ESR studies of the dynamics of dipalmitoylglycerophosphocholine-sulfatide model membranes

The effects of sulfatide on the fluidity and surface dynamics of bilayered and micellar model membranes of dipalmitoylglycerophosphocholine containing sulfatide were studied as a function of gel-to-liquid-crystalline state of the lipids by electron spin resonance. 5- and 15-nitroxystearic acid were employed as spinlabel probes for the region close to the surface and that close to the nonpolar core of lipid structures. The sulfatide effect is completely different above and below the gel-to-liquid-crystalline phase transition point, the glycolipid promoting a more disordered state below it and having a condensing effect above the phase transition temperature.     

Electron spin resonance studies on the dynamics of phosphatidylcholine — sulfatide model membranes

The effects of sulfatide on the fluidity and surface dynamics of bilayered and micellar model membranes of egg phosphatidylcholine containing sulfatide were studied by electron spin resonance (ESR). 5-Nitroxystearic acid and 15-nitroxystearic acid were employed as spin-label probes for the region close to the surface and that close to the hydrophobic core of lipid structures. In the vesicular structures, the signals generated by 5-nitroxystearic acid showed that the presence of sulfatide reduced the mobility of the hydrocarbon chains around the probe. The effect increased with increasing glycolipid concentration. The decrease in membrane fluidity was also monitored with the 15-nitroxystearic acid probe, although to a lesser extent. We think that sulfatide causes strong side-to-side head-group interactions on the bilayer surface, causing the lipid chains to assemble in a more rigid fashion, though this effect may be balanced in part by the disordered mechanical coupling of glycolipid acyl chains in theapposite faces of the hydrophobic core of the bilayer. Reduction of this mechanical coupling between apposite lipids when there was transition from a bilayered to a micellar structure resulted in a further increase in the order of the system.     

Thermal and structural behavior of natural cerebroside 3-sulfate in bilayer membranes

Differential scanning calorimetry (DSC), polarizing microscopy and X-ray diffraction studies have been performed on dry and hydrated natural bovine brain sulfatides. Dry sulfatide fractions exhibit a high temperature transition (delta H = 6.6 kcal/mol sulfatide) at 87.3 degrees C. X-ray diffraction shows this transition to be associated with a hydrocarbon chain order-disorder transformation between two lamellar phases. Hydrated sulfatide dispersions undergo a complex chain order-disorder transition (delta H = 7.5 kcal/mol sulfatide) at 32 degrees C with two peak temperatures at 35 degrees C and 47 degrees C. Structural studies performed on hydrated liquid-crystal sulfatide dispersions at 75 degrees C verify the existence of a bilayer structure over the 16 wt.% to 50 wt.% phosphate buffer (pH = 7.4) range. The interbilayer separation between galactosyl-3-sulfate groups averages 48 A as the multilamellar bilayers swell with the addition of phosphate buffer. The formation of micellar phases is not observed at high water contents. The comparison of the structural characteristics of dry and hydrated sulfatides with structural data for dry and hydrated bovine brain non-sulfated glycolipid (cerebroside) is discussed in molecular terms.     

Structure and phase behavior of lipid suspensions containing phospholipids with covalently attached poly(ethylene glycol).

Liposomes containing phospholipids with covalently attached poly(ethylene glycol) (PEG-lipids) are being developed for in vivo drug delivery. In this paper we determine the structure and phase behavior of fully hydrated distearoylphosphatidylcholine (DSPC) suspensions containing PEG-lipids composed of distearoylphosphatidylethanolamine with attached PEGs of molecular weights ranging from 350 to 5000. For DSPC:PEG-lipid suspensions containing 0-60 mol % PEG-lipid, differential scanning calorimetry shows main endothermic transitions ranging from 55 to 64 degrees C, depending on the size of the PEG and concentration of PEG-lipid. The enthalpy of this main transition remains constant for all PEG-350 concentrations but decreases with increasing amounts of PEG-750, PEG-2000, or PEG-5000, ultimately disappearing at PEG-lipid concentrations greater than about 60 mol %. Low-angle and wide-angle x-ray diffraction show that tilted gel (L beta') phase bilayers are formed for all PEG-lipid molecular weights at concentrations of about 10 mol % or less, with the distance between bilayers depending on PEG molecular weight and PEG-lipid concentration. At PEG-lipid concentrations greater than 10 mol %, the lipid structure depends on the size of the PEG moiety. X-ray diffraction analysis shows that untilted interdigitated (L beta I) gel phase bilayers form with the incorporation of 40-100 mol % PEG-350 or 20-70 mol % PEG-750, and untilted gel (L beta) phase bilayers are formed in the presence of about 20-60 mol % PEG-2000 and PEG-5000. Light microscopy, turbidity measurements, x-ray diffraction, and 1H-NMR indicate that a pure micellar phase forms in the presence of greater than about 60% PEG-750, PEG-2000, or PEG-5000.     

