Dependence of the surface expression of the glycolipid cerebroside sulfate on its lipid environment: comparison of sphingomyelin and phosphatidylcholine

The influence of the membrane lipid environment on the reactivity with antibody of the acidic glycolipid cerebroside sulfate (CBS) was examined by using a spin membrane immunoassay. Fewer antibodies in a polyclonal anti-CBS antiserum recognized the antigen in a bovine brain sphingomyelin/cholesterol (SM/CHOL) environment than in dipalmitoylphosphatidylcholine/cholesterol (DPPC/CHOL). Changes in the CBS ceramide group appeared to have less influence on antibody recognition of CBS in SM/CHOL than in DPPC/CHOL [Crook et al. (1986) Biochemistry 25, 7488-7494]. Although the fatty acid chain length of phosphatidylcholine strongly influences CBS recognition, the fatty acid chain length of sphingomyelin had only a moderate effect on CBS recognition and did not account for the decreased recognition in SM compared to in DPPC. Inhibition studies revealed that the antibodies which recognize CBS in SM/CHOL (S antibodies) form a population distinct from those which recognize CBS in DPPC/CHOL (P antibodies). The specificity of the P and S antibodies was examined further by comparing the efficacy of various substances, which share chemical features with the components of CBS in a SM/CHOL or DPPC/CHOL environment, to inhibit lysis of liposomes containing CBS. Intact CBS, cholesterol, and a phosphocholine lipid, at certain antigen densities, were required for optimal recognition of the antigen, especially by the P antibodies, suggesting that a complex of all three lipids in a multivalent array may be recognized by these antibodies. The S antibodies may recognize a smaller complex or monomers of CBS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fatty acids of glycosphingolipids from pig blood fractions

Four glycolipids have been isolated from three fractions of pig blood. The glycolipids were presumably cerebroside, diglycosyl ceramide, triglycosyl ceramide, and globoside. The blood fractions were erythrocytes and plasma high and low density lipoproteins. Fatty acid distributions were determined for each glycolipid as a means to assist in identifying relationships among the several glycolipids. Normal fatty acids predominated in all glycolipids except the globosides from erythrocytes in which the amount of hydroxy acids was slightly greater than the amount of normal acids. Hydroxy acids appeared to be present in all the glycolipids, but the concentration was very low in cerebrosides isolated from high density lipoproteins and erythrocytes, and in diglycosyl ceramide and globoside of the low density lipoproteins. In general, the average fatty acid chain length increased from cerebroside to globoside. This was most apparent in erythrocytes and also greater for normal acids than for hydroxy acids. Fatty acid distributions of erythrocyte glycolipids had sufficient variation to make a metabolic relationship by simple addition of a hexose appear doubtful. While the fatty acid distributions found in plasma lipoproteins were more similar, some means of acyl group selection is probably present for either the synthesis or degradation of these glycolipids.     

Evidence that trans-bilayer interdigitation of glycosphingolipid long chain fatty acids may be a general phenomenon

'Interdigitation' is a term coined to describe the phenomenon whereby pure phosphatidylcholines with intramolecular fatty acid chain length heterogeneity when hydrated to form bilayers may insert the methyl ends of long fatty acids from one side across more than half of the membrane thickness to protrude amongst the acyl chains of the opposite side of the bilayer (Keough, K.M.W. and Davis, P.J. (1979) Biochemistry 18, 1453-1459; Huang, C. and Mason, J.T. (1986) Biochim. Biophys. Acta 864, 423-470). In this article we address the fate of long fatty acid chains of glycosphingolipids present as minor components in membranes of non-interdigitating phosphatidylcholines. In this pursuit, derivatives of galactosyl ceramide, lactosyl ceramide, globoside and GM1 were synthesized having either 18-carbon or 24-carbon fatty acid with a spin label covalently attached at C-16. Labelled glycolipids were incorporated at 1-2 mol% into bilayers of synthetic phosphatidylcholines, their mixtures with cholesterol, or natural egg phosphatidylcholine. In each case the C-16 carbon of the glycolipid long chain fatty acid showed considerably greater 'order' and immobility than did C-16 of the fatty acid which was similar in length to the host matrix phospholipids. We interpret this as strong evidence that the long chain fatty acid interdigitates across the mid point of the bilayer in the systems studied. Clearly this phenomenon did not require that the phospholipid host matrix have mixed chain lengths. Furthermore it was totally independent of glycolipid family: for a given host matrix and (glycolipid) fatty acid chain length the order parameter values found were the same amongst all four glycolipid families tested.     

Interdigitated lipid bilayers of long acyl chain species of cerebroside sulfate. A fatty acid spin label study

The metastable phase behavior of semi-synthetic species of cerebroside sulfate (CBS), with hydroxy and non-hydroxy fatty acids from 16 to 26 carbons in length, was compared in Li+ and K+ using differential scanning calorimetry. The structure of the metastable and various stable phases formed in the presence of these two cations was investigated using a fatty acid spin label, 16-doxylstearate. A number of stable phases with successively higher phase transition temperatures and enthalpies occur in the presence of K+ (see the preceding paper). Li+ prevents formation of the most stable phases with the highest transition temperatures and enthalpies for all species of CBS. However, it does not prevent a transition from the metastable phase to the first stable phase of the longer chain C24 and C26 species. Furthermore, it allows C24:0h-CBS to undergo a similar transition, in contrast to a high K+ concentration, which prevents it. The spin label has anisotropic motion in the metastable gel phase formed by all species of CBS on cooling from the liquid crystalline phase. The spectra resemble those in gel phase phospholipids. The spin label is partially insoluble in the most stable phases formed by all the lipids, including the unsaturated C24:1 species, preventing further elucidation of their structure using this technique. However, the spin label is soluble in the first stable phase formed on cooling by the longer chain C24:0 and C26:0-CBS in Li+ and K+ and by C24:0h-CBS in Li+, and is motionally restricted in this phase. The motional restriction is similar to that observed in the mixed interdigitated bilayers of asymmetric species of phosphatidylcholine and fully interdigitated bilayers formed by symmetric phospholipids. It strongly suggests that the highly asymmetric long chain species of CBS form a mixed interdigitated bilayer in their first stable gel phases while the metastable phase of these and the shorter chain lipids may be partially interdigitated. The metastable phase of C24:1-CBS is more disordered suggesting that it may not be interdigitated at all. Thus the results suggest that (i) the hydroxy fatty acid inhibits but does not prevent formation of a mixed interdigitated bilayer by long chain species of CBS, (ii) an increase in non-hydroxy fatty acid chain length from 24 to 26 carbons promotes it, and (iii) a cis double bond probably prevents any form of interdigitation. These results may be relevant to the physiological and pathological roles of these structural modifications of CBS.     

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.     

Lipid domain formation and ligand-induced lymphocyte membrane changes

Spectral parameters of spin-labelled phosphatidylcholine, ceramide and cerebroside in the plasma membranes of human blood lymphocytes were measured before and after treatment with various ligands, which included concanavalin-A and phytohaemagglutinin. It was found that ligand treatment led to a significant decrease in order of the hydrocarbon chains of the phospholipids. This was accompanied by a clustering of the labelled sphingolipids, as estimated by spin-spin interaction, and an increase in the order of their hydrocarbon chains. In the untreated cells the cerebroside fatty acid chain was more ordered than that of the phosphatidylcholine.It was considered that the decrease in phospholipid order was brought about by the sequestration of the more rigid sphingolipids into the patches and caps formed by receptor-ligand complexes. The significance of these changes in lipid distribution and ordering is discussed in relation to the activation of membrane enzyme systems by mitogenic ligands.     

Kinetic Changes in Free Ceramide and Cerebroside during Germination of Black Gram

Free ceramide and cerebroside were isolated from black gram sprouts of all germinating stages. Free ceramide and cerebroside were found to increase during germination.

The major sphingosine bases of free ceramide were 4-hydroxysphingenine and 4-hydroxy-sphinganine (trihydroxy type) while that of cerebroside was sphinga-4,8-dienine (dihydroxy type). A change in the component sphingosine base was that 4-hydroxysphingenine in free ceramide and cerebroside increased slightly after germination.

The major fatty acid of free ceramide was α-hydroxylignoceric acid while that of cerebroside was α-hydroxypalmitic acid. Changes in component fatty acid were that α-hydroxylignoceric acid in both sphingolipids increased after germination.     

Interaction of myelin basic protein and polylysine with synthetic species of cerebroside sulfate

The effect of myelin basic protein on the myelin lipid cerebroside sulfate was studied by differential scanning calorimetry and use of the fatty acid spin label, 16-S-SL, in order to determine (i) the effect of basic protein on the metastable phase behavior experienced by this lipid, and (ii) to determine if basic protein perturbs the lipid packing as it does with some acidic phospholipids. The effects of basic protein on the thermodynamic parameters of the lipid phase transition were compared with those of polylysine which has an ordering effect on acidic phospholipids as a result of its electrostatic interactions with the lipid head groups. Different synthetic species of cerebroside sulfate of varying fatty acid chain length and with and without a hydroxy fatty acid were used. The non-hydroxy fatty acid forms of cerebroside sulfate undergo a transition from a metastable to a more ordered stable state while the hydroxy fatty acid forms remain in the metastable state at the cation concentration used in this study (0.01 M Na+ or K+). The non-hydroxy fatty acid forms were still able to go into a stable state in the presence of both basic protein and polylysine. At low concentrations, basic protein increased the rate of the transition to the stable state, while polylysine decreased it for the longest chain length form studied. However, at high concentrations, basic protein probably prevented formation of the stable state. The hydroxy fatty acid forms did not go into the stable state in the presence of basic protein and polylysine. It is argued that the increased rate of formation of the stable state in the presence of basic protein and decreased rate in the presence of polylysine are consistent with interdigitation of the lipid acyl chains in the stable state. Basic protein also had a small perturbing effect on the lipid. It decreased the total enthalpy of the lipid phase transition. When added to the non-hydroxy fatty acid forms it increased the temperature of the liquid crystalline to metastable phase transition and decreased the temperature of the stable to liquid crystalline phase transition. It significantly decreased the transition temperature of the hydroxy fatty acid forms but only a portion of the lipid was affected. In contrast, polylysine increased the transition temperature of the metastable and stable states of all forms of cerebroside sulfate but had a greater effect on the non-hydroxy fatty acids forms than on the hydroxy fatty acid forms.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of fatty acid chain length,fatty acid hydroxylation,and various cations on phase behavior of synthetic cerebroside sulfate

Synthetic species of CBS containing palmitic, stearic, lignoceric, D-2-hydroxy palmitic, or D-2-hydroxy stearic acid were prepared and their phase behavior in the presence of a number of mono- and divalent cations was studied by differential scanning calorimetry and the use of fatty acid spin labels. The results showed that both the non-hydroxy fatty acid (NFA) and hydroxy fatty acid (HFA) forms of cerebroside sulfate (CBS) can occur in two different gel states, a metastable state and a lower entropy stable state. The phase behavior is more sensitive to the type and concentration of cation present than in the case with acidic phospholipids. The sensitivity of the transition temperature (T_m) to cation concentration reflects, in part, increased participation of the lipid in intermolecular hydrogen bonding interactions as the negative charge of the sulfate is shielded. The extra hydroxyl group on the HFA also contributes to the intermolecular hydrogen bonding network causing a significant increase in the T_m.The HFA has an even more significant effect in causing inhibition of formation of the stable state. Formation of the stable state is also inhibited by Li⁺ and divalent cations. A similar mechanism may be involved, i.e.; cross-linking of adjacent lipids or increased intermolecular interactions inhibit the molecular rearrangement necessary to form the stable state. This inhibition is counteracted by an increase in fatty acid chain length. The results suggest that the stable state may be interdigitated as a result of the unequal chain length between the sphingosine base and the fatty acid.     

Oxidation of glycolipids in liposomes by galactose oxidase

Small unilamellar phosphatidylcholine vesicles containing globo-series glycolipids were labeled by the galactose oxidase/NaB[3H]4 procedure. The major glycolipid of human red cells, globoside, was the best substrate for galactose oxidase both in vesicles and in tetrahydrofuran-containing buffer. The oxidation rates of membrane-bound ceramide trihexoside and Forssman glycolipid were one-fourth and one-tenth, respectively, of the oxidation rate of globoside. Membrane-bound ceramide dihexoside was not a substrate for galactose oxidase, although it was readily oxidized in tetrahydrofuran-containing buffer. Soluble sialoglycoproteins and membrane-incorporated glycophorin A stimulated the oxidation of globoside-containing vesicles, whereas membrane-bound GD1a ganglioside had no effect on globoside oxidation.     

Characterization of two monoclonal antibodies specific for lacto-series type 1 chain Gal beta 1----3GlcNAc-terminal structures

Murine monoclonal antibodies, TE-1 and TE-3, generated by immunization with a biosynthetic reaction product containing a terminal Gal beta 1----3GlcNAc structure have been produced and found to react specifically with underivatized type 1 chain lacto-series carbohydrate structures. Detailed analysis of these antibodies, both IgM, indicates two differing classes of epitope specificity. Antibody TE-1 was found to bind preferentially to longer chain carbohydrate structures containing a terminal Gal beta 1----3GlcNAc disaccharide, indicating that optimal antibody binding involved more than recognition of this disaccharide. In contrast, antibody TE-3 was found to bind strongly carbohydrate structures containing terminal Gal beta 1----3GlcNAc structures irrespective of chain length. Modification of core chain structures by addition of fucose and/or sialic acid residues completely abolished antibody binding with either antibody. TLC immunostaining of neutral glycolipids isolated from a variety of human colonic adenocarcinoma cell lines indicated intensely stained bands, particularly with antibody TE-3, which correlated with the level of expression of type 1 chain based glycolipid derivatives. These antibodies are applied to the detailed study of the regulation of synthesis of lacto-series type 1 chain based carbohydrate structures.     

Glycosphingolipids from rabbit aorta, plasma, and red blood cells: effects of high cholesterol-high fat diets on fatty acid distribution and quantity of glycosphingolipids

Four glycosphingolipids were isolated from rabbit aorta, plasma, and red blood cells. They were identified, by thin-layer chromatography and by quantitative analysis of hexose and fatty acid, as cerebroside, diglycosyl ceramide, triglycosyl ceramide, and globoside. The rabbits had been maintained on a normal diet or on one of three high cholesterol diets for 180 days. The quantities of the glycosphingolipids and their fatty acid distributions were determined, and comparisons were made between the control and experimental animals. Aorta and plasma glycosphingolipids were more affected by the high cholesterol diets than were those from red blood cells. The effects on aorta and plasma glycosphingolipids were similar. The amount of cerebroside was increased in aorta and plasma in all animals in the experimental groups. The amount was also increased in red blood cells in rabbits from two of the experimental groups. The average fatty acid chain length was greater in the lipids from the experimental animals than in those from the control animals for all measured glycosphingolipids from aorta. The average chain length was also greater in cerebrosides from the experimental animals from all three tissues. Probably the most notable differences in the experimental animals were the increased 24:1/24:0 ratios and the increased concentrations of 24:2. These increases occurred in nearly all samples from plasma and aorta, but not in red blood cells. There was also an increase of total unsaturated fatty acids in aorta cerebrosides from the experimental animals. Except for the increase in 24:2, lard generally caused more deviation from normal than did cottonseed oil when the level of cholesterol in the diet was 1%.     

The specific glycosphingolipid composition of human ureteral epithelial cells

Total non-acid and acid glycolipid fractions were isolated from epithelial cell scrapings and the non-epithelial residue of a human upper ureter. The glycolipid fractions were structurally characterized as total mixtures by thin-layer chromatography, mass spectrometry, and proton NMR spectroscopy. Selected structural information was also obtained on binding of monoclonal antibodies and bacteria to the thin-layer chromatograms. The major epithelial cell glycolipids were Glc beta 1-1ceramide (75%), dihexosylceramide (10%) and NeuAcLacceramide (10%). In addition, 8 minor glycolipids belonging to the blood group P, Lewis and ABO systems were identified. The major glycolipids of the non-epithelial residues were mono- and dihexosylceramides together with globotriaosyl- and globotetraosylceramides. The epithelial mono- and diglycosylceramide compounds had an unusual ceramide composition with mainly C18 and C20 trihydroxy long chain bases in combination with C22-C24 hydroxy fatty acids in contrast to the non-epithelial glycolipids which contained mainly C18 dihydroxy long chain bases in combination with C16-C24 non-hydroxy fatty acids.     

Glycosphingolipid fatty acid arrangement in phospholipid bilayers: cholesterol effects.

Deuterium wide line NMR spectroscopy was used to study cholesterol effects on the ceramide portions of two glycosphingolipids (GSLs) distributed as minor components in fluid membranes. The common existence of very long fatty acids on GSLs was taken into account by including one glycolipid species with fatty acid chain length matching that of the host matrix, and one longer by 6 carbons. N-stearoyl and N-lignoceroyl galactosyl ceramide with perdeuterated fatty acid (18:0[d35] GalCer and 24:0[d47] GalCer) were prepared by partial synthesis. They were dispersed in bilayer membranes having the 18-carbon-fatty-acid phospholipid, 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), as major component. Glycolipid fatty acid chain behavior and arrangement were analyzed using order profiles derived from their 2H-NMR spectra. Cholesterol effects on order parameter profiles for 18:0[d35] GalCer, with chain length equal to that of the host matrix, followed the pattern known for acyl chains of phospholipids. The presence of sterol led to restriction of trans/gauche isomerization along the length of the chain, with the largest absolute increase in order parameters being toward the surface, but somewhat greater relative effect just below the "plateau" region. In cholesterol-containing membranes, order parameter profiles for the long chain species, 24:0[d47] GalCer, showed a characteristic secondary "plateau" associated with carbon atoms C14 to C23, a feature also present in SOPC bilayers without cholesterol and in pure hydrated 24:0[d47] GalCer. Cholesterol-induced ordering effects on the long chain glycolipid were similar to those described for the shorter chain species, but were minimal at the methyl terminus.(ABSTRACT TRUNCATED AT 250 WORDS)     

A series of human erythrocyte glycosphingolipids reacting to the monoclonal antibody directed to a developmentally regulated antigen SSEA-1

A series of glycolipid antigens reacting with the monoclonal antibody directed to the stage-specific embryonic antigen 1 was isolated and characterized from group O human erythrocyte membranes. A ceramide heptasaccharide (Structure 1), ceramide nonasaccharide (Structure 2), and ceramide decasaccharide (Structure 3) have been characterized (formula, see text) The main feature of this glycolipid series is its long core sugar chain with a nonbranched repeating N-acetyllactosamine (norpolylactosamine). This characteristic is in contrast to that of co-existing H-active glycolipid series in which the longer core structures are branched type repeating N-acetyllactosamine (isopolylactosamine). The reactivity of these glycolipids to monoclonal anti-stage-specific embryonic antigen 1 antibody varied proportionately to the length of their core sugar chains. A possible significance of these glycolipids as developmentally regulated antigens and as cancer-associated antigens was discussed.     

Interactions of phospholipid vesicles with rat hepatocytes in vitro influence of vesicle-incorporated glycolipids

We examined the interaction of glycolipid-containing phospholipid vesicles with rat hepatocytes in vitro. Incorporation of either $\text{N-}\text{lignoceroyldihydrolactocerebroside}$ or the monosialoganglioside, G_M1, enhanced liposomal lipid uptake 4–5-fold as judged by the uptake of radioactive phosphatidylcholine as a vesicle marker. Cerebroside enhanced phospholipid uptake only when incorporated into dimyristoyl, but not into egg phosphatidylcholine vesicles. The lack of cerebroside effect in egg phosphatidylcholine-containing vesicles appeared to be due to a limited exposure of the carbohydrate part of the glycolipid as suggested by the reduced agglutinability of those vesicles by Ricinus communis agglutinin.In contrast to the results with radioactive phosphatidylcholine, we observed only a 20% increase in vesicle-cell association as a result of glycolipid incorporation, when a trace amount of [¹⁴C]cholesteryloleate served as a marker of the liposomal lipids or when using the fluorescent dye, carboxyfluorescein, as a marker of the aqueous space of the vesicles. By the same token, intracellular delivery of vesicle-contents was only slightly enhanced (approx. 10%).The discrepancy between the association with the cells of phosphatidylcholine on the one hand and cholesteryoleate or entrapped marker on the other suggests different mechanisms of uptake for these markers. Our results are compatible with the notion that the main effect of incorporation of glycolipids into the vesicles is the enhancement of exchange or transfer of phospholipid molecules between vesicles and cells. Incubation of the cells with galactose or lactose, prior to addition of vesicles, suggests that this enhanced phospholipid exchange or transfer involves specific recognition of the terminal galactose residues of the glycolipid vesicles by a receptor present on the plasma membranes of hepatocytes.     

Characterization of neutral sphingolipids from chicken erythrocytes

The neutral sphingolipids from chicken erythrocytes were characterized. The total concentration of neutral sphingolipids was found to be 480 nmol/g of dry stroma. They were isolated and purified by droplet counter-current chromatography, Iatrobeads column chromatography, and preparative thin-layer chromatography. The major neutral sphingolipids were free ceramide, ceramide monohexoside, ceramide dihexoside, and ceramide pentahexoside, which represented 43%, 23.5%, 10.0%, and 3.6% of the long chain bases, respectively. Thus, free ceramide was the most abundant neutral sphingolipid in chicken erythrocytes. Ceramide monohexoside was composed of more galactosylceramide than glucosylceramide. Galabiosylceramide was found in the ceramide dihexoside fraction together with lactosylceramide. Ceramide pentahexoside was a Forssman glycolipid. There were two groups of neutral sphingolipids; one had mainly C16 fatty acid and the other had C22 and C24 fatty acids. In both groups sphingosine (d18:1) was predominant as a long chain base. 2-Hydroxy-C16 fatty acid was a major component of one of the ceramide monohexosides.     

Effects of cell density on the neutral glycolipid composition of cultured human brain and glioma cells

Density dependent chain elongation of neutral glycosphingolipids (NGSL) is associated with contact inhibition of mitosis in several normal cultured cell lines. Transformed non-neural cell lines which have impaired contact inhibition frequently lose this biochemical response. To determine if either of these phenomena occur in human neural cells we determined NGSL compositions of cultured glioblastoma multiforme and normal fetal brain cells. Fetal cells generally had more total NGSL than the tumor cells. As a percentage of total NGSL, both cell lines at higher cell densities had larger proportions of ceramide trihexoside and globoside, but smaller proportions of cerebroside. This decrease was mainly in nonhydroxy fatty acid cerebroside of glioma cells, but in hydroxy fatty acid cerebroside of normal fetal brain cells. These results demonstrate that although glioblastoma multiforme cells have markedly impaired growth control, they still preserve density dependent chain elongation of NGSL. A role for this phenomenon in normal cellular growth control has yet to be established.     

The lipid composition of the electric organ of the ray, Torpedo marmorata, with specific reference to sulfatides and Na+-K+-ATPase.

The lipids from the electric organ of the ray, Torpedo marmorata, have been isolated and characterized. The major lipids were cholesterol, choline phospholipids, ethanolamine phospholipids, and sphingomyelins. The major fatty acids of ethanolamine phospholipids were 18:1, 18:0, 22:6, and 20:4. More than 50% of the acids in choline phospholipids were 16:0. The sphingomyelins consisted of five major ceramide species, all with sphingosine and the fatty acids 14:0, 15:0, 16:0, 22:1, and 24:1. The fatty acid 15:0 was mostly branched (n-2), a fatty acid earlier identified in sphingomyelins of the rectal gland of spiny dogfish. All long-chain bases were dihydroxy bases with a small percentage of branched chains. Sulfatides (cerebroside sulfate) made up the largest glycolipid fraction. The polar moiety wase galactose-3-sulfate. The fatty acids were normal and 2-hydroxy; the homologue 24:1 was the most abundant in both types of fatty acids. Most fatty acids were higher homologues of mono-unsaturated acids, but normal 18:0 fatty acid was also found. The long-chain bases were both dihydroxy and trihydroxy, with very small amounts of branched chains. The two major ceramide species of sulfatides were sphingosine combined with normal and hydroxy 24:1 fatty acids, respectively. Smaller amounts of trihydroxy base (18:0) were found linked to hydroxy 24:1 fatty acid, but not to its normal homologue. The cerebrosides contained the two major species mentioned above but lacked the trihydroxy base-hydroxy fatty acid species. The ratio of the activity of Na+-K+-dependent ATPase (EC 3.6.1.3) and the concentration of sulfatides was similar to ratios found for other tissues with normal and increased Na+ and K+ transporting capacity. The significance of this finding is discussed.     

A sulfated glucosylceramide from rat kidney

A novel sulfated glycosphingolipid containing a sulfated glucosyl residue was isolated from rat kidney and purified to homogeneity by column chromatographies with DEAE-Sephadex and silica beads. By compositional analyses, permethylation studies, one- and two-dimensional proton magnetic resonance spectroscopy, infrared spectroscopy, negative secondary ion mass spectrometry, solvolysis, and immunostaining on thin layer chromatogram, the structure of this glycolipid was proposed to be HSO3-3Glc beta 1-1Cer (where Cer is ceramide). The ceramide portion consisted of 4-D-hydroxysphinganine as the sole long chain base, and the fatty acid consisted of predominantly tetracosanoic acid, deduced from both composition analysis and negative secondary ion mass spectrometry. The yield of glucosyl sulfatide was about 5 nmol/g of tissue, being about three times as much as that of lactosylceramide sulfate.