Reconstitution of lipoproteins. II. Lipid-protein interaction between dimyristoyl-lecithin and dimyristoyl-lecithin:cholesterol vesicles and purified apolipoprotein C-I and C-III2 studied by isomeric spin-labelled lecithins

The reconstitution of purified apolipoprotein C-I and C-III2 with sn-3-dimyristoyl-lecithin and sn-3-dimyristoyl-lecithin:cholesterol (10:1) vesicles was studied by electron spin resonance spectroscopy using isomeric 5'-, 12'-, and 16'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithin probes. Results obtained from the temperature-induced changes of lipoprotein recombinants showed the hydrophilic nature of the lipid-protein interactions. The temperature-induced phospholipid phase transition, as measured by 5'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithin probe in recombinants containing apoprotein C-1 or apoprotein C-iii2, is very broad and has a small cooperative unit indicative of extensive lipid-protein interactions occurring at the head group region of the phospholipid bilayer. When 12"- and 16'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithins are used as probes in the same system, similar sharper and more cooperative lipid phase changes are detected. These results indicate a surface location for both apoprotein C-I and apoprotein C-III2 with respect to the phospholipid bilayer in lipoprotein recombinants with and without cholesterol.     

Rotational correlation time of spin-labeled alpha-chymotrypsin

α-Chymotrypsin has been spin labeled with (I), 3-carboxy 2,2,5,5-tetramethyl pyrrolidine-N-oxyl-p-nitrophenyl ester, and (II), the N-oxyl-4′,4′-dimethyl oxazolidine of 5-α-androstan-3-one-17-β-methyl phosphono-fluoridate. The positions of the high field hyperfine components in the paramagnetic resonance spectra of the spin-labeled enzymes have been measured as a function of solvent viscosity and have been used to determine a rotational correlation time of 12 nanoseconds for α-chymotrypsin in water at 20° C.     

A spin label study of E. coli membrane vesicles

The phase transition in E. coli membrane vesicles has been investigated by the spin labeling technique. N-oxyl-4′,4′-dimethyloxazolidine derivatives of stearic acid were incorporated into the vesicles. The results suggest that there are two phase transitions in these bacterial membrane vesicles (one at ≈20°C and the other at ≈30°C). These two phase transitions may be related to some of the functional properties of the membranes.     

Domain formation in sphingomyelin/cholesterol mixed membranes studied by spin-label electron spin resonance spectroscopy

Interactions of palmitoylsphingomyelin with cholesterol in multilamellar vesicles have been studied over a wide range of compositions and temperatures in excess water by using electron spin resonance (ESR) spectroscopy. Spin labels bearing the nitroxide free radical group on the 5 or 14 C-atom in either the sn-2 stearoyl chain of phosphatidylcholine (predominantly 1-palmitoyl) or the N-stearoyl chain of sphingomyelin were used to determine the mobility and ordering of the lipids in the different phases. Two-component ESR spectra of the 14-position spin labels demonstrate the coexistence first of gel (L(beta)) and liquid-ordered (L(o)) phases and then of liquid-ordered and liquid-disordered (L(alpha)) phases, with progressively increasing temperature. These phase coexistences are detected over a limited range of cholesterol contents. ESR spectra of the 5-position spin labels register an abrupt increase in ordering at the L(alpha)-L(o) transition and a biphasic response at the L(beta)-L(o) transition. Differences in outer splitting between the C14-labeled sphingomyelin and phosphatidylcholine probes are attributed to partial interdigitation of the sphingomyelin N-acyl chains across the bilayer plane in the L(o) state. In the region where the two fluid phases, L(alpha) and L(o), coexist, the rate at which lipids exchange between phases (<7 x 10(7) s(-)(1)) is much slower than translational rates in the L(alpha) phase, which facilitates resolution of two-component spectra.     