Preparation of asymmetric,cerebroside sulfate-containing phospholipid vesicles

A procedure is described which inserts asymmetrically cerebroside sulfate (‘sulfatide’) into the outer leaflet of bilayered phospholipid vesicles. Cerebroside sulfate is adsorbed onto a cellulose, filter-paper support and, when incubated with phosphatidylcholine vesicles is transferred to and inserted into the outer leaflet of these vesicles. This transfer occurs at, or above the transition temperature of the phospholipid and follows a similar pattern with small or larger (‘fused’) unilamellar vesicles. The transfer is linear with time for 1–2 h and is maximal after about 6 h, when the sulfatide content reaches about 6 mol% of the total quantity of phospholipid, corresponding to about 10 mol% of the phospholipids present in the outer layer. Initial rates of sulfatide transfer were somewhat increased when the vesicles contained a positively charged lipid (e.g. stearylamine) and decreased when this lipid was negatively charged (e.g. dicetyl phosphate) or hydrophobic (e.g. cholesterol). Divalent ions markedly inhibited sulfatide transfer and monovalent ions did so to a lesser degree. Once incorporated into the outer leaflet of the vesicle, the sulfatide could not be removed by washing with buffer, 1 M NaCl or 1 M urea.     

N-pyrene dodecanoyl sulfatide as membrane probe: a study of glycolipid dynamic behavior in model membranes

An N-linked pyrene-dodecanoyl sulfatide was employed to measure the ratio of excimer fluorescence to monomer fluorescence intensities (E/M). The E/M values provided information about both the dynamic behavior and the structural distribution of the labelled glycolipid in note dispersion of micellar sulfatides and multilamellar vesicles of different phospholipids. Most of the labelled sulfatide seems to be located in domains sequestered from the surrounding phospholipids still above the phase transition temperature of the vesicles. The glycolipids sequestered in these domain environments are less sensitive to the structural changes that the addition of cholesterol or Ca2+ can induce in the phospholipid regions during the phase transition.     

Thermotropic behavior of mixtures of glycosphingolipids and phosphatidylcholine: effect of monovalent cations on sulfatide and galactosylceramide

The thermotropic behavior of both sulfatide (3-sulfogalactosylceramide) and galactosylceramide in dielaidoylphosphatidylcholine (DEPC) liposomes was studied, using steady-state fluorescence polarization of parinaric acid isomers. The glycosphingolipid (GSL) concentration of the liposomes was varied from 0 to 100%, and phase diagrams were constructed. The data indicate that sulfatide and DEPC are immiscible in the gel phase at sulfatide mole ratios of less than 0.30. The temperature of onset of the gel-liquid-crystalline phase transition is higher in K+ -containing buffer than in osmotically equal Na+ -containing buffer. Similar measurements, using galactosylceramide, a neutral GSL, indicated that this lipid and DEPC are immiscible in the gel phase at galactosylceramide mole ratios of less than 0.40. In contrast to the results obtained with sulfatide, onset temperatures are identical in Na+- or K+-containing buffers. The phase properties of sulfatide/DEPC mixtures are shown to depend on the cation only when the sulfatides contain hydroxy fatty acids. Our observations indicate that physiologically relevant concentrations of monovalent cations affect motion and distribution of sulfatide in biological membranes and further implicate this GSL as an important determinant of function of the Na+,K+-ATPase. A preliminary report of these data [Rintoul, D.A., Welti, R., & Song, W. (1988) Biophys. J. 53, 126a].     

