which serves to maintain its chemical and physico-chemical properties for reliable utilization in the baking, cosmetic and pharmaceutical industries. The great uniformity in the composition of the egg yolk phospholipids makes them desirable starting materials for partial chemical resynthesis of glycerophospholipids. Partial hydrogenation of the egg yolk lipids promises to further increase the utility of the product as a desirable material for the manufacture of liposomes and liposome based drug products. In contrast, the constancy of the egg yolk composition and the inability to alter it significantly by dietary or genetic means also renders egg yolk undesirable for unlimited human consumption. Excessive ingestion of egg yolk raises plasma lipid and cholesterol levels which are believed to contribute to the development of heart disease. The physico-chemical and biological properties of egg yolk apoproteins have been less extensively investigated and their function is less well understood. The finding that phosvitin is a effective chelator of metal ions and thus an effective antioxidant demonstrates that egg yolk lipoproteins possess as yet unexplored potential for beneficial nutritional, medical and industrial application.