Relationships between the Transition of the Physical Phase of Membrane Lipids and Photosynthetic Parameters in Anacystis nidulans and Lettuce and Spinach Chloroplasts

The transition of the physical phase of lipids in membrane fragments of a blue-green alga Anacystis nidulans was studied by a spin labeling technique. The maximum hyperfine splitting of the electron spin resonance spectrum of the N-oxyl-4′, 4′-dimethyloxazolidine derivative of 5-ketostearic acid plotted against the reciprocal of the absolute temperature gave a discontinuity point that was characteristic of a transition of the physical phase of the hydrocarbon region of membrane lipids. The phase transition appeared at approximately 13 or 24 C in the organisms grown at 28 or 38 C, respectively.     

Structure and cohesive properties of sphingomyelin/cholesterol bilayers.

Thermal, structural, and cohesive measurements have been obtained for both bovine brain sphingomyelin (BSM) and N-tetracosanoylsphingomyelin (C24-SM) in the presence and absence of cholesterol. A goal of these experiments has been to clarify the mechanisms responsible for the strong interaction between sphingomyelin and cholesterol. Differential scanning calorimetry shows that fully hydrated bilayers of BSM and C24-SM have main endothermic phase transitions at 39 and 46 degrees C, respectively, that reflect the melting of the acyl chains from a gel to a liquid-crystalline phase. For each lipid, the addition of cholesterol monotonically reduces the enthalpy of this transition, so that at equimolar cholesterol the transition enthalpy is zero. The addition of equimolar cholesterol to either BSM or C24-SM coverts the wide-angle X-ray diffraction reflection at 4.15 A to a broad band centered at 4.5 A. Electron density profiles of gel-phase C24-SM bilayers contain two terminal methyl dips in the center of the bilayer, indicating that the lipid hydrocarbon chains partially interdigitate so that the long saturated 24-carbon acyl chains in one monolayer cross the bilayer center and appose the shorter sphingosine chains from the other monolayer. The incorporation of cholesterol adds electron density to the hydrocarbon chain region near the head group and removes the double terminal methyl dip. These wide- and low-angle X-ray data indicate that cholesterol packs into the hydrocarbon chain region near the sphingomyelin head group, fluidizes the methylene chains near the center of the bilayer compared to the gel phase, and reduces the extent of methylene chain interdigitation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological transitions of brain sphingomyelin are determined by the hydration protocol: ripples re-arrange in plane, and sponge-like networks disintegrate into small vesicles.

The phase transition of hydrated brain sphingomyelin occurs at around 35 degrees C, which is close to the physiological temperature. Freeze-fracture electron microscopy is used to characterize different gel state morphologies in terms of solid-ordered and liquid-ordered phase states, according to the occurrence of ripples and other higher-dimensional bilayer deformations. Evidently, the natural mixed-chain sphingomyelin does not assume the flat L beta, phase but instead the rippled P beta, phase, with symmetric and asymmetric ripples as well as macroripples and an egg-carton pattern, depending on the incubation conditions. An unexpected difference was observed between samples that are hydrated above and below the phase transition temperature. When the lipid is hydrated at low temperature, a sponge-like network of bilayers is formed in the gel state, next to some normal lamellae. The network loses its ripples during cold-incubation, which indicates the formation of a liquid-ordered (lo) gel phase. Ripples re-appear upon warming and the sponge-like network disintegrates spontaneously and irreversibly into small vesicles above the phase transition.     

Cholesterol favors phase separation of sphingomyelin

The phase behavior of mixed lipid dispersions representing the inner leaflet of the cell membrane has been characterized by X-ray diffraction. Aqueous dispersions of phosphatidylethanolamine:phosphatidylserine (4:1 mole/mole) have a heterogeneous structure comprising an inverted hexagonal phase H(II) and a lamellar phase. Both phases coexist in the temperature range 20-45 degrees C. The fluid-to-gel mid-transition temperature of the lamellar phase assigned to phosphatidylserine is decreased from 27 to 24 degrees C in the presence of calcium. Addition of sphingomyelin to phosphatidylethanolamine/phosphatidylserine prevents phase separation of the hexagonal H(II) phase of phosphatidylethanolamine but the ternary mixture phase separates into two lamellar phases of periodcity 6.2 and 5.6 nm, respectively. The 6.2-nm periodicity is assigned to the gel phase enriched in sphingomyelin of molecular species comprising predominantly long saturated hydrocarbon chains because it undergoes a gel-to-fluid phase transition above 40 degrees C. The coexisting fluid phase we assign to phosphatidylethanolamine and phosphatidylserine and low melting point molecular species of sphingomyelin which suppresses the tendency of phosphatidylethanolamine to phase-separate into hexagonal H(II) structure. There is evidence for considerable hysteresis in the separation of lamellar fluid and gel phases during cooling. The addition of cholesterol prevents phase separation of the gel phase of high melting point sphingomyelin in mixtures with phosphatidylserine and phosphatidylethanolamine. In the quaternary mixture the lamellar fluid phase, however, is phase separated into two lamellar phases of periodicities of 6.3 and 5.6 nm (20 degrees C), respectively. The lamellar phase of periodicity 5.6 nm is assigned to a phase enriched in aminoglycerophospholipids and the periodicity 6.3 nm to a liquid-ordered phase formed from cholesterol and high melting point molecular species of sphingomyelin characterized previously by ESR. Substituting 7-dehydrocholesterol for cholesterol did not result in evidence for lamellar phase separation in the mixture within the temperature range 20-40 degrees C. The specificity of cholesterol in creation of liquid-ordered lamellar phase is inferred.     

Phase transitions and hydrogen bonding in a bipolar phosphocholine evidenced by calorimetry and vibrational spectroscopy.

As a model for natural archaebacterial bolalipids, we have synthesized omega-hydroxybehenylphosphocholine (HBPC, HO-(CH(2))(22)-OP(O(-)(2))O-(CH(2))(2)-N+(CH(3))(3)) and investigated it, by Fourier-transform infrared and Raman spectroscopy and differential scanning calorimetry, both as fully hydrated dispersions (varying temperature) and as aligned films (varying hydration) in terms of particular structural features predestining such bipolar lipids for their occurrence in extremophilic organisms. The phase behavior of HBPC in dispersions depends on sample pretreatment as it comprises metastabilities in annealed samples. However, main transition proceeds consistently near 81 degrees C. Some (extra) deal of headgroup (phosphate) hydration accompanying a gel-gel phase transition near 66 degrees C appears to precede chain melting. Studies with HBPC films revealed lamellar interdigitated-like solid phases with an extraordinarily strong omega-OH--OPO(-) omega-OH--OPO(-) omega-OH hydrogen-bond pattern formed along both sides of the resulting monolayers. The "clamping" effect inherent to such structures provides a clue to explain the relatively high main-transition temperature of HBPC assemblies.     

Interaction of cholesterol with various glycerophospholipids and sphingomyelin

The influence of cholesterol on the phase behavior of glycerophospholipids and sphingomyelins was investigated by spin-label electron spin resonance (ESR) spectroscopy. 4-(4,4-Dimethyl-3-oxy-2-tridecyl-2-oxazolidinyl)butanoic acid (5-SASL) and 1-stearoyl-2-[4-(4,4-dimethyl-3-oxy-2-tridecyl-2-oxazolidinyl)butanoy l]-sn- glycero-3-phosphocholine (5-PCSL) spin-labels were employed for this purpose. The outer hyperfine splitting constants, Amax, measured from the spin-label ESR spectra as a function of temperature were taken as empirical indicators of cholesterol-induced changes in the acyl chain motions in the fluid state. The Amax values of 5-PCSL exhibit a triphasic dependence on the concentration of cholesterol for phosphatidylcholines and bovine brain sphingomyelin. We interpret this dependence as reflecting the existence of liquid-disordered, ld, liquid-ordered, lo, and coexistence regions, ld + lo. The phase boundary between the ld and the two-phase region and the boundary between the lo and the two-phase region in the phosphatidylcholine-cholesterol systems coalesce at temperatures 25-33 degrees C above the main-chain melting transition temperature of the cholesterol-free phosphatidylcholine bilayers. In the case of bovine brain sphingomyelin, the ld-lo phase coalescence occurs about 47 degrees C above the melting temperature of the pure sphingomyelin. The selectivity of interaction of cholesterol with glycerophospholipids of varying headgroup charge was studied by comparing the cholesterol-induced changes in the Amax values of derivatives of phosphatidylcholine, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine spin-labeled at the fifth position of the sn-2 chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the lack of a specific interaction between cholesterol and sphingomyelin

The putative specific interaction and complex formation by sphingomyelin and cholesterol was investigated. Accordingly, low contents (1 mol % each) of fluorescently labeled derivatives of these lipids, namely 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PyrPC), n-[10-(1-pyrenyl)decanoyl]sphingomyelin (PyrSM), and increasing concentrations of cholesterol (up to 5 mol %), were included in large unilamellar vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dinervonoyl-sn-glycero-3-phosphocholine (DNPC), and the excimer/monomer fluorescence emission ratio (I(e)/I(m)) was measured. In DNPC below the main phase transition, the addition of up to 5 mol % cholesterol reduced I(e)/I(m) significantly. Except for this, cholesterol had only a negligible effect in both matrices and for both probes. We then compared the efficiency of resonance energy transfer from PyrPC and PyrSM to 22-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBDchol). An augmenting colocalization of the latter resonance energy transfer pair with temperature was observed in a DMPC matrix below the main phase transition. In contrast, compared to PyrSM the colocalization of PyrPC with NBDchol was more efficient in the longer DNPC matrix. These results could be confirmed using 5,6-dibromo-cholestan-3beta-ol as a collisional quencher for the pyrene-labeled lipids. The results indicate lack of a specific interaction between sphingomyelin and cholesterol, and further imply that hydrophobic mismatch between the lipid constituents could provide the driving force for the cosegregation of sphingomyelin and cholesterol in fluid phospholipid bilayers of thicknesses comparable to those found for biomembranes.     

Dehydration induces lateral expansion of polyunsaturated 18:0-22:6 phosphatidylcholine in a new lamellar phase.

To gain a better understanding of the biological role of polyunsaturated phospholipids, infrared (IR) linear dichroism, NMR, and x-ray diffraction studies have been conducted on the lyotropic phase behavior and bilayer dimensions of sn-1 chain perdeuterated 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC-d35), a mixed-chain saturated (18:0)-polyunsaturated (22:6 omega 3) lipid. SDPC films were hydrated at definite values of temperature (T) and relative humidity (RH). In excess water, the lipid forms exclusively lamellar phases in the temperature range 0--50 degrees C. Upon dehydration the lipid undergoes the main phase transition between the liquid-crystalline (L(alpha)) and gel (L(beta)) phase at T < 15 degrees C. Both the saturated and polyunsaturated chains adopt a stretched conformation in the L(beta) phase, presumably the all-trans (stearoyl) and angle iron or helical (docosahexaenoyl) one. A new fluid lamellar phase (L(alpha)') was found in partially hydrated samples at T > 15 degrees C. SDPC membranes expand laterally and contract vertically in the L(alpha)' phase when water was removed. This tendency is in sharp contrast to typical dehydration-induced changes of membrane dimensions. The slope of the phase transition lines in the RH-T phase diagram reveal that the lyotropic L(alpha)'-L(alpha) and L(beta)-L(alpha) transitions are driven by enthalpy and entropy, respectively The possible molecular origin of the phase transitions is discussed. The properties of SDPC are compared with that of membranes of monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d31).     

Negatively charged phospholipids and their position in the cholesterol affinity sequence

The effects of low concentrations of cholesterol in mixtures of a negatively charged phospholipid (phosphatidylserine or phosphatidylglycerol) and another phospholipid (phosphatidylcholine, sphingomyelin or phosphatidylethanolamine) have been studied by differential scanning calorimetry. Only mixtures which showed a gel phase miscibility gap have been employed. It was demonstrated that in mixtures with phosphatidylethanolamine, cholesterol was preferentially associated with the negatively charged phospholipid, regardless whether this species represented the component with the high or with the low transition temperature in the mixture. In mixtures of a negatively charged phospholipid and phosphatidylcholine, cholesterol associated with the negatively charged phospholipid; when the phosphatidylcholine was the species with the low transition temperature, cholesterol had an affinity for the phosphatidylcholine and for the negatively charged phospholipid as well. Cholesterol, in a mixture of sphingomyelin with a high and phosphatidylserine with a low transition temperature, was preferentially associated with sphingomyelin.From these experiments it is concluded that phospholipids show a decrease in affinity for cholesterol in the following order: sphingomyelin ? phosphatidylserine, phosphatidylglycerol > phosphatidylcholine ? phosphatidylethanolamine.     

X-ray diffraction studies of lipid phase transitions in cholesterol-rich membranes at sub-zero temperatures

In X-ray diffraction studies of hydrated (greater than 60%) cholesterol/dioleoylphosphatidylcholine mixtures the lipid packing band showed an abrupt transition from liquid crystal-type to gel-type position and definition at a temperature which decreased progressively to almost -50 degrees C as the proportion of cholesterol was increased to a saturation level of about 50 mol%. Plots of transition temperature against composition (mol% cholesterol) and of peak position against composition provided evidence of a significant change in phospholipid configuration at about 20 mol% cholesterol. However, the data overall suggested a uniform dispersion of the cholesterol molecules in the phospholipid bilayer at all concentrations up to the saturation point. Parallel studies of hydrated lipid extract of erythrocyte membranes and of several cholesterol-rich membrane preparations showed a similar overall change from liquid crystal-type packing at +20 degrees C to a gel-type packing at -30 degrees C to -40 degrees C but without displaying a defined transition temperature.     

Partial purification and characterization of phospholipase C from Yersinia enterocolitica

About 34% of the strains of Yersinia enterocolitica isolated from raw milk were found to produce lecithinase. A selected strain produced phospholipase C at 22°C and 37°C; production was optimum at 37°C in the stationary phase (14–16 h). A decrease in phospholipase C activity at various storage temperatures (—5°C, 4°C, 37°C) was also observed, although the enzyme was active over a wide range of temperature (5–65°C) and pH (3mD5–7mD5). The phospholipase C was partially purified by ammonium sulphate precipitation and Sephadex column chromatography, and characterized.     

Composition and physical properties of lipids from plasma membranes of dog kidney

Lipid composition, physical state of major phospholipid classes and transbilayer migration of phosphatidylcholine have been determined in plasma membranes of the dog kidney. The lipid composition of brush-border membranes markedly differs from that of antiluminal membranes with respect to: (a) the total phospholipid content; (b) the cholesterol to phospholipid ratio (C/P); (c) the distribution of the major phospholipid classes. Sphingomyelin present in large amounts in both luminal and antiluminal membranes extracts exhibits a transition of phase between 20 and 44 degrees C approximately. In the range of temperature studied (5-55 degrees C) no phase transitions were detected for the other phospholipid species. Our data suggest that: (1) at physiological temperature the higher C/P ratio of brush-border membranes is in large part responsible for their lower fluidity; (2) both the relatively low cholesterol and high sphingomyelin contents contribute to the thermotropic transitions observed in intact membranes. Finally transbilayer migration of phosphatidylcholine in brush-border membranes is a very slow process with a half time of 6.5 h at 37 degrees C which compares with that of other biological membranes.     

Orientational order in the choline headgroup of sphingomyelin: A 14N-NMR study

An aqueous dispersion of fully hydrated bovine sphingomyelin was studied using ¹⁴N-NMR spectroscopy. Spectra were obtained as a function of temperature over the range 15–80°C, in both the liquid crystal and gel phases. In the liquid crystal phase, powder pattern lineshapes were obtained, whose quadrupolar splitting slowly decreases with increasing temperature. The spectra are increasingly broadened as the temperature is lowered through the phase transition into the gel phase. The linewidths and the second moments of these spectra indicate that the onset of a broad phase transition occurs at approx. 35°C, in agreement with previous calorimetric and ³¹P-NMR measurements. There is no evidence from the lineshapes for an hexagonal phase in this system, and this conclusion is supported by X-ray diffraction measurements carried out on aqueous dispersions of sphingomyelin in both phases. Assuming that the static nitrogen quadrupole coupling constant is the same for both sphingomyelin and dipalmitoyl-l-α-phosphatidylcholine (DPPC), the decrease observed in the quadrupolar splitting of sphingomyelin compared to that of DPPC indicates that the orientational order of the choline headgroup in liquid crystalline sphingomyelin is not the same as that of its counterpart in DPPC. Preliminary relaxation time measurements of $\text{T}{}_1$ and $\text{T}{}_2$ are presented which suggest that there are also dynamic differences between sphingomyelin and DPPC in the choline headgroup.     

A synchrotron X-ray diffraction characterization of the structure of complexes formed between sphingomyelin and cerebroside

Specific lipid-lipid interactions are believed to be responsible for lateral domain formation in the lipid bilayer matrix of cell membranes. The miscibility of glucocerebroside and sphingomyelin extracted from biological tissues has been examined by synchrotron X-ray powder diffraction methods. Fully hydrated binary mixtures of egg-sphingomyelin codispersed with glucosylceramide rich in saturated C22 and C24 N-acyl fatty acids were subjected to heating scans between 20 and 90 °C at 2 °C·min(-1). X-ray scattering intensity profiles were recorded at 1 °C intervals simultaneously in both small-angle and wide-angle scattering regions. A gel phase characterized by a single symmetric peak in the wide-angle scattering region was transformed in all mixtures examined to a fluid phase at about 40 °C, similar to dispersions of pure egg-sphingomyelin. A coexisting lamellar structure was identified at temperatures up to about 75 °C which was characterized by a broad Bragg reflection. The scattering intensity of this structure increased relative to the structure assigned as bilayers of pure sphingomyelin with increasing proportions of glucosylceramide in the mixture. The relationship between the scattering intensities of the two peaks and the relative mass fractions of the two lipids showed that the bilayers assigned to a glucosylceramide-rich structure were composed of sphingomyelin and glucosylceramide in molar ratios of 1 : 1 and 2 : 1, respectively, at temperatures below and above the order-disorder phase transition temperature of the sphingomyelin (40 °C).     

On the biosynthesis of cerebrosides containing non-hydroxy acids. 1. Mass spectrometric evidence for the psychosine pathway

[1, 1, 1′, 2′, 3′, 4′, 5′, 6′, 6′-²H₉]1-O-(β-galactopyranosyl) DL-sphinganine and [4, 5-³H₂]1-O-(β-D-galactopyranosyl) D-sphinganine^∗ were prepared, and the conversion to cerebroside of a mixture of these compounds was studied with rat brain microsomes. The product was characterized by thin layer radiochromatography in several solvent systems and, as the trimethylsilyl ether derivative, by gas-liquid chromatography — mass spectrometry. The mass spectrometric analyses conclusively showed that the glycosidic bond of the substrate remained intact during the transformation to cerebroside.