Effect of calcium ions on the thermotropic behaviour of neutral and anionic glycosphingolipids

In the concentration range of 10(-5) to 10(-1) M Ca2+ modulates the thermotropic properties of several neutral and anionic glycosphingolipids (galactosylceramide, asialo-GM1, sulfatide, GM1, GD1a, GT1b) and of their mixtures with dipalmitoylphosphatidylcholine. The transition temperature of gangliosides is not appreciably changed while the transition enthalpy increases by 20% in the presence of Ca2+. The more marked effect of Ca2+ is on the thermotropic behavior of systems containing sulfatide. Increasing concentrations of Ca2+ between 10(-5) and 10(-3) M (up to a molar ratio of Ca2+/sulfatide 1:2) induce a progressive increase of both the transition temperature and enthalpy. Further increases up to 10(-1) M Ca2+ induce a new phase transition at a lower temperature. No evidence is found for induction of phase separation of pure glycosphingolipid-Ca2+ domains in mixtures of any of the glycosphingolipids with dipalmitoylphosphatidylcholine. The modification of the phase behavior of anionic glycosphingolipids by Ca2+ does not involve detectable variations of the intermolecular packing but is accompanied by marked modifications of the dipolar properties of the polar head group region.     

Myelin Proteins in Reverse Micelles: Tight Lipid Association Required for Insertion of the Folch-Pi Proteolipid into a Membrane-Mimetic System

The solubility and reactivity of the Folch-Pi proteolipid from bovine CNS have been studied in reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate, isooctane, and water. Such a membrane-mimetic system resembles the aqueous spaces of the native myelin sheath in terms of its physicochemical properties. Although the proteolipid is completely insoluble in water, it can be inserted into the water-containing micellar system. In contrast, the lipid-depleted protein failed to be incorporated into these organized assemblies. The lipid requirements for insertion of the proteolipid were studied, therefore, after delipidation by several precipitations with isooctane, a nondenaturing solvent. Novel extraction procedures and quantitative analyses by HPLC of the protein-bound lipids revealed the persistence of a lipid-protein complex (6 +/- 1 mol of lipid/mol of protein) displaying optimal micellar solubilization. Competition experiments carried out with brain lipids provide evidence for a preference of the myelin protein for sulfatide, phosphatidylinositol, and phosphatidylserine, in that order. The resulting proteolipid, although differing in relative composition, showed good solubility in the membrane-mimetic system. In contrast, reconstitution experiments carried out with the lipid-depleted protein resulted in weak lipid binding and poor micellar incorporation. These results suggest that the tightly bound acidic lipids may stabilize a protein conformation required for insertion into the micellar system.     

Structure and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine bilayer membranes.

The structural and thermotropic properties of 1-stearoyl-2-acetyl-phosphatidylcholine (C(18):C(2)-PC) were studied as a function of hydration. A combination of differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the phase behavior of C(18):C(2)-PC. At low hydration (e.g., 20% H2O), the differential scanning calorimetry heating curve shows a single reversible endothermic transition at 44.6 degrees C with transition enthalpy delta H = 6.4 kcal/mol. The x-ray diffraction pattern at -8 degrees C shows a lamellar structure with a small bilayer periodicity d = 46.3 A and two wide angle reflections at 4.3 and 3.95 A, characteristic of a tilted chain, L beta' bilayer gel structure. Above the main transition temperature, a liquid crystalline L alpha phase is observed with d = 53.3 A. Electron density profiles at 20% hydration suggest that C(18):C(2)-PC forms a fully interdigitated bilayer at -8 degrees C and a noninterdigitated, liquid crystalline phase above its transition temperature (T > Tm). Between 30 and 50% hydration, on heating C(18):C(2)-PC converts from a highly ordered, fully interdigitated gel phase (L beta') to a less ordered, interdigitated gel phase (L beta), which on further heating converts to a noninterdigitated liquid crystalline L alpha phase. However, the fully hydrated (> 60% H2O) C(18):C(2)-PC, after incubation at 0 degrees C, displays three endothermic transitions at 8.9 degrees C (transition I, delta H = 1.6 kcal/mol), 18.0 degrees C (transition II), and 20.1 degrees C (transition III, delta HII+III = 4.8 kcal/mol). X-ray diffraction at -8 degrees C again showed a lamellar gel phase (L beta') with a small periodicity d = 52.3 A. At 14 degrees C a less ordered, lamellar gel phase (L beta) is observed with d = 60.5 A. However, above the transition III, a broad, diffuse reflection is observed at approximately 39 A, consistent with the presence of a micellar phase. The following scheme is proposed for structural changes of fully hydrated C(18):C(2)-PC, occurring with temperature: L beta' (interdigitated)-->L beta (interdigitated)-->L alpha(noninterdigitated)-->Micelles. Thus, at low temperature C(18):C(2)-PC forms a bilayer gel phase (L beta') at all hydrations, whereas above the main transition temperature it forms a bilayer liquid crystalline phase L alpha at low hydrations and a micellar phase at high hydrations (> 60 wt% water).     

Properties of ganglioside GM1 in phosphatidylcholine bilayer membranes.

Gangliosides have been shown to function as cell surface receptors, as well as participating in cell growth, differentiation, and transformation. In spite of their multiple biological functions, relatively little is known about their structure and physical properties in membrane systems. The thermotropic and structural properties of ganglioside GM1 alone and in a binary system with 1,2-dipalmitoyl phosphatidylcholine (DPPC) have been investigated by differential scanning calorimetry (DSC) and x-ray diffraction. By DSC hydrated GM1 undergoes a broad endothermic transition TM = 26 degrees C (delta H = 1.7 kcal/mol GM1). X-ray diffraction below (-2 degrees C) and above (51 degrees C) this transition indicates a micellar structure with changes occurring only in the wide angle region of the diffraction pattern (relatively sharp reflection at 1/4.12 A-1 at -2 degrees C; more diffuse reflection at 1/4.41 A-1 at 51 degrees C). In hydrated binary mixtures with DPPC, incorporation of GM1 (0-30 mol%; zone 1) decreases the enthalpy of the DPPC pretransition at low molar compositions while increasing the TM of both the pre- and main transitions (limiting values, 39 and 44 degrees C, respectively). X-ray diffraction studies indicate the presence of a single bilayer gel phase in zone 1 that can undergo chain melting to an L alpha bilayer phase. A detailed hydration study of GM1 (5.7 mol %)/DPPC indicated a conversion of the DPPC bilayer gel phase to an infinite swelling system in zone 1 due to the presence of the negatively charged sialic acid moiety of GM1. At 30-61 mol % GM1 (zone 2), two calorimetric transitions are observed at 44 and 47 degrees C, suggesting the presence of two phases. The lower transition reflects the bilayer gel --> L alpha transition (zone 1), whereas the upper transition appears to be a consequence of the formation of a nonbilayer, micellar or hexagonal phase, although the structure of this phase has not been defined by x-ray diffraction. At > 61 mol % GM1 (zone 3) the calorimetric and phase behavior is dominated by the micelle-forming properties of GM1; the presence of mixed GM1/DPPC micellar phases is predicted.     

Metamorphic Changes in Glycolipids and Myelin Proteins and 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase in Bullfrog and Axolotl Brains

Abstract: The metamorphic changes in levels of glycolipids and myelin proteins and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) in the brains of bullfrog tadpoles, adult frogs, and axolotls were investigated, with particular emphasis on myelin maturation. The concentrations of cerebroside. sulfatide, and galactosyldiacylglycerol gradually increased from the onset of prometamorphosis throughout the active metamorphic period and then greatly increased after metamorphosis was completed. The ratio of glucocerebroside to galactocerebroside increased greatly in the prometamorphic period and then rapidly decreased to the frog level during the climax period. The fatty acid compositions of cerebroside and sulfatide showed a developmental change, with 24:1 being more predominant in the later metamorphic stage. The proportion of hydroxy fatty acids increased up to the onset of the prometamorphic stage and thereafter remained constant at ∼ 50% of the total. The CNP activity remained unchanged throughout metamorphosis at 60% that in frog myelin and increased in the adult frog. The composition of tadpole myelin proteins remained constant during metamorphosis, with large basic protein being the most abundant, and in the frog, proteolipid protein and large basic protein were present in comparable amounts. The two adult forms of axolotl, i.e., the neotenous and metamorphosed forms, exhibited almost identical myelin constituents, and CNP activity in the neotenous form amounted to one-fifth that in the bullfrog. These results indicate that active biosynthesis of myelin marker components occurs as metamorphosis proceeds, but more pronounced changes of myelin components occur after metamorphosis is completed.     

Thermotropic phase properties of 1,2-di-O-tetradecyl-3-O-(3-O-methyl- beta-D-glucopyranosyl)-sn-glycerol.

The hydration properties and the phase structure of 1,2-di-O-tetradecyl-3-O(3-O-methyl-beta-D-glucopyranosyl)-sn-glycerol (3-O-Me-beta-D-GlcDAIG) in water have been studied via differential scanning calorimetry, 1H-NMR and 2H-NMR spectroscopy, and x-ray diffraction. Results indicate that this lipid forms a crystalline (Lc) phase up to temperatures of 60-70 degrees C, where a transition through a metastable reversed hexagonal (Hll) phase to a reversed micellar solution (L2) phase occurs. Experiments were carried out at water concentrations in a range from 0 to 35 wt%, which indicate that all phases are poorly hydrated, taking up < 5 mol water/mol lipid. The absence of a lamellar liquid crystalline (L alpha) phase and the low levels of hydration measured in the discernible phases suggest that the methylation of the saccharide moiety alters the hydrogen bonding properties of the headgroup in such a way that the 3-O-Me-beta-D-GlcDAIG headgroup cannot achieve the same level of hydration as the unmethylated form. Thus, in spite of the small increase in steric bulk resulting from methylation, there is an increase in the tendency of 3-O-Me-beta-D-GlcDAIG to form nonlamellar structures. A similar phase behavior has previously been observed for the Acholeplasma laidlawii A membrane lipid 1,2-diacyl-3-O-(6-O-acyl-alpha-D-glucopyranosyl)-sn-glycerol in water (Lindblom et al. 1993. J. Biol. Chem. 268:16198-16207). The phase behavior of the two lipids suggests that hydrophobic substitution of a hydroxyl group in the sugar ring of the glucopyranosylglycerols has a very strong effect on their physicochemical properties, i.e., headgroup hydration and the formation of different lipid aggregate structures.     

Physical chemical studies of short-chain lecithin homologues. II. Micellar weights of dihexanoyl- and diheptanoyllecithin

The micellar weights of dihexanoyl- and diheptanoyllecithin in aqueous solutions are calculated from light scattering and ultracentrifugation data. A monomer-micelle assocation model is used and corrections for the thermodynamic nonideality, on the basis of rigid noninteracting particles, are applied. A few experiments on the influence of high NaCI concentrations (up to 3 M) are described. Dihexanoyllecithin forms micelles with micellar weight of 15 000 to 20 000 and with rather narrow weight distributions. Diheptanoyllecithin micelles however, have broad size distributions with micellar weights of 20 000 up to about 100 000 in the concentration range studied. Micelles are assumed to be spherical or to have sphero-cylindrical shapes depending on the molecular weights. Two models are used: (1) a compact structure, where no attention is paid to the hydrocarbon-water contact (2) micelles with as little hydrocarbon-water contact as possible.     

Micelle-vesicle transition in phospholipid-bile salt mixtures. A study by precision scanning calorimetry

A systematic study of the micelle-vesicle transformation in phospholipid-bile salt mixtures using differential scanning calorimetry (DSC) indicates that the lipid undergoes a variety of changes in its thermal properties as mixed micellar solutions are diluted to concentrations at which vesicles form. In the experiments, micellar solutions of 50 mg/mL total lipid, containing sodium taurocholate (TC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), are diluted to concentrations corresponding to differing extents of aggregation of the TC with phospholipid. Turbidity and equilibrium dialysis measurements are used to establish boundaries between where micelles persist and where vesicles are formed and to determine the extent of aggregation of the TC with DPPC. At molar ratios Re of bound TC to DPPC greater than 0.3, micellar solutions are formed, while at Re less than 0.15 vesicles are evident upon dilution. As the transformation from micelles to vesicles occurs, the thermal transitions in the lipid change from broad, low Cp (max) peaks in the micelle region to multiple peaks of high cooperativity in regions of composition where lamellar structures and vesicles form. The DSC curves show that in the composition region corresponding to where bilayer micelles exist a new thermal phase forms, which has a melting transition near 32 degrees C, if the solutions are allowed to equilibrate for 48 h at 21 degrees C. Furthermore, at compositions between Re = 0 and 0.25, there is metastability in the lipid when equilibrated at 21 degrees C, but heating the lipid through the thermal transitions leads to reversible behavior.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of cholesterol with galactocerebroside and galactocerebroside-phosphatidylcholine bilayer membranes

The interaction of the galactocerebroside, N-palmitoylgalactosylsphingosine (NPGS), with cholesterol has been studied by differential scanning calorimetry (DSC) and x-ray diffraction. Thermal and structural studies demonstrate complex behavior characterized by two endothermic transitions: transition I (TI approximately equal to 50-60 degrees C) corresponding to an NPGS-cholesterol bilayer gel----bilayer liquid crystal transition II (TII where TI less than TII less than TNPGS) corresponding to an NPGS bilayer crystal (stable E form)----bilayer liquid crystal transition. For mixtures containing from 6 to 80 mol % cholesterol, x-ray diffraction studies at 22 degrees C (T less than TI) indicate two separate lamellar phases; an NPGS crystal bilayer phase and a cholesterol monohydrate phase. For cholesterol concentrations less than 50 mol % at TI less than T less than TII, NPGS-cholesterol liquid crystal bilayer and excess NPGS crystal bilayer phases are observed. For greater than 50 mol % cholesterol concentrations at these temperatures, an excess cholesterol monohydrate phase coexists with the NPGS-cholesterol liquid crystal bilayers. At T greater than TII, complete NPGS-cholesterol miscibility is only observed for less than 50 mol % cholesterol concentrations, whereas at greater than 50 mol % cholesterol an excess cholesterol phase is present. The solid phase immiscibility of cerebroside and cholesterol at low temperatures is suggested to result from preferential NPGS-NPGS associations via hydrogen bonding. The unique thermal and structural behavior of NPGS-cholesterol dispersions is contrasted with the behavior of cholesterol-phosphatidycholine and cholesterol-sphingomyelin bilayers. Thermal and structural studies of NPGS in dipalmitoylphosphatidylcholine (DPPC)/cholesterol (1:1, molar ratio) bilayers have been performed. For dispersions containing less than 20 mol % NPGS at 22 degrees C there are no observable calorimetric transitions and x-ray diffraction studies indicate complete lipid miscibility. At greater than 20 mol % NPGS, a high temperature transition is observed that is shown by x-ray diffraction studies to be due to an excess NPGS crystal bilayer----liquid crystal bilayer transition. Complete miscibility of NPGS in DPPC/cholesterol bilayers is observed at T greater than TNPGS. The properties of NPGS/DPPC/cholesterol bilayers are discussed in terms of the lipid composition of the myelin sheath.     

Sulfatide prolongs blood-coagulation time and bleeding time by forming a complex with fibrinogen

Sulfatides (galactosylceramide I³-sulfate), which are found in serum lipoproteins of various mammals, effectively increased prothrombin time (anticoagulant effect) and also effectively prolonged bleeding time (anti-platelet effect). When equal volumes of a homogeneous micellar solution of sulfatide and fibrinogen in phosphate-buffered saline were mixed, an insoluble complex precipitated. Analysis of the precipitated complex showed that the molar ratio of sulfatide to fibrinogen was about 400:1. These results indicate that the sulfatide micelle binds tightly to fibrinogen and thereby interferes with both fibrin gel formation (anticoagulant activity) and platelet function.Abbreviations BSA bovine serum albumin - PBS phosphate-buffered saline - PT prothrombin time Dedicated to Dr Sen-itiroh Hakomori in celebration of his 65th birthday.     

The glycosphingolipid sulfatide in the islets of Langerhans in rat pancreas is processed through recycling: possible involvement in insulin trafficking

In previous studies we have shown that sulfatide (galactosylceramide-3-O-sulfate), in various species, is present in the insulin-producing cells in pancreatic islets of Langerhans. In this study the synthesis of sulfatide in the islets has been investigated by pulse chase labeling at varying glucose levels and in the presence or absence of the glycosphingolipid synthesis inhibitory agents, Brefeldin A, fumonisin B1 and chloroquine and the distribution of sulfatide by immune-electronmicroscopy. The data showed that (1) sulfatide was produced in islets of Langerhans, (2) the main pathway for synthesis was through recycling involving partial degradation in the lysosome, and that (3) high glucose levels, although not primarily reflected in an increased synthesis of sulfatide, lead to an increased expression of mRNA for the UDP-galactose:ceramide galactosyltransferase, producing the immediate precursor of sulfatide. Furthermore, mass spectrometry analyses revealed a high proportion of short chain fatty acids, C16:0 (50%) and no hydroxylated forms and thus special physicochemical properties, indicating important differences between pancreatic and brain/neural sulfatide. Immune electron microscopy revealed an intracellular expression of sulfatide in the secretory granules, the Golgi network and the lysosomes of the islets. These results indicate that sulfatide follows the same intracellular route as insulin and suggest a functional association between these molecules. We have raised the hypothesis that sulfatide possibly plays a role in the trafficking of insulin in the islets of Langerhans in rat pancreas.     

Studies on the anomalous thermotropic behavior of aqueous dispersions of dipalmitoylphosphatidylcholine-cholesterol mixtures

Examination of the thermotropic behavior of aqueous dispersions of dipalmitoylphosphatidylcholine-cholesterol mixtures by high-sensitivity scanning calorimetry has revealed that the phospholipid gel to liquid-crystalline phase transition consists of two components. One, a relatively sharp transition centered at 39.6-40.7 degrees C, exhibits a transition enthalpy change which decreases linearly with increasing cholesterol content, approaching zero at a cholesterol content of about 25 mol %. The other, a broad, lower intensity transition centered at approximately 41.5 degrees C for cholesterol concentrations of 20 mol %, displays an enthalpy change which is maximal at about 20-25 mol % cholesterol and which decreases as the cholesterol content decreases to zero or increases above 25 mol %. The origin of these two transitions is discussed in terms of a separation of these lipid mixtures into cholesterol-rich and cholesterol-poor domains.     

Modulation of the physical state of cellular cholesteryl esters by 4,4'-(isopropylidenedithio)bis(2,6-di-t-butylphenol) (probucol).

The effect of 4,4'-(isopropylidenedithio)bis(2,6-di-t-butylphenol) (probucol) on cholesteryl ester physical state was examined in dry mixtures, phospholipid-containing dispersions, and cells. Probucol has little effect on the solid to isotropic transition of cholesteryl oleate, but broadens and decreases the enthalpy of the liquid-crystalline transitions at concentrations as low as 1-2 mol %. A probucol transition is only observed at concentrations greater than 20 mol %. The mesomorphic phases of the cholesteryl oleate/probucol mixtures were identified by visual inspection and polarized light microscopy. Mixtures are liquid at probucol concentrations in excess of 5 mol % at 37 degrees C. Probucol also dramatically reduces the enthalpy of the liquid-crystalline transitions of the cholesteryl oleate core of dispersions of the ester with phospholipids at a concentration of 10 mol %, reducing the enthalpy by greater than 80% and the transition temperatures by approximately 2 degrees C. The phase state of cholesteryl esters in Fu5AH rat hepatoma cells was examined after incubation with cholesterol/phospholipid dispersions that caused the accumulation of anisotropic cholesteryl ester droplets. Differential scanning calorimetry scans of cells incubated with cholesterol-rich phospholipid dispersions indicated a phase transition near 48 degrees C, which was abolished when the cells were co-incubated with 50-100 micrograms/ml of probucol in the loading medium. Subsequent to the formation of isotropic cholesteryl ester droplets in the presence of probucol, the rate of efflux of cholesterol from the cells to phosphatidylcholine-containing acceptors in the medium was increased. These data show that probucol is relatively soluble in cholesteryl esters and that probucol changes the phase state of cholesteryl ester droplets in cells to a more fluid phase in which the cholesteryl esters are more readily mobilized.