Possible role of sulphatide in the K+-activated phosphatase activity

A microsomal fraction rich in (Na+ + K+)-ATPase has been isolated from the outer medulla of pig kidney. (Mg2+ + K+)-activated ouabain-sensitive phosphatase activity was studied in this preparation treated with arylsulphatase, an enzyme that specifically hydrolyzes ceramide galactose-3-sulphate. The activity of phosphatase was inactivated in proportion to the amount of sulphatide hydrolyzed. A maximum inactivation of ouabain-sensitive activity was obtained with 60% of the sulphatide content hydrolyzed. The inactivation caused by arylsulphatase was partially reversed by the sole addition of sulphatide. The evidence offered in this paper about sulphatide function in the sodium pump mechanism supports the idea that sulphatides are involved in the K+-activated phosphatase, a partial reaction of the (Na+ + K+)-ATPase.     

Kinetic mechanism of pulmonary carbonyl reductase.

An IgG1 monoclonal antibody, Sulph I, reacting with sulphatide (3'-sulphogalactosylceramide), was produced by immunizing Balb/c mice with that glycolipid coated on Salmonella minnesota bacterial membrane. Radioimmunodetection of the binding of the monoclonal antibody to structurally related glycolipids adsorbed to microtitre plates or chromatographed on thin-layer plates was used to determine its binding epitope. The antibody showed similar binding avidity to three sulphated glycolipids: sulphatide, sulpholactosylceramide and seminolipid. Lysosulphatide did bind the antibody, but, compared with sulphatide, 30 times more antigen was needed for half-maximal binding. Bis(sulphogangliotriosyl)ceramide and bis-sulphogangliotetraosylceramide did not bind the antibody. These results suggest that terminal galactose-3-O-sulphate and part of the hydrophobic region of the glycolipid are recognized by the Sulph I antibody.     

Different metabolic recycling of the lipid components of exogenous sulphatide in human fibroblasts.

Cultured human fibroblasts were fed with two differently labelled sulphatide molecules [one labelled on C-3 of the sphingosine (Sph) moiety [( Sph-3H]sulphatide), the second on C-1 of stearic acid [( stearoyl-14C]sulphatide)], and the intracellular metabolic fate of radioactivity was monitored. Incorporated radioactivity was almost all recovered in the total lipid extract, regardless of the labelling position of the added sulphatide; however, large differences in the level of incorporation occurred among labelled glycosphingolipids. For example, sphingomyelin was present as the major radiolabelled lipid after [Sph-3H]-sulphatide incubation, but was detectable only in trace amounts after [stearoyl-14C]sulphatide administration; in the latter case the radioactivity was located predominantly in glycerophospholipids. From this finding it can be inferred that the free long-chain base (sphingosine) that originates from lysosomal catabolism of sulphatide is mainly, and quite specifically, utilized for sphingomyelin biosynthesis, whereas the ceramide moiety is not; conversely the fatty acid released from ceramide is non-specifically re-utilized for phospholipid biosynthesis.     

Cultured oligodendrocytes metabolize a fluorescent analogue of sulphatide; inhibition by monensin.

It has been suggested that oligodendrocytes can actively phagocytose myelin debris during active myelination or after injury and experimental demyelination. Therefore, we have used a fluorescent analogue (N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulphate) to study the metabolic fate of sulphatide, a galactosphingolipid that is highly enriched in myelin membranes. The fluorescent sulphatide was incorporated in small unilamellar vesicles and administered to cultured oligodendrocytes. The association of the lipid probe to the cells in culture was saturable in time and with the concentration of the probe. The processes of association, internalization and subcellular distribution were followed by confocal scanning laser microscopy and appeared to be very rapid. Within 20 min a marked perinuclear staining was seen. After prolonged incubation the fluorescence distributed gradually over the cytoplasm and into cellular branches along structures suggestive of cytoskeletal elements. Lipid analysis demonstrated that ceramide was the major metabolite present in the cells but galactosylceramide, sphingomyelin and free fatty acid were also detected. In the culture medium only free fatty acid and sphingomyelin were found. Monensin did not affect the cellular association and internalization of the fluorescent sulphatide but markedly reduced its conversion to metabolic products. These results indicate that exogenous sulphatide is targeted to the Golgi apparatus prior to its lysosomal degradation.     

Possible role of sulphatide in the K+-activated phosphatase activity

A microsomal fraction rich in $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has been isolated from the outer medulla of pig kidney. $\text{(}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{activated}$ ouabain-sensitive phosphatase activity was studied in this preparation treated with arylsulphatase, an enzyme that specifically hydrolyzes ceramide galactose-3-sulphate. The activity of phosphatase was inactivated in proportion to the amount of sulphatide hydrolyzed. A maximum inactivation of ouabain-sensitive activity was obtained with 60% of the sulphatide content hydrolyzed. The inactivation caused by arylsulphatase was partially reversed by the sole addition of sulphatide. The evidence offered in this paper about sulphatide function in the sodium pump mechanism supports the idea that sulphatides are involved in the K⁺-activated phosphatase, a partial reaction of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$.     

The structure of keratan sulphates from various sources.

Quantitative structural comparisons were made between keratan sulphates isolated from various sources, namely pig nucleus pulposus, bovine cornea, and the costal cartilages of children, a young adult with Marfan syndrome and of old human autopsies. In human costal cartilage the amount of keratan sulphate increases markedly with age, although total mucopolysaccharide decreases to some extent, concomitant with a decrease in chondroitin 4-sulphate and an increase in chondroitin 6-sulphate. Comparison of molecular weights estimated by gel chromatography with those calculated from the molar ratio of galactose to mannose indicates that keratan sulphates of human costal cartilages of children and of a young adult with Marfan syndrome, and of pig nucleus pulposus, contain one mannose residue per chain, whereas keratan sulphates of old human costal cartilage and of bovine cornea contain one to two, and two, per chain respectively. After mild acid-catalysed desulphation of pig nucleus pulposus keratan sulphate, approx. 12% of the mucopolysaccharide aggregates irreversibly once the water is removed from the polysaccharide. The following conclusions have been drawn from a methylation analysis of keratan sulphates of various sources, aided by g.l.c.-mass spectrometry. (1) Fucose and N-acetylneuraminic acid are non-reducing terminal residues and the sialic acid is linked to the 3-position of galactose residues. (2) Pig nucleus pulposus keratan sulphate has approximately 4 non-reducing terminal groups per molecule and appears to be slightly less branched than the costal-cartilage keratan sulphate of children. The branching in human costal-cartilage keratan sulphates decreases with age. Bovine corneal keratan sulphate appears to be unbranched. (3) Mannose residues are linked by 3 different substituents in human costal-cartilage and bovine corneal keratan sulphates, and by two different substituents in pig nucleus pulposus keratan sulphate. (4) The sulphate ester groups are all on the 6-position of N-acetyl-glucosamine and galactose residues. The degree of sulphation increases with age in costal keratan sulphates with the increase mainly of the galactose 6-sulphate residues.     

Ca<Superscript>2+</Superscript>-dependent aggregation and permeabilization of erythrocytes by ω-hydroxypalmitic and α, ω-hexadecandioic acids

This paper presents the data and describes the Ca²⁺-dependent effect of the products of ω-oxidation of palmitic acid, as well as ω-hydroxypalmitic and α, ω-hexadecandioic acids, on rat erythrocytes. It is shown that in the presence of Ca²⁺ these acids induce aggregation of erythrocytes, which is accompanied by a reduction in the number of single cells in suspension. As well, a release of K⁺ from the cells occurs, which indicates the permeabilization of the plasma membrane. However, ω-hydroxypalmitic and α, ω-hexadecandioic acids are inferior to palmitic acid in their ability to induce Ca²⁺-dependent erythrocyte permeabilization. Bovine serum albumin and blood serum inhibit the effects of palmitic acid. At the same time, the influence of these agents on the effects of ω-hydroxypalmitic and α, ω-hexadecandioic acids appears to be much weaker. It is shown that ω-hydroxypalmitic and α, ω-hexadecandioic acids in the presence of Ca²⁺ induce an increase in the hydrodynamic diameter of single-walled lecithin liposomes, which indicates their fusion and (or) aggregation. The mechanisms of ω-hydroxypalmitic acid/Ca²⁺- and α, ω-hexadecandioic acid/Ca²⁺-induced effects on rat erythrocytes are discussed.     

Brain sphingoglycolipids in Krabbe's globoid cell leucodystrophy

Abstract— Seven sphingoglycolipids were isolated from the white matter of a patient with globoid cell leucodystrophy (Krabbe's disease). After purification by saponification and column and preparative thin-layer chromatography, these compounds were analysed for the carbohydrate composition and sequence and for fatty acid composition by paper and gas-liquid chromatography. The compounds were identified as gluco- and galactocerebrosides, lactosyl-ceramide, digalactosy I-glucosyl-ceramide, two types of tetrahexosyl-ceramides (asialo-ganglioside and globoside), and sulphatide. Glucocerebrosideconstituted 13 percent of total cerebroside in white matter, but sulphatide contained only galactose. Galactocere-broside and sulphatide exhibited compositions of fatty acids similar to those in normal white matter, with only minor abnormalities. Other sphingoglycolipids showed fatty acid patterns with relatively high proportions of longer-chain fatty acids, rather than the predominant C_18:0 acid usually found in ceramide hexosides of the brain. Hematoside, also found in the white matter in a significant amount, similarly contained a large proportion of longer-chain fatty acids, whereas other gangliosides contained predominantly C_18:0 acid. The abnormal ceramide hexoside pattern was restricted mostly to white matter except for glucocerebroside, which constituted 32 per cent of grey matter cerebroside. We postulate that the visceral type of sphingoglycolipids may be constituents of globoid cells, abundantly present in white matter and considered to be cells of mesenchymal origin.     

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.     

Analysis of gangliosides using fast atom bombardment mass spectrometry

The native gangliosides GM3, GM1, Fuc-GM1, GD1a, GD1b, Fuc-GD1b, GT1b and GQ1b were analysed by fast atom bombardment mass spectrometry (FAB-MS) in the negative ion mode in a matrix of thioglycerol. After permethylation the same gangliosides were analysed by electron impact (EI) and FAB-MS in the positive ion mode. The negative ion mass spectra furnished information on the molecular weight, the ceramide moiety and the sequence of carbohydrate residues. The sites of attachment and the number of sialic acids present could be deduced directly from the pattern of sequence ions. After addition of sodium acetate positive ion FAB-spectra of the permethylated samples show intense pseudomolecular ions M + Na, that provide evidence on the homogeneity of the samples. In addition, the ceramide part, the oligosaccharide moiety obtained after cleavage of the glycosidic bond of the hexosamine residue, the whole carbohydrate chain and the sialic acids are represented by specific fragment ions. With EI-MS further information can be obtained on the sphingosine and fatty acid components of the ceramide residue. The data show, that the combination of soft ionization mass spectrometry with classical EI-MS gives valuable information on the structure and homogeneity of gangliosides. The method is also applicable to the structural elucidation or quantitation of more complex gangliosides or glycolipid mixtures using only micrograms of material.     

The structure of the polysaccharide of Pachymenia carnosa

The sulphated polysaccharide of Pachymenia carnosa and its desulphated derivative have been studied by methylation analysis. Depolymerization during the desulphation process has been shown to occur mainly through the cleavage of (1→3) linkages. The methylation results indicate that the ratio of (1→4) to (1→3) linkages in the native polysaccharide is 1:2.26. The sulphate groups occur on positions 2, 4, and 2,6 of (1→3)-linked galactose residues. Methylations carried out in methyl sulphoxide with the Purdie reagents lead to extensive desulphation; 2-sulphate units appear to be more susceptible to desulphation than 4- or 6-sulphate units. Desulphation does not occur during methylation by the Hakomori method.     

Aberrant fatty acyl alpha-hydroxylation in human neuroblastoma tumor gangliosides

Abnormalities of ganglioside structure characterize the neoplastic state, and aberrant glycosylation has been implicated as underlying many new tumor ganglioside structures. However, variations in ceramide structure can also result in novel tumor gangliosides. To address systematically this aspect of ganglioside metabolism, we have initiated a study of the structures of the ceramide species of an oligosaccharide-homogeneous human tumor-derived ganglioside, GM2. The ganglioside was isolated from neuroblastoma tissue and purified by normal-phase high pressure liquid chromatography. Marked ceramide heterogeneity was observed; 18 individual ceramide species of neuroblastoma GM2 were separated by reversed-phase high pressure liquid chromatography and collected. Their structures were determined by a combination of negative- and positive-ion fast atom bombardment mass spectrometry and collisionally activated dissociation tandem mass spectrometry of the underivatized gangliosides. The striking finding was the detection of alpha-hydroxylation of a significant fraction of each of the major fatty acid species (16:0, 18:0, 20:0, 22:0, and 24:1); alpha-hydroxylated species quantitatively represented almost one-fifth of the total tumor GM2 species. Fatty acyl hydroxylation was also detected in the ceramide of several other human tumor gangliosides. In contrast, as previously known, fatty acyl hydroxylation was not detected in the normal human brain gangliosides GM3, GM2, and GM1. We propose that aberrant fatty acid alpha-hydroxylation is a novel and sometimes quantitatively significant characteristic of human tumor ganglioside metabolism.     

Absence of effect of aldosterone on sodium efflux catalyzed by the human erythrocyte Na+, K+-ATPase in vitro

The effect of physiological and pharmacological concentrations of aldosterone on Na+ efflux catalyzed by the human erythrocyte Na+,K+-ATPase in vitro were studied. Aldosterone had no significant effect on ouabain-sensitive Na+ efflux from fresh erythrocytes. In addition, aldosterone did not alter Na+ transport activity of stimulated Na+,K+-ATPase of Na+ loaded erythrocytes. Finally, Na+ efflux from Na+ loaded erythrocytes was not changed by preincubation of the cells with aldosterone. It is concluded that aldosterone in vitro does not modify pump activity of the human erythrocyte Na+, K+-ATPase.     

The determination of phytosphingosine-containing globotriaosylceramide from human kidney in the presence of lactosylceramide

Globotriaosylceramide, the natural substrate of alpha-galactosidase A (the enzyme deficient in Fabry's disease) was prepared from human kidney by repeated medium pressure chromatography on Lichroprep Si 60 (E. Merck) before and after peracetylation. The apparently homogeneous preparation migrating as a single band on HPTLC was analysed by fast atom bombardment mass spectrometry and 1H-NMR at 500 MHz. It was found that in this fraction two major molecular species were comigrating: Gal alpha 1-4Gal beta 1-4Glc beta 1-1ceramide with nervonic and lignoceric acid linked to phytosphingosine and Gal beta 1-4Glc beta 1-1 ceramide with palmitic acid linked to sphingosine.     

Fingerprinting of large oligosaccharides linked to ceramide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: highly heterogeneous polyglycosylceramides of human erythrocytes with receptor activity for Helicobacter pylori.

Highly microheterogeneous polyglycosylceramides (PGCs) of human erythrocytes with an average composition of about 25 monosaccharides linked to ceramide were analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). The human gastric pathogen Helicobacter pylori was earlier shown to bind this glycosphingolipid mixture by thin-layer chromatogram binding assay. The receptor activity was present along the whole nonresolved chromatographic interval. Mass spectra of intact PGCs were compared with corresponding spectra of oligosaccharides enzymatically released from the ceramides. Two subfractions of PGCs containing less than one and more than one sialic acid residue per molecule were used. MALDI-MS spectra were recorded in both linear and reflectron mode with the accuracies of 相似文献   

Formation of 20-oxoleukotriene B4 by an alcohol dehydrogenase isolated from human neutrophils

When 20-hydroxyleukotriene B4 (20-OH-LTB4) is incubated at pH 10.5 in the presence of NAD+ with an alcohol dehydrogenase isolated from human neutrophils, a polar product is formed as detected on reverse-phase high-performance liquid chromatography (RP-HPLC). The product is identified as 20-oxo-LTB4 (20-CHO-LTB4) on the basis of its co-elution with the authentic compound on HPLC, ultraviolet spectrometry and gas chromatography-mass spectrometry. The 20-CHO-LTB4-forming activity requires NAD+, but NADP+ scarcely replaces NAD+. The apparent Km for 20-OH-LTB4 is 83 microM and the Vmax is 2.04 mumol/min per mg of protein. The activity is inhibited by omega-hydroxy fatty acids such as 12-hydroxylauric acid, 16-hydroxypalmitic acid and 12(S), 20-dihydroxyeicosatetraenoic acid, but not by 4-methylpyrazole. At pH 7.0 with NADH, the purified dehydrogenase catalyzes the reverse reaction, the reduction of 20-CHO-LTB4 to 20-OH-LTB4.     

Characterisation of the potassium influx in rat erythrocytes.

In the rat erythrocyte membrane five different transport pathways for K+ are present. In addition to the well characterised K+ transport via the Na+ pump, the Na,K,Cl cotransport and the Ca(2+)-activated K+ channel, there are a K,Cl cotransport and a residual (leak) K+ transport. The K,Cl cotransport is already present under physiological conditions, and can be stimulated by N-ethylmaleimide treatment but not by a cell volume increase. A low ionic strength stimulated increase of the residual K+ influx can be demonstrated in rat erythrocytes after suppressing the K,Cl cotransport pathway. Between 11 and 19 weeks of age, rats show significant differences in all transport pathways of the erythrocyte potassium influx. Using influx data from individual rats a significant correlation between the total K+ influx and the ouabain-sensitive K+ influx has been found. Maintaining the rats on a diet poor in essential fatty acids leads to a significant change of the linoleic acid content of the erythrocyte membrane phospholipids. However, no significant effect on the various K+ transport pathways has been found. An analysis of the fatty acid composition of the erythrocyte membrane phospholipids showed significant correlations between the content of oleic acid, and arachidonic acid, and the ouabain-sensitive K+ influx (as well as the total K+ influx).     

Formation of omega- and (omega-1)-hydroxyfatty acids and plausible formation of ketofatty acid from microsomal phospholipids

Rat liver microsomes labeled with spin-labeled phosphatidylcholine release the label into the aqueous phase during the aerobic incubation with NADPH (Biochem. Biophys. Res. Commun. (1979) 87, 300-307). To establish the chemical nature of the released moiety, microsomes were labeled with [14C]phosphatidylcholine. When the 14C-labeled microsomes were incubated with NADPH under aerobic conditions, a few percent of the radioactivity was liberated into the aqueous phase within 60 min. Thin-layer chromatographic analysis of the radioactive substance liberated showed the presence of hydroxylated fatty acids derived from the 2-position of glycerol moiety. About one-third of the fatty acids formed from [14C]phosphatidylcholine during the incubation were converted into hydroxy-derivatives. Gas chromatography/mass spectrometry analysis further confirmed an NADPH-dependent formation of 16-hydroxypalmitic acid, 15-hydroxypalmitic acid, and hydroxy-derivatives of other fatty acids from the phospholipids of the microsomal membrane. Evidence was also obtained indicating the formation of ketopalmitic acid.     

Structural analysis of neutral glycosphingolipids from Ascaris suum adults (Nematoda: Ascaridida)

The neutral glycosphingolipid fraction from adults of the pig parasitic nematode, Ascaris suum, was resolved into four components on thin-layer chromatography. The high-performance liquid chromatography-isolated components were structurally analysed by: methylation analysis; exoglycosidase cleavage; gas-liquid chromatography/mass spectrometry; liquid secondary-ion mass spectrometry; and, in particular, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Their chemical structures were determined as: Glc(β1-1)ceramide, Man(β1-4)Glc(β1-1)ceramide, GlcNAc(β1-3)Man(β1-4)Glc(β1-1)ceramide and Gal(α1-3)GalNAc(β1-4)GlcNAc(β1-3)Man(β1-4)Glc(β1-1)ceramide; and were characterized as belonging to the arthro-series of protostomial glycosphingolipids. No glycosphingolipid component corresponding to ceramide tetrasaccharide was detected during these analyses. The ceramide composition of the parent glycosphingolipids was dominated by the 2-(R)-hydroxy C24:0 fatty acid, cerebronic acid, and C17 sphingoid-bases: 15-methylhexadecasphing-4-enine and 15-methylhexadecaphinganine in approximately equal proportions. The component ceramide monohexoside was characterized by an additional 15-methylhexadecaphytosphingosine. Abbreviations: CDH, ceramide dihexoside; Cer, ceramide; CMH, ceramide monohexoside; CPH, ceramide pentahexoside; CTH, ceramide trihexoside; CTetH, ceramide tetrahexoside; Hex, hexose; HexNAc, N-acetylhexosamine; HPTLC, high-performance thin-layer chromatography; LSIMS, liquid secondary-ion mass spectrometry; MALDI-TOF-MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; N-, Nz- and A-glyco(sphingo)lipids, neutral, neutralzwitterionic and acidic glyco(sphingo)lipids, respectively This revised version was published online in November 2006 with corrections to the Cover Date.     

Separation and mass spectrometric characterization of covalently bound skin ceramides using LC/APCI-MS and Nano-ESI-MS/MS

Ceramides covalently bound to keratinocytes are essential for the barrier function of the skin, which can be disturbed in diseases, such as psoriasis and atopic dermatitis. These ceramides of the classes omega-hydroxyacyl-sphingosine and omega-hydroxyacyl-6-hydroxysphingosine contain an omega-hydroxy fatty acid. For their separation and identification, a new analytical approach based on normal phase liquid chromatography coupled to atmospheric pressure chemical ionization mass spectrometry and tandem nano-electrospray mass spectrometry, respectively, is presented here. Tandem mass spectrometry provided structural information about the sphingoid base as well as the fatty acid moieties. The chain lengths of the bases ranged from C12 to C22, the chain lengths of the fatty acids varied between C28 and C36. In total, 67 ceramide species have been identified in human skin. The analytical methods presented in this work can be helpful for investigating alterations in the ceramide composition of the skin as seen in psoriasis, atopic dermatitis, and diseases with impaired epidermal barrier